APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES

Transcription

1 APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES

2 F.1 SCHLUMBERGER GUAR BASED SYSTEMS APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES

3 Toxicity Profile Acetic acid (CAS No ) Acetic acid dissociates in aqueous media to H + and the acetate anion (CH3CO2 - ). It is naturally occurring as the acid in apple cider vinegar and other fruit-derived products. It and several of its salts are commonly used as food additives (e.g., as flavor enhancers) and are listed as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA). ph: 2.5 at 50 g/l and 20 o C pka: 20 o C Acute Toxicity The oral LD50 in rats is 4,950 mg/kg, and the dermal LD50 in rabbits is 1,060 mg/kg. The 4-hour LC50 in rats is 11.4 mg/l. Irritation In the EU, acetic acid is classified as a skin and eye irritant at concentrations <25%. At >25%, it is classified as corrosive to the skin. Sensitization No data are available. Repeated Dose Toxicity In an 8-month study, rats dosed with 60 mg/kg acetic acid (three times per week) developed hyperplasia in the esophagus and forestomach. Genotoxicity Acetic acid was not mutagenic to Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 in an in vitro bacterial mutation assay with or without metabolic activation. Acetic acid (in media adjusted to a ph of 8.5) was also not genotoxic in an in vitro chromosomal aberration test using Chinese Hamster Ovary cells with or without metabolic activation. 1

4 Carcinogenicity No adequate carcinogenicity studies have been conducted on acetic acid. Reproductive Toxicity No studies could be located. Developmental Toxicity There were no effects on implantations or on maternal or fetal survival in mice, rats, or rabbits at doses up to 1,600 mg/kg. Key Study/Critical Effect for Screening Criteria There are no repeat dose toxicity studies that were considered adequate for human health risk assessment. The only study reported on the toxicity of acetic acid by oral gavage showed effects of irritation (probably due to the ph of acetic acid) at the site of contact in the gastrointestinal tract. No systemic effects were reported. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has maintained a group ADI of not limited for acetic acid and its potassium and sodium salts. The Australian drinking water guidance value for ph may apply to acetic acid. References ECHA REACH: JECFA: U.S. EPA HPVIS database: 2

5 Toxicity Profile Ammonium C6-C10 Alcohol Ethoxysulfate (CAS No ) Alcohol ethoxysulfates (AES) are a widely used class of anionic surfactants. No data could be located on alcohol ethoxysulfates in the C6 to C10 range. Thus, the C12-C14 alcohol ethoxysulfates were used to read-across to the C6-C10 alcohol ethoxysulfates. In the HERA report, the alcohol ethoxysulfate family covers commercial grades of lineartype primary alcohol ethoxysulfates of either sodium, ammonium or triethanolamine (TEA) salts. Sodium salts of AES are by far the commonly used grades. Acute Toxicity Alcohol ethoxysulfates are considered to have a low order of acute oral toxicity in the rat. In two guideline compliant acute oral toxicity studies, the oral LD50s were >2,000 mg/kg. Irritation The irritation potential of AES is concentration dependent. Materials with concentrations >70% are moderately to severely irritating to rabbit skin. At concentrations between 10 and 30%, the AES solutions exhibit mild to moderate irritancy under the conditions of an occluded patch test. AES concentrations below 1% are virtually non-irritating under the conditions of the acute skin irritation testing protocol. The triisopranolammonium salt of C12-14E2S (90% active material) and NaC12-14E2S (28% active material) were shown to be moderately to severely irritating to rabbit eyes. It has been shown that solutions containing less than 1-10% AES are slightly to moderately irritating to eyes and below 1%, AES solutions are virtually non-irritating. Sensitization AES are not considered to be skin sensitizers. Repeated Dose Toxicity Rats were given by oral gavage 0, 25, 75 or 225 mg/kg NaC12-14AE2S for 90 days. Four animals died during the treatment period and were considered incidental, and three animals died due to experimental procedures. There were no treatment-related effects on total body weight gain, food and water consumption, and systemic effects. In the 225 1

6 mg/kg animals, hyperplasia, submucosal edema and chronic ulceration were seen in the forestomach. In groups 25 and 75 mg/kg groups, 3/10 animals showed small eosinophilic foci in the stratified epithelium of the forestomach. The LOAEL appears to be 25 mg/kg/day and the NOAEL was not determined. Rats were fed in their diet 0, 40, 200, 500, 1,000 or 5,000 ppm NaC12-15E3S for 90 days. All animals survived until the end of the study. There were no treatment-related effects on clinical signs and body weights in females. Male body weights were significantly higher than controls in the 500 ppm group from week 10 onwards and at 200 ppm at weeks 11 and 13. At higher concentrations, there was no difference in body weights from the control values. At 5,000 ppm, male and female liver weights were significantly increased, and absolute testes weights were also increased; however, there was no significant difference with control animals when adjusted for terminal body weight. These organ weight changes were not accompanied by any histopathological, clinical chemical or hematological changes and were therefore considered to be adaptive in nature and not a toxic effect of the test substance. The NOAEL for this study is 5,000 ppm (250 mg/kg/day). Rats were given in their diet 0, 0.1 or 0.5% C12 AE3S for 2 years. There were no treatment-related effects (including tumor incidence). The results of this study suggests that the NOAEL was 0.5% (250 mg/kg/day). Rats were given in their drinking water 0 or 0.1% C12AE3S for two years. There were no treatment-related effects on survival, growth, food consumption, body weights, clinical laboratory findings, hematology and urinalyses. The only unusual finding was slight, but consistently higher water consumption by all rats receiving the test compound in their drinking water and a significant difference in the empty cecum to body weight ratio of females. Absolute organ weights were all comparable to controls and no consistent gross or histopathology was found. The incidence and types of tumors observed in the treated group was similar to that of control animals. The NOAEL for this study is 0.1% (75 mg/kg/day). Genotoxicity AES are not genotoxic when tested either in vitro or in vivo. Carcinogenicity The available oral and dermal long term toxicity/carcinogenicity studies, even if not performed according to accepted guidelines for carcinogenicity bioassays, appear to be conducted and documented in an acceptable manner. There is sufficient evidence that AES was carcinogenic in these studies. 2

7 Reproductive Toxicity AES did not adversely affect reproduction in the rat and the NOAEL for reproductive effects was > 300 mg/kg. Slight systemic effects were observed in the parental and F1 generation with a NOAEL of 86 and 149 mg/kg, respectively. Developmental Toxicity Based on the available information, there is sufficient evidence to conclude that AES are not teratogenic or a developmental toxicants. A NOAEL of >1,000 mg/kg/day can be estimated for teratogenicity and embryotoxicity, and the NOAEL for developmental toxicity appears to be >750 mg/kg/day. Key Study/Critical Effect for Screening Criteria A LOAEL of 225 mg/kg/day was reported in the 90-day oral gavage study on NaC12-14AE2S based on forestomach lesions in the rat. These lesions appear to be a localized effect due to the irritation properties of the test substance. No systemic toxicity was observed in this study. In contrast, when the test AES were given in the diet or in drinking water, not such irritation effects of the gastrointestinal tract was noted. This study will not be considered for the derivation of a drinking water guidance value since repeated dosing by oral gavage (a single daily oral bolus) is not a realistic human exposure scenario. Furthermore, 90-day and 2-year dietary and drinking water studies exist. The lowest NOAEL from the 90-day and 2-year dietary and drinking water studies is 75 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 75/100 = 0.75 mg/kg/day Drinking water guidance value = 2.6 ppm References Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Alcohol Ethoxysulphates, Human Risk Assessment (2003), Draft 3

8 Toxicity Profile Amorphous/Non-crystalline Silica (CAS No ) Silica Gel (CAS No ) Amorphous silica and silica gel have been reviewed in the OECD-SIDS program (OECD, 2004a,b). The CAS No is the general CAS No. for silicon dioxide which includes all forms of silicas (e.g. also crystalline and natural forms). Only the silica sub-classes, the synthetic amorphous silicas, are the subject of this assessment. Synthetic amorphous silicas (SAS) are in powder form which have a low water solubility: 70 mg/l Toxicokinetics After oral ingestion, there is no accumulation of SAS in body tissues. Upon cessation of exposure, rapid elimination occurs. Intestinal resorption appears to be insignificant in animals and humans. In a human study, the small apparent increases in the urine output of human volunteers were remarkably low as compared with the high dose of 2,500 mg SiO2 applied. After daily oral administration of 1,500 mg/kg SAS (FK 700) as aqueous suspension to rats for one month, there was no accumulation of SiO2 in the body: the average SiO2 content was: 1.5 μg in the liver, 6.4 μg in the kidney, and 5.3 μg in the spleen. The corresponding control values were 1.8, 7.2 and 7.8 μg SiO2, respectively. In a similar experiment in rats receiving 20 daily oral doses of 100 mg SAS (HDK V15) per animal (about 500 mg/kg) each, tissue values were slightly increased in liver and kidney: in liver 4.2 μg (control value 1.8 μg), in the spleen 5.5 μg (7.2 μg) and in the kidneys 14.2 μg (7.8 μg). In 12 human volunteers, no significant increased renal excretion of SiO2 was found following single oral ingestion of 2,500 mg (AEROSIL 175 and FK 700). Acute Toxicity The acute oral administration of various forms of SAS (aqueous suspension or gel) failed to produce signs of toxicity or deaths in treated animals with LD50 values greater than the 1

9 top doses applied, either by gavage: >3100 to >20000 mg/kg in mice and rats; or in the diet for 24 hours. Irritation Synthetic amorphous silicas are not irritating to the skin of rabbits exposed to 0.19 g (one case) or 0.5 g of dry or moistened test item under occlusive conditions for 4 or 24 hours. All products tested as a powder (0.1 g) have shown no or only weak and transient irritating effects on the conjunctivae of the eyes of rabbits with the iris and cornea not affected at all. Sensitization No experimental data are available on the synthetic amorphous silicas. Medical surveillance records on workers gave no evidence of skin sensitization over decades of practical experience. Repeated Dose Toxicity/Carcinogenicity Fischer rats and B6C3F1 mice were administered SAS in the diet at levels of 0, 1.25, 2.5, and 5% for 102 weeks. The animals were in good condition throughout and showed high survival. The tumor responses in all organs of SAS-fed rats and mice were not statistically significantly different from the controls. The NOAEL for rats and mice were 2,500 and 6,500 mg/kg/day, respectively. Genotoxicity There is no evidence for synthetic amorphous silica to induce mutations either in vitro or in vivo using standard genotoxicity tests. Reproductive Toxicity An early limited one-generation study on rats gave no evidence of adverse effects on reproduction performance at 500 mg/kg/day, the highest dose tested (NOAEL). But the reliability is poor due to the small group size of animals. Developmental Toxicity 2

10 SAS was examined for embryotoxic and developmental effects during the gestation phase in various animals species, rat, mouse, rabbit and hamster, at oral doses up to 1,600 mg/kg/day.. There were no significant signs of maternal or embryotoxic/developmental toxic effects in any species tested. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the frequencies occurring spontaneously in the control animals. Key Study/Critical Effect for Screening Criteria The lowest NOAEL from the two-year dietary study was 2,500 mg/kg/day for rats. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 2,500/100 = 2.5 mg/kg/day Drinking water guidance value = 0.09 ppm References OECD (2004a). IUCLID Data Set for Synthetic Amorphous Silica and Silicates: Silicon Dioxide (CAS No , , ; Silicic Acid, Aluminum Sodium Salt (CAS No ); Silicic Acid, Calcium Salt (CAS No ), UNEP Publications. OECD (2004b). Screening Information Dataset (SIDS) Initial Assessment Report for Synthetic Amorphous Silica and Silicates: Silicon Dioxide (CAS No , , ; Silicic Acid, Aluminum Sodium Salt (CAS No ); Silicic Acid, Calcium Salt (CAS No ), UNEP Publications. 3

11 Toxicity Profile Reaction mass of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2Hisothiazolin-3-one [3:1] (CAS No ) Acute Toxicity The oral LD50 of Kathon WT (1.5% a.i.) is >5,000 mg/kg (>75 mg a.i./kg) in male rats and >3,310 mg/kg (~49.6 mg a.i./kg) in female rats. The oral LD50 of Acticide 14 (14% a.i.) is mg/kg (~66 to 69 mg a.i./kg) in rats (combined sexes); and 465 mg/kg (69 mg a.i./kg) in male rats and 393 mg/kg (59 mg a.i./kg) in female rats. The dermal LD50 of Kathon CG is >5,000 mg/kg (>75 mg a.i./kg) in female rabbits. The dermal LD50 of Acticide 14 (14% a.i.) is 1,008 mg/kg (141 mg a.i./kg) in rats (combined sexes). The inhalation LC50 of Kathon 886F (13.9% a.i.) in male and female rats is 2.36 mg/l (0.33 mg a.i./l). Irritation Undiluted Kathon MW (1.5% a.i.) and Acticide 14 (14% a.i.) were corrosive to rabbit skin. Undiluted Kathon CG was severely irritating to the eyes of rabbits. Kathon RH 886T at 100 ppm (0.01% a.i.) was non-irritating to the eyes of rabbits. Sensitization CMI/MI has been tested in the guinea pig Buehler and Magnusson-Kligman tests and has been shown to be a strong skin sensitizer. It is also a strong contact allergen in humans. Repeat Dose Toxicity Rats were exposed to CMI/MI in a powdered commercial diet. CMI/MI concentrations were increased over the 13-week period (initial concentration up to week 2, intermediate concentration week 3-4, final concentration week 5 to 13). Concentrations in the control and CMI/MI groups were: 0/0/0, 40/57/80, 132/187/260, 400/570/800 ppm. There were no mortalities and no effects on body weight or food consumption. In each group, some animals showed slight alopecia or reddened raw or scabbed areas on the skin. There were no other differences in general behavior or appearance. There were no treatment-related changes in hematological, biochemical, urinary parameters nor any pathology. No systemic toxicity was observed up to and including the highest dose of either CMI/MI 1

12 (800 ppm, equivalent 29.1 mg a.i./kg/day). The NOAEL in this study is 800 ppm (29.1 mg a.i. /kg/day). Rats were exposed to CMI/MI via their drinking water at concentrations of 25, 75 or 225 ppm a.i. for three months (equivalent to an average intake of 2.38, 6.28 and 16.3 mg/kg/day in males and 4.06, 10.8, and 24.7 mg/kg/day in females). Two additional groups of rats were given tap water or tap water containing the inorganic ions present in the CMI/MI solution, at a concentration equal to that in the high dose group (225 ppm). This solution is referred to as the ion control solution. There were no mortalities in either sex at any dose level. There were no treatment-related effects on body weight up to the mid dose. A significant decrease in body weight was seen in males at the high dose during the first two weeks of the study. Food consumption was significantly decreased in males at all dose levels and in females mid and high dose groups during the first few weeks of dosing. Water consumption was significantly decreased at all concentrations. No overt clinical signs or ophthalmic were seen in any group throughout the 13 week toxicity or the reproductive phase. No adverse effects were seen in the examinations. No hematological treatment-related changes were seen in either sex at any dose level. There was a significant decrease in globulin and an increase in the albumin/globulin (A/G) ratio in males in both the high concentration and in the ion control groups, after 13 weeks of treatment. A significant decrease in total protein was also seen high concentration group, but was not seen in the ion control group. Females in the high concentration group showed a modest (40%) increase in SGOT (AST, aspartate aminotransferase) levels after 13 weeks of dosing. No changes were observed at any dose in mixed-function oxidase activities of the liver. At the end of 13 weeks of treatment, there was a significant increase in relative liver weight in males and in relative kidney weight in females at the high concentration but without any correlative changes in organ pathology. Histology revealed a local irritation of the glandular mucosa of the stomach in 7 of 15 males and 5 of 15 females at the high concentration. These subtle low level changes did not occur at the low or mid concentration nor were they present in either control group. No other compound related changes were seen. Reproductive organs at all doses were comparable to the control. The NOEL in this study was considered to be 75 ppm a.i. (equivalent to 6.28 and 10.8 mg/kg body weight/day in males and females, respectively), based primarily on irritation of the glandular stomach at the high dose. The No Observed Adverse Effect Level (NOAEL) is 225 ppm a.i. (equivalent to 16.3 and 24.7 mg/kg body weight/day in males and females, respectively), the highest dose tested, since no adverse effects were observed on the histopathology of any tissues or organs distant from the site of dosing. Genotoxicity The results of the in vitro mutagenicity tests were equivocal. In bacterial reverse mutation tests, CMI/MI was positive in Salmonella strain TA100 but was predominantly negative in other strains commonly tested strains. CMI/MI was mutagenic in the mouse lymphoma assay with and without metabolic activation. Effects at the Tk locus was seen both with and without S-9, but were enhanced with S9 mix. CMI/MI was not genotoxic 2

13 in the in vitro unscheduled DNA synthesis (UDS) assay with primary rat hepatocytes or in an in vitro chromosomal aberration test with Chinese hamster lung cells. The in vivo studies indicated that CMI/MI does not have relevant mutagenic potential in vivo. The positive mutagenic effect of CMI/MI found in in vitro gene mutation assays was not confirmed in the sex-linked recessive lethal test in Drosophila melanogaster nor in two unscheduled DNA synthesis (UDS) studies in the rat. CMI/MI also did not show any increase in cells with micronuclei in mice nor did it induce chromosomal aberrations in rat bone marrow cells. Carcinogenicity In a two-year rat chronic study, Kathon 886 is given as 14.2% active ingredient with a ph between 2-3. In the document with compiled batch information for Kathon the active ingredient is 13.2, [10.13 % CMI/ 3.85% MI] with 15.4% magnesium nitrate and 9.0% magnesium chloride. The dose levels were 0, 30, 100 and 300 ppm (equivalent to: 0, 2.0, 6.6, 17.2 mg a.i./kg/day in males and 0, 3.1, 9.8, 25.7 mg a.i./kg/day in females. There were no deaths. There were no treatment-related effects on body weight or body weight gain at doses or food consumption up to and including the mid dose group. A treatmentrelated and concentration-dependent decrease in water consumption was seen in both sexes in all treated groups throughout the study. These decreases ranged from 0-22% at low dose 3-30% at mid dose and 15-40% at high dose. These decreases appear to be due to the unpalatability of the CMI/MI and not its inorganic stabilizer salts since the water consumption in the salt control was comparable to the tap water control throughout the study. Based on the average daily water consumption, the high dose was judged to be a maximum tolerated dose. The decreases in body weight and body weight gain were seen in high dose animals throughout the study and may be secondary to decreased water consumption. No treatment-related clinical effects were recorded. No treatment-related ophthalmic, hematological. biochemical or urinary changes were noted. Organ weights were comparable to the control. No effects on type or incidence of neoplasms were seen at up to and including the high dose (males: 17.2 a.i.; females 25.7 mg a.i./kg/day). Slight to moderate forestomach hyperplasia was seen at both mid and high dose groups. Gastric irritation was the primary effect observed. No adverse effects on the histopathology of any other tissues/organs were observed away from the site of dosing. No systemic effects were observed. The NOEL in this study was considered to be 30 ppm a.i. (2.0 to 3.1 mg a.i./kg/day), based primarily on gastric irritation of the stomach at 100 and 300 ppm a.i.. The NOAEL was considered to be 300 ppm a.i. (17.2 to 25.7 mg/a.i./kg/day), since no evidence of systemic toxicity was observed at any dose and there was no adverse effects on the histopathology of any tissues/organs distant from the site of dosing at any dose. Reproductive/Developmental Toxicity A one-generation reproductive toxicity study was combined with the 13-week drinking 3

14 water study reported above in the repeated dose toxicity section. Reproductive capability was similar in all groups. Litter size and survival at birth was also similar in all groups. One dam at the high concentration lost the entire litter by day 4 due to a lactation problem. This is not uncommon and was not considered treatment related. Pups of the other high concentration group dams, except one, survived and thrived to day 21. Thus, no adverse effects were observed on reproductive capability of male and female rats and no effects were observed on fetal health or pup survival (to day 21) up to and including the high dose (equivalent to 16.3 and 24.7 mg/kg/day in males and females, respectively]. In a two-generation reproductive toxicity study, rats were dosed with CMI/MI (Kathon 886F: 11.1% CMI, 3.7% MI a.i.) at concentrations of 0 (control), 0 (magnesium salt control), 30, 100 or 300 ppm a.i. For the P1 generation, this was equivalent to 0, ; , and mg a.i./kg/day; and in the P2 generation 0, , , and mg a.i./kg/day. There were no treatment related effects on survival, food consumption or overt signs of toxicity. A decrease in bodyweight gain was noted initially in the P1 generation. This was thought to be linked to reduced water consumption since significant dose-related reduction in water consumption was seen at all concentrations in both the P1 and P2 generations, during the premating, gestation and lactation stages. Treatment-related histopathological changes were seen in the stomach in the P1 and P2 generation. These included erosions of the glandular mucosa, edema and inflammation in the submucosa of the glandular and nonglandular stomach, with hyperplasia and hyperkeratosis of the nonglandular stomach at the 100 and 300 ppm a.i. Other histopathological changes were seen but were not dose dependent.the estrus cycle in P1 or P2 females at any treatment level was comparable with the controls, as was the sperm motility, morphology, testicular sperm count or caudal epididymal reserves of P1 or P2 males. All other endpoints (gestation index, gestation length, number of pups per litter or treatment-related gross findings in F1 or F2 pups) were similar to those in the controls in either generation. The study authors considered that rats exposed to CMI/MI in the drinking water through two generations had a NOAEL of 30 ppm a.i. ( mg/kg/day in the P1 animals; mg/kg/day in the P2 animals) for parental animal toxicity, based on the gastric irritation of stomach at higher doses. The NOEL for reproductive toxicity is 300 ppm a.i. ( mg/kg/day in the P1 animals; mg/kg/day in the P2 animals), the highest dose tested. There were no effects on fertility or fetal development at any dose level. In a rat oral gavage developmental toxicity study on Kathon 886 (13.9% a.i.), the developmental NOEL for CMI/MI is >15 mg a.i./kg during organogenesis (highest dose tested). In a rat oral gavage developmental toxicity study on Acticide 14 (10.2% CMi/ 4% MI), the NOAEL for maternal toxicity is <3.95 mg a.i./kg; the NOAEL for teratogenicity is >19.6 mg a.i./kg; and the NOAEL for embryotoxicity is >19.6 mg a.i./kg. In a rabbit oral gavage developmental toxicity study on Acticide 14 (10.2% CMI/ 4% MI), the developmental NOAEL is >5.49 mg a.i./kg; the NOAELs for maternal toxicity and fetal toxicity are 1.41 mg a.i./kg. In a rabbit oral gavage developmental toxicity study on Kathon MW (13.9% a.i.), the NOEL for maternal toxicity is 2 mg a.i./kg; the developmental NOEL is 8 mg a.i./kg, the highest dose based on severe 4

15 maternal toxicity at 20 mg a.i./kg. Key Study/Critical Effect for Screening Criteria A two-year dietary study was conducted in rats with CMI/MI (13.2% active ingredient). No systemic effects were observed. There was, however, gastric irritation of the stomach at 100 and 300 ppm. The NOEL for this study is 30 ppm (equivalent to 2.0 and 3.1 mg a.i./kg/day for males and females, respectively). The NOEL of 2.0 mg/kg/day will be used for deriving a drinking water guidance value. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 2/100 = 0.02 mg/kg/day Drinking water guidance value = 0.07 ppm Reference EU SCCS (2009). Opinion on the mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2H-isothiazol-3-one [3:1], Colipa N o P56, Scientific Committee on Consumer Safety, SCCS/1238/09. 5

16 Toxicity Profile C6-C10 Alcohol Ethoxylates Alcohol ethoxylates are a class of non-ionic surfactants. The AEs have the basic structure Cx-yAEn. The subscript (x-y) following the C indicates the range of carbon chain units. AEs with carbon unit range between C8 to C18 are most commonly used in household detergent products. The hydrocarbon chain can be either linear or branched. AEs also contain an ethylene oxide (E) chain attached to the alcohol. The degree of ethylene oxide polymerization is indicated by the subscript (n) which indicates the average number of ethylene oxide units. In household products the average ethylene oxide chain length commonly ranges between 3 and 12 units. The toxicity profile of C6-C10 alcohol ethoxylates has been evaluated as part of the class of alcohol ethoxylates ranging from C8 to C15 in the HERA report. Acute Toxicity The acute oral LD50 of C9-11AE2.5 in rats was calculated to be >4,000 mg/kg and <10,000 mg/kg. The acute oral LD50 of C7-9AE6 in rats was determined to be <2,000 mg/kg. The acute oral LD50 of C9-11AE8 in fasted rats was found to be 1,200 mg/kg. The oral LD50 in rats for C12-13AE6.5 is 2,100 mg/kg. The oral LD50 in rats for C12-15AE7 is 1,700 mg/kg. For C14-15AE11, the acute oral LD50 values were reported to be 720 mg/kg (neat) and 1,800 mg/kg (given as 50% (m/v) solution in corn oil). The oral LD50 in rats for C12-15AE11 is >2,00 mg/kg for males and between 1,000 and 2,000 mg/kg for females. The oral LD50 in rats for C14-15AE13 is 1,100 and 1,000 mg/kg in two separate studies. The dermal LD50 of C7-9AE6 was >2 g/kg. The dermal LD50 values for AEs with an alkyl chain length of 9 11 carbon atoms in three different rat studies were determined to be >2,000 mg/kg and >4,000 mg/kg. An acute dermal LD50 value of >2,000 mg/kg was determined for C12-14AE3 and C12-14AE6 in two separate studies. The acute dermal LD50 of C12-15AE7 was determined to be >2,000 mg/kg. The acute rat 4h-LC50 of C9-11AE5 generated as a mist was determined to be >0.22 mg/l. Talmage (1994) reported that alcohol ethoxylates were not acutely toxic to rats at concentrations less than or equal to their saturated vapor concentrations in air. Acute toxic thresholds were reached only when animals were exposed to the undiluted test chemical in the form of a respirable mist or aerosol. Under these conditions, 1- or 4-hour inhalation LC50 values ranged from 1.5 to 20.7 mg/l. Some studies reported no mortalities (1-hours LC50 -study) occurred at concentrations as high as 52 mg/l. Irritation 1

17 Alcohol ethoxylates with varying alkyl chain lengths and ethoxylation degree were found to be slightly to severely irritating to skin in rabbits and rats. The degree of irritation was dependant on the type of patches used (open application versus full occlusion), the exposure time as well as the concentration of the test material. In humans, AEs are less irritating to skin than in animals. Neat applications of a range AEs in a 4h human patch test did not warrant these chemicals to be classified as skin irritants under EU legislation, while the same AEs would have been classified for skin irritation on the basis of animal data. Alcohol ethoxylates range from mildly to severely irritating to rabbit eyes. Rinsing the eyes directly after exposure with water for 20 to 30 seconds greatly reduced the severity of the effects such that these products produced only mildly irritating effects. The degree of irritation is concentration-dependent as dilutions in water cause proportionally lower irritation. Generally, concentration of 0.1% were non-irritating, and concentrations of 1 to 10% ranged from slight to moderately irritating. No relationship could be established between the chemical structures of the tested AEs and their eye irritation responses. Sensitization Alcohol ethoxylates should not be considered as skin sensitizers. A substantial amount of skin sensitization studies in guinea pigs following either the Magnusson-Kligman maximization or the Buehler testing protocol are available to evaluate the skin sensitization potential of AEs. Although a mild skin sensitization reaction was observed in a study following the Magnusson-Kligman protocol, the weight of evidence clearly supports the assessment that AEs should not be considered as skin sensitizers. This is further supported by clinical and market data that demonstrate the absence skin sensitization responses to AEs when tested under the conditions of the HRIPT or when used in AE containing consumer products. Repeated Dose Toxicity C10AE5 was fed to rats at doses of 0, 125, 250 or 500 mg/kg for 90 days. There were not treatment-related clinical signs, body weight gain, food consumption or feed efficiency. There was a slight increase in absolute liver weights as well as a trend toward a dose-dependent increase in the liver weight/body weight ratio, with a statistically significant increase in the high dose group. However, the histological evaluation did not reveal any indication of hepatotoxicity and therefore the increase in liver weights was not interpreted to be a toxicological effect. It can be considered to be an adaptive response as a result of extensive metabolism of the test compound by the liver. There were no other gross or histopathological changes that were considered treatment-related. The NOAEL for this study can be considered to be 500 mg/kg/day. Rats were given in their diet 0, 125, 250, 500, 1,000 and 3,000 ppm C9-11AE6 for 90 2

18 days. There were no signs of toxicity at any dose level. The NOAEL is 3,000 ppm (approximately 150 mg/kg/day). Rats were given in their diet 0. 04, 0.2 and 1.0% C9-11AE8 for 90 days. Lower body weight gain and decreased food consumption were noted in the 1% males and females and in the 0.2% females from week 1 through the end of the study. Further statistical analyses revealed a significant decrease in the mean body weight gain noted in the 1% females and the decreases in mean food consumption noted in the 1% males and females. The differences noted in the 0.2% females were not statistically significant. The investigators considered these observations to be the result of poor palatability of the test substance. There were no other treatment-related effects. The NOAEL for this study is 1.0% (about 400 mg/kg/day). Rats were fed C12-15AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% and 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the 0.25% and higher dose groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the 0.25% groups and higher. There were not treatment-related changes in hematology, urinary and clinical chemistry parameters. The histological examination of the liver at necropsy revealed hepatocytic enlargement at 0.125% and higher, suggesting an increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (102 mg/kg/day). Rats were fed C12-14AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% and 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the 0.25% and higher dose groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the 0.25% groups and higher. There were not treatment-related changes in hematology, urinary and clinical chemistry parameters. The histological examination of the liver at necropsy revealed hepatocyte cell enlargement at 0.125% and higher, suggesting an increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (110 mg/kg/day). Wistar rats were fed C14-15AE7 in the diet at concentrations of 0, 300, 1,000, 3,000, and 10,000 ppm for 90 days. All animals survived until their scheduled necropsy date. Significant treatment-related effects on body weight (i.e., reduced mean body weights in males at 10,000 ppm and in females at 3,000 ppm), food intake (i.e., reduced intake in both sexes at 10,000 ppm and at 3,000 ppm for females), organ weights (i.e., increased relative liver weight in both sexes at 3,000 and 10,000 ppm and in females also at 1,000 ppm; increased spleen weight in males at 10,000 ppm; clinical chemistry (i.e., confined to 10,000 ppm dose groups; significantly higher urea, chloride and potassium levels in males; significantly higher urea, chloride and cholesterol levels in females) and hematology (i.e., in both sexes at 10,000 ppm and in males also at 3,000 ppm increased total leukocytes and lymphocytes; females at 10,000 ppm showed depression in numbers 3

19 of neutrophils, mean cell volume and mean cell hemoglobin) were identified in one or both sexes fed with dietary concentrations of 3,000 and 10,000 ppm. There were no compound-related histopathological effects at any dose level. Minor, but statistically significant changes in liver weight, kidney weights and plasma urea concentration were recorded in female rats in the 1,000 ppm group were not of toxicological significance. The NOAEL for this study is 1,000 ppm (50 mg/kg/day). Rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1%, 0.5% and 1% for 90 days. There were no treatment-related changes in body weight, food intake, and organ weights including those of the reproductive system, clinical chemistry and hematology at any treatment level. The NOAEL is 1% in the diet, which corresponded to 700 and 785 mg/kg/day for males and females, respectively. Rats were fed C12-13AE6.5 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Rats were fed C14-15AE7 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Charles River rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1, 0.5 and 1% for two years. There was a dose-related decrease in body weights in the 0.5 and 1% females and in the 1% males, the likely cause being poor palatability of the diet. At study termination, elevated organ-to-body weight ratios were noted for the liver, kidney, heart and thyroid/parathyroid glands in the 1% dietary group. The only significant histopathological finding prevalent in all dose groups was a dose-related increase in incidence of focal myocarditis at 12 months but not at study termination at 2 years. No other treatment-related histopathology was noted. The NOAEL was established at the 0.5% dietary level, which corresponds to about 162 and 190 mg/kg/day for males and females, respectively. Genotoxicity In all available in vitro and in vivo genotoxicity assays, there was no indication of genetic toxicity of broad range of structurally different alcohol ethoxylates. 4

20 Carcinogenicity It can be concluded that alcohol ethoxylates as a class are not carcinogenic by the oral route based of available oral long term toxicity/carcinogenicity studies on AEs. Charles River rats were given in their diet 0, 0.1, 0.5 or 1% C14-15AE7 for two years. There was no treatment-related changes in general behavior and appearance. The survival rate of the test animals was comparable if not better than the controls. Body weights of 0.5% females and 1% males and females had significantly lower weight gains than the control. There were no treatment-related effects on organ weights and tumor incidence. Sprague-Dawley rats were fed C14-15AE7 at 0.1, 0.5 and 1% in the diet for two years. A treatment-related body weight depression was observed in females at the two highest treatment levels and in males at the 1% dose level, probably due to the poor palatability of the diet. There was no evidence for any carcinogenic activity. Sprague-Dawley rats were fed C12-13AE6.5 in the diet at doses up to 1% (500 mg/kg/day). Reduced food consumption was noted at the higher dose levels (i.e., 0.5 and 1% for females and 1% for males), resulting in a lower body weight gain compared to the control group. No treatment-related histopathology was found and no increase in tumor incidence was observed. Reproductive Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% ( about 0, 25, 50 and 250 mg/kg/day). No treatment related effects in the parents or pups on general behavior, appearance or survival were observed. Fertility of treated groups was comparable with the controls. The only observation was related to a reduced weight gain of parental rats and pups relative to the control at the 0.5% dose level. The NOAEL for reproduction was therefore set at the highest dose level which was 0.5% dietary level (250 mg/kg/day). In a two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% (about 0, 25, 50 and 250 mg/kg). The control group and the six treated groups comprised of 25 male and 25 female animals. Three of the groups received the compound continuously during the study. In the other three groups the females received the compound only during gestational days 6-15 and the males were untreated. No treatment-related changes in behavior or appearance were observed in the parental rats or pups throughout the study. Female rats from the 0.5% continuous treatment group gained slightly less body weight compared to control females. No other consistent differences in body weight were observed. Food consumption was similar for control and treated rats. No compound related differences were seen between control and treated rats with respect to fertility, gestation or viability indices. The average 21-day body weights for pups at the 0.5% continuous treatment group were significantly lower as compared to the average pup body weights in the 5

21 controls. No other compound-related changes in body weight were observed. None of the deaths of parental rats during the study was considered to be compound-related. Examination of organ weight values revealed that compound-related effects were limited to increased group mean relative liver weights of male and female F1 from the 0.5% continuous feeding group at the 91-day sacrifice, and increases in group mean relative liver weights of males from the 0.5% continuous feeding group of the F2 generation at the 60-day and caesarean section sacrifices. No compound-related histopathological lesions were observed in any of the tissues examined from rats for the F0 and F1 generations. The NOAEL for reproductive toxicity is at least 0.5% in the diet (250 mg/kg/day). Developmental Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% ( about 0, 25, 50 and 250 mg/kg/day). General behavior, appearance and survival were not affected by treatment. At the 0.5% dose level, adults and pups gained less weight than the control rats. In the 0.5% dose group, there was a statistical increase in embryo lethality and soft tissue anomalies and at the 0.1% there was a statistical decrease in mean fetal liver weight. Neither of these effects was considered to be treatment-related by the authors as they showed no dose response characteristics. The NOAEL for maternal toxicity is 50 mg/kg/day. The NOAEL for developmental and teratogenicity is 0.1% (50 mg/kg/day). Pregnant rabbits were given by oral gavage 0, 50, 100 or 200 mg/kg C12AE6 from GD 2 to16. Nine control rabbits and 31 treated rabbits died during the study. Surviving rabbits at the 200 mg/kg dose level generally showed slight losses of body weight. At 100 and 200 mg/kg, ataxia and a slight decrease in body weight was observed in the pregnant animals. In seven treated and two control rabbits early deliveries were recorded. There were no treatment-related effects on corpora lutea, implantations, number of live fetuses and spontaneous abortions. The NOAEL for maternal toxicity is 50 mg/kg/day; the NOAEL for developmental toxicity is 200 mg/kg/day. In two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% (about 0, 25, 50 and 250 mg/kg). On the 13th day of the gestation period a representative number of female rats from each treatment group of the FC generation (i.e., pups from the 3rd mating of the F0 and F1 parental generation) were sacrificed. Laparotomies were performed and the uterus was examined for uterine abnormalities, normal implantation and resorption sites. Remaining females were sacrificed on gestational day 21. Various maternal and fetal parameters showed occasional values that were significantly different from the corresponding controls. However these were not considered related to the material tested as none occurred at the high feeding level and no dose response for these parameters was apparent. With respect to body weight gains, parental female rats and pups of the high dose group did not gain as much body weight as the control rats. Examination of organ weight values reveal compound related effects were limited to increased group mean liver weighs of male and female P1 generation from the 0.5% continuous feeding group at the 6

22 91 day sacrifice and increase in group mean relative liver weights of males of the 0.5% continuous feeding group of the P2 generation at the 60 day section sacrifices. The NOAEL for maternal and developmental toxicity was established at the 0.1% in the diet (50 mg/kg/day). Key Study/Critical Effect for Screening Criteria AEs of different structures with regard to the length of the alkyl chain and the degree of ethoxylation have been evaluated in a number of 90-day and two-year oral toxicity studies. The lowest NOAEL of AEs for systemic toxicity was established at 50 mg/kg/day in two chronic (two-year) dietary studies on C12-13AE6.5 and C14-15AE7. Effects observed at the LOAEL were related to significantly elevated organ-to-body weight ratios for liver, kidney and heart, although there were no adverse histopathological changes. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 50/100 = 0.5 mg/kg/day Drinking water guidance value = 1.75 ppm References Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Alcohol Ethoxylates (2009) Talmage, S.S. (1994). Environmental and Human Safety of Major Surfactants Alcohol Ethoxylates and Alkylphenol Ethoxylates. The Soap and Detergent Association. Lewis Publishers, Boca Raton, Florida, USA page 35; cited in the HERA report. 7

23 Toxicity Profile Crystalline Silica, Cristobalite (CAS No ) Crystalline Silica, Quartz (CAS No ) Silica is an off-white granule that occurs naturally in various crystalline and amorphous or other non-crystalline forms. Crystalline silica is characterized by silicon dioxide (SiO2) molecules oriented in fixed, periodic patterns to form stable crystals. The primary crystalline form of silica is quartz. Other crystalline forms of silica include cristobalite, tripoli and tridymite. Particle size is a key determinate of silica toxicity, since toxicity is restricted to particles that are small enough to be deposited into the target regions of the respiratory tract. Oral Exposure No oral studies were located; however, crystalline silica is not expected to exhibit toxicity by the oral route. Although absorption studies were not found for crystalline silica, kinetic studies on amorphous silica show no absorption from the gastrointestinal tract. Dermal Exposure No dermal studies were located; however, crystalline silica is not expected to exhibit toxicity by the dermal route. Inhalation Exposure See attached OECD-SIDS Initial Targeted Assessment Profile on Quartz and Cristobalite, SIAM 32, April 2011.

24 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

25 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

26 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

27 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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28 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

29 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

30 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

31 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

32 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

33 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

34 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

35 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

36 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

37 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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38 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

39 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

40 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

41 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

42 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

43 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

44 Toxicity Profile Diammonium peroxidisulfate Ammonium persulfate (CAS No ) Diammonium peroxisulfate or ammonium persulfate has been reviewed in the OECD- SIDS program (OECD, 2005a,b). Ammonium persulfate is distributed into the water compartment in the ionic form of the ammonium cation and persulfate ion. The persulfate anion will readily hydrolyze (decompose) into sulfate ions. Acute Toxicity The acute oral LD50 in rats is 495 mg/kg, and the acute dermal LD50s in rats and rabbits are >5,000 mg/kg. In acute inhalation studies in rats, the 4-hour LC50 was generally greater than the maximum attainable concentration (>2,950 mg/m 3 for ammonium persulfate). Irritation Ammonium persulfate is slightly irritating to the eye and skin of rabbits. Studies in humans indicate that aqueous solutions of 5% persulfate or higher can cause skin irritation. Sensitization Results of animal skin sensitization tests (Buehler Test and Maximization Test) were negative when persulfate was applied topically, but was positive when persulfate was injected intradermally in induction and challenge phases in a non-standard Maximization Test. Numerous dermal challenge tests indicate that all persulfates are dermal and respiratory sensitizers in humans occupationally exposed to persulfates in hairdressing salons and, in one case, in a production facility. In controlled clinical trials with nonoccupationally exposed-subjects (ammonium and sodium salts), no sensitization reactions were observed. Repeated Dose Toxicity Rats were fed in their diet 0, 300, 1,000 or 3,000 ppm sodium persulfate (0, 23, 100 and 225 mg/kg/day) for 90 days. On day 48 of the study, the concentration of the group 1

45 receiving 1,000 ppm was increased to 5,000 ppm for the remainder of the study. Body weights was decreased in the two highest dose groups during the last six weeks of treatment. There were no treatment-related effects on urinalysis, clinical chemistry or hematology parameters. Pathological findings were limited to the 3,000 ppm group only and consisted of necrosis and atrophy of the gastrointestinal tract epithelial lining. The absence of the gastrointestional lesions in the group receiving 1,000 ppm for 8 weeks, followed by 5000 ppm for 5 weeks, indicates that the lesions are related both to concentration in diet (dose) and length of exposure. There were no treatment-related pathological findings in reproductive organs or any other organ system or tissue. Genotoxicity Sodium persulfate was not genotoxic in a bacterial reverse mutation (Ames) assay or in an unscheduled DNA synthesis (UDS) assay with rat hepatocytes. It was also not active in a rat bone marrow micronucleus assay and when tested in vivo in a rat hepatocyte UDS assay. Carcinogenicity A 51-week dermal study in female SENCAR mice exposed to 0.2 ml of a 200 mg/ml solution of ammonium persulfate showed that ammonium persulfate is neither a tumor promoter nor a complete carcinogen when applied to the skin. Reproductive/Developmental Toxicity In a developmental/reproduction study with ammonium persulfate in rats (OECD 421), no effects on reproductive performance, fertility, fetal anomalies, fetal viability, spermatogenesis, spermatogenic cycle were reported up to 250 mg/kg/day. Dose levels were chosen based on the acute lethality studies for the ammonium salt and on a 90-day repeat-dose study in rats with the sodium salt (high dose: 225 mg/kg/day). In the developmental/reproduction study, animals were dosed prior to and during mating through gestation until lactation day 4. There was a transient depression in pup body weight at the 250 mg/kg dose level on lactation day 0 which resolved by day 4. This effect was not considered adverse. Based on the available data, the persulfates do not show evidence of reproductive or developmental toxicity. The NOAEL is 250 mg/kg/day. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for sulfate may apply to sulfate (500 ppm, health; 250 ppm, aesthetic). There are no existing drinking water guideline values for ammonium ions. 2

46 Reference OECD (2005a). IUCLID Data Set for Ammonium persulfate (CAS No ); Potassium persulfate (CAS No ); Sodium persulfate (CAS No ), UNEP Publications. OECD (2005b). Screening Information Dataset (SIDS) Initial Assessment Report for Ammonium persulfate (CAS No ); Potassium persulfate (CAS No ); Sodium persulfate (CAS No ), UNEP Publications. 3

47 Toxicity Profile Diatomaceous Earth, Calcined (CAS No ) Silica is an off-white granule that occurs naturally in various crystalline and amorphous or other non-crystalline forms. Crystalline silica is characterized by silicon dioxide (SiO2) molecules oriented in fixed, periodic patterns to form stable crystals. The primary crystalline form of silica is quartz. Other crystalline forms of silica include cristobalite, tripoli and tridymite. Particle size is a key determinate of silica toxicity, since toxicity is restricted to particles that are small enough to be deposited into the target regions of the respiratory tract. Uncalcined diatomaceous earth (CAS No ) typically contains around 1% crystalline silica. When diatomaceous earth is subjected to pressure or is processed ("calcined") at temperatures above 1000 C some of the amorphous silica is converted to crystalline silica in the form of cristobalite. Calcined diatomaceous earth can contain anywhere from 1% to 75% cristobalite. Oral Exposure No oral studies were located; however, crystalline silica is not expected to exhibit toxicity by the oral route. Although absorption studies were not found for crystalline silica, kinetic studies on amorphous silica show no absorption from the gastrointestinal tract. Dermal Exposure No dermal studies were located; however, crystalline silica is not expected to exhibit toxicity by the dermal route. Inhalation Exposure See attached OECD-SIDS Initial Targeted Assessment Profile on Quartz and Cristobalite, SIAM 32, April 2011.

48 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

49 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

50 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

51 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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52 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

53 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

54 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

55 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

56 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

57 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

58 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

59 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

60 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

61 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 4 of 10

62 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

63 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

64 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

65 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

66 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

67 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

68 Toxicity Profile 2-Butoxyethanol (CAS No ) 2-Butoxyethanol has been reviewed by U.S. EPA (2010) and an oral RfD and inhalation RfC is available in IRIS. An EU Risk Assessment Report is also available on 2- butoxyethanol (2006), an the information in this toxicity profile has been obtained from this report. Toxicokinetics/Metabolism Uptake of EGBE by the oral route is rapid and is assumed to be complete (100% absorption). In toxicokinetic studies involving radiolabeled EGBE, the forestomach, liver and kidneys were the organs where most of the radiolabel was found. The major metabolite of EGBE is butoxy acetic acid (BAA). EGBE is metabolized to butoxy aldehyde (BAL) by alcohol dehydrogenases, which is then further metabolized to BAA by aldehyde dehydrogenases. The metabolism of EGBE to BAA appears to be a saturable process. The other metabolites of EGBE are (in order of magnitude): the glucuronide conjugate of EGBE (a competing pathway to BAA formation and whose percentage increases relative to dose), the sulfate-conjugate of EGBE and ethylene glycol. Elimination is rapid and occurs mainly by urinary excretion. EGBE does not accumulate in tissues, and the metabolic profile does not change after repeated exposures compared to acute exposures. The in vitro dermal uptake studies, measuring the rate of absorption of liquid EGBE through human skin, gave results that varied by a factor of 25 (0.064 mg/cm 2 /hr for the lowest and 1.66 mg/cm 2 /hr for the highest). In vitro, rate of absorption is highly dependent on the concentration of the aqueous solution of EGBE used. In vivo, in one study, an estimation of the skin penetration gave results in the following range: 7 to 96 nmol/min/cm 2 (0.008 mg/cm 2 /hr to mg/cm 2 /hr) for pure liquid EGBE. Another study performed with liquid EGBE has showed that greater absorption was found with EGBE 50% in water than neat EGBE (dermal flux 1.34 mg/cm 2 /hr and 0.26 mg/cm 2 /hr, respectively). This is consistent with data available in vitro and in animals. In the EU RAR, where required, a permeation rate of 0.63 mg/cm 2 /hr will be used. This is the upper figure from the in vivo data and is consistent with the in vitro data. PBPK models has been developed for EGBE and can be used to derive animal to human toxicokinetic extrapolation factors for the inhalation route. These factors are based on the toxokinetics of BAA since this is the metabolite that causes the critical toxic effects. Acute Toxicity 1

69 The oral LD50 values are >1,000 mg/kg, and the dermal LD50 for rabbits (the most sensitive species) is approximately 500 mg/kg. The 4-hour LC50 in rats is approximately 450 ppm (2,214 mg/m 3 ), with higher values seen in other species. Suicide cases suggest that the LOEL in humans is about 400 mg/kg. It should be noted that this is a worst case estimation of the LOEL derived from McKinney et al. (2000) in which the possible range of exposure was between 400 and 1200 mg/kg. EGBE causes hematotoxicity in vivo in rats, and that the metabolite BAA is considered responsible for this effect. The rat, mouse, hamster and baboon are very sensitive to the hemolytic effects of EGBE; whereas the dog, guinea pig, pig, cat, rabbit and humans (30- times less sensitive than rats) are resistant to EGBE-induced hematoxicity. The hemolysis from EGBE exposure is due to a decrease of erythrocyte deformability due to erythrocyte swelling (this also explains the formation of thrombosis). Newly formed erythrocytes are more resistant than old ones. Irritation EGBE is moderately irritating to the skin of rabbits when applied under occlusive conditions for 4 hours. It is also an irritant or severely irritant to the eyes of rabbits. Sensitization EGBE is not a skin sensitizer to guinea pigs. A 10 % (v/v) aqueous solution of EGBE was not sensitizing in a human repeat patch test. Repeated Dose Toxicity (Oral Studies Only) Male and female Sherman rats were fed in their diets 0, 0.03, 0.125, 0.5 or 2% EGBE for 90 days. The average daily intakes were 0, 18, 76, 310 or 1,540 mg/kg-day. There were no treatment-related deaths and appetite was not affected. Tests for blood in pooled urine sample after 3 and 6 days of testing from the 0, 0.5 and 2% groups were negative. The mean weight gain was lower in the 2% group compared to controls. Relative kidney and live weights were increased in the 2% group, and relative liver weights were only increased in the 0.5% group. No adverse histopathological effects were observed The NOAEL can be considered to be % (76 mg/kg-day) (Mellon Institute of Industrial Research, 1952; EU, 2006). Male and female DW albino rats were given in their diet 0, 0.01, 0.05, 0.25 or 1.25% EGBE (corresponding to 7, 38, 188 or 919 mg/kg-day for males; and 0, 9, 41, 222, or 976 mg/kg-day for females) for three months. There was one death in the 1.25% group on day 73 of the study. Food consumption was decreased in the 1.25% group (both sexes). There was also a significant decrease of food consumption in the 0.25% males. Body 2

70 weight gain was significantly decreased in the 1.25 % animals (both sexes) and in the 0.25% males. Mean liver and kidney weight of males and females were significantly increased in the 1.25% group. The testes were atrophied in the 0.25 and 1.25% males. The NOAEL for this study can be considered to be 0.05%. (Mellon Institute of Industrial Research, 1963; reviewed in EU, 2006). Rats (CR, COBS, CD-BR) were dosed by oral gavage with 0, 222, 443 or 885 mg/kg-day EBGE, five days/week for 6 weeks. In the high-dose animals, there was mortality (2/10) and significantly decreased body weight gain and food consumption (from days 3 to 20 of the study). Bloody urine, which persisted through the third week of treatment, was observed in the mid- and high-dose group; only one rat in the low-dose group had bloody urine. Other clinical signs in the mid- and high-dose groups were lethargy, unkempt hair coats, piloerection, rales, slight weakness and inactivity. In all treatment groups, there were significant effects on red blood cell (RBC) parameters, indicating hemolytic anemia. No effects were seen on white blood cells (WBC). There was a significant increase of alkaline phosphatase was seen from the mid-dose group. In the high-dose group, there was a significant increase of SGPT and a decrease of glucose. Absolute and relative spleen weights were increased for the middle and high dose of EGBE. Liver weights were slightly increased (only significant for relative weight) in the high-dose group. No effects were seen on testes weight (relative or absolute) at any dose group. Gross pathology showed only enlarged, dark spleens in the mid- and high-dose groups. Histopathological lesions were hyperkeratosis and acanthosis in the stomach epithelium in all treated animals. Hepatomegaly was also seen in 4 out of 10 animals of the high dose group. Hemosiderin deposition was seen in some animals (6/10 and 7/10 in the mid- and high-dose groups, respectively). Thymus atrophy was seen in one animal of the high-dose group. Congestion of the spleen was seen in all treated animals. Extramedulary hematopoiesis was reported for one animal in the high-dose group and hemosiderin deposit in the majority of animals of the mid- and high-dose group animals. The LOAEL for this study is considered to be 222 mg/kg-day based on effects in the spleen and on RBC parameters. A NOAEL was not obtained (Eastman Kodak, 1982). Male and female F344/N rats were given in their drinking water 0, 750, 1,500, 3,000, 4,500 or 6,000 ppm EGBE for 13 weeks. Based on water consumption, the average daily intake was 0, 69, 129, 281, 367 or 452 mg/kg-day for males; and 0, 82, 151, 304, 363 or 470 mg/kg-day for females. Supplemental groups were included for hematology and clinical chemistry observations at weeks 1 and 3. There was no mortality and no clinical signs of toxicity. Reduced body weight gain was seen in the >4,500 ppm animals. There were reductions in drinking water consumption in the higher dose groups (both sexes), this being clearly concentration-related in females, from a mean of 18.8 ml/day in the control group to 10.7 ml/day in the 6,000 ppm group. A markedly macrocytic and mildly hypochromic anemia was observed at each time point and reticulocyte counts were moderately increased in weeks 1 and 13. For males there was a decrease in erythrocyte counts at all time points in the >3,000 ppm groups, while in females the decrease occurred in the >1,500 ppm groups. A consistent thrombocytopenia was observed at all time points in >4,500 ppm males and females; it also occurred in the 3,000 ppm females at week 13. The most consistent blood chemistry observation was an 3

71 increase in alkaline phosphatase, particularly in the high dose groups. This observation is consistent with mild cholestasis. Thymus weights were significantly reduced in 4,500 ppm males of the 4,500 ppm and the 6,000 ppm males and females. Other organ weight changes were found, but these appeared to be secondary to body weight gain reduction (spleen was not weighed). Histopathological effects were seen in the liver, spleen and bone marrow of both male and female rats. The liver changes were primarily centrilobular hepatocellular degeneration and centrilobular Kupffer cell pigmentation. These changes were present in the majority of dosed rats, but they were more prevalent in the >3,000 ppm animals and were slightly more severe in females. In addition, the cytoplasm of hepatocytes of treated rats was more eosinophilic and lacked the ampholytic-to-basophilic granularity typical of the controls. In the spleen there was an increase in hematopoiesis and deposition of hemosiderin. In bone marrow there was an hyperplasia characterized by an increase of hematopoietic cells and decrease in marrow fat cells. All of these lesions were present in the majority of dosed rats, but they were more prominent in the >3,000 ppm animals. Testis weights were unaffected by treatment, but the size of the uterus in the >4,500 ppm groups were reduced. Changes in uterine weight were considered by the authors to be secondary to the reduction in body weight gain rather than a direct effect of EGBE. Sperm concentration was slightly decreased in all treated males (not dose-related); all other sperm measurements were similar to controls. Estrous cycle length was unaffected by treatment, although the >4500 ppm females spent more time in diestrous than the other groups. This correlated with the smaller uterine size, which was attributed to a secondary consequence of reduced body weight gain. The LOAEL for this study is 750 ppm (69 and 82 mg/kg-day for males and females, respectively). A NOAEL was not obtained (NTP, 1993). Male and female B6C3F1 mice were given in their drinking water 0, 750, 1,500, 3,000, 4,500 or 6,000 ppm EGBE for 14 weeks. Based on water consumption, the average daily intake was estimated to be 0, 118, 223, 553, 676 or 694 mg/kg-day for males; and 0, 185, 370, 676, 861 or 1,306 mg/kg-day for females. There was no mortality and no significant clinical signs of toxicity. Reduction in body weight gain was seen in the >3,000 ppm males and females. Water consumption did not appear to be affected by treatment. Organ weight changes were considered secondary to body weight gain reduction. No treatment-related gross or microscopic lesions in male or female mice were observed. The NOAEL for this study is 223 and 370 mg/kg-day for males and females, respectively. However, this study did not include hematology measurements (NTP, 1993). Genotoxicity Below are the conclusions from the EU Risk Assessment Report on 2-butoxyethanol. EGBE is not mutagenic in bacteria. One report showed a significant response in S. typhimurium strain TA97a (Hoflack et al., 1995). This finding could not be substantiated by Gollapudi et al. (1996). The metabolites butoxy aldehyde (BAL) and butoxy acetic acid (BAA) are also not mutagenic to bacteria. No mutagenic activity was observed in 4

72 two of three in vitro mammalian cell mutation assays; the other study, which was poorly reported, showed a positive finding at very high concentrations. The same study also reported a positive finding with a very high concentration of BAL, whereas another study showed not effects at slightly lower concentrations. No in vitro mammalian cell mutation assays have been conducted with BAA. Inconsistent findings have been reported for SCE induction with EGBE. Inhibition of gap-junctional intercellular communication was reported in a single study with EGBE and its two major metabolites. A significant response was seen in a single assay for UDS induction with a technique that is now considered to be invalid. EGBE has not been shown to induce chromosomal aberrations in a number of mammalian cell culture studies, or in one study with BAL or BAA. Weak aneugenic effects were seen in one study with EGBE and BAL, but not with BAA. In vitro studies with long exposure to BAL, and to a much less extent with EGBE itself, appear to induce micronuclei from aneuploidy rather than from chromosomal breakage. There is no evidence for micronucleus induction in bone marrow cells or interaction with DNA in several organs of rats from in vivo exposure. The possibility of non-disjunction occurring and not being detected in these assays appears to be remote, because BAA produced no evidence of aneugenicity in vitro. BAA is rapidly formed in vivo and is by far the most prevalent blood metabolite of EGBE; thus, exposure of possible target cells to either EGBE or BAL at high concentrations is brief. Overall, the evidence suggests that EGBE does not pose a significant mutagenic potential in vivo. Carcinogenicity Male and female F344/N rats were exposed by inhalation to 0, 31.2, 62.5 or 125 ppm (0, 151, 302 or 604 mg/m 3 ) EGBE vapor for 6 hours/day, five days/week for 104 weeks (NTP, 2000). For hematological and bone marrow analyzes, additional groups of animals were exposed to 0, 62.5 or 125 ppm for evaluation at 3, 6 and 12 months; and to 31.2 ppm for 3 months (hematological examination only) and 6 months. Survival of exposed male and female rats was similar to the control animals, and no clinical signs were attributed to EGBE exposure. Body weights of male and female rats in the low- and middose groups and the high-dose males were generally similar to the controls throughout the study. Body weights of the high-dose female rats were generally lower than those of the controls from week 17 until the end of the study. The incidence of benign or malignant pheochromocytoma (combined) of the adrenal medulla in females exposed to 125 ppm EGBE was not significantly increased compared to the chamber controls, but it did exceed the historical control range. There was only one malignant phaeochromocytoma, which occurred in the 125 ppm group. It was concluded that there was no evidence for carcinogenicity in male rats and equivocal evidence for carcinogenicity in female rats. 5

73 Male and female B6C3F1 mice were exposed by inhalation to 0, 62.5, 125 or 250 ppm (0, 302, 604 or 1,208 mg/m 3 ) EGBE vapor for 6 hours/day, five days/week for 104 weeks (NTP, 2000). For hematological and bone marrow analyzes, additional groups of animals were exposed to 0, 62.5, 125 or 250 ppm for evaluation at 3, 6 and 12 months. Survival of male mice exposed to 125 or 250 ppm was significantly less than that of the controls, whereas survival in all other treated groups was similar to the controls. No clinical signs were attributed to EGBE. Body weights of exposed male mice were generally less than the controls during the last 25 weeks of the study. Body weights of the high-dose females were generally lower than the controls from week 30 until the end of the study, the difference being about 20% for much of that time. Body weights of the low- and mid-dose groups were generally lower than the controls from about week 60 until the end of the study. There was a positive trend in the incidences of forestomach squamous cell papilloma and squamous cell papilloma or carcinoma combined in female mice. The incidences were significantly increased in the 250 ppm group, in which the only squamous cell carcinoma occurred. These incidences exceeded the historical control range for female mice. There was no significant increase in the incidence of these neoplasms in male mice, but they did exceed the historical control range for male mice. Again, there was one squamous cell carcinoma, but in the 125 ppm group. There was a positive trend in the incidence of hemangiosarcomas in male male mice, which was statistically significant in the 250 ppm group. The incidence at 250 ppm also exceeded the historical control range for this tumor in male mice. There was also a positive trend in the incidence of hepatocellular carcinomas, which was statistically significant in the 250 ppm group. There was, however, no change in the incidence of hepatocellular adenomas and carcinomas combined, because of a reduced incidence of hepatocellular adenomas in the treated groups. In female mice there was a single haemangiosarcoma, in the 62.5 ppm group, which was considered to be an incidental finding. Furthermore, the incidences of hepatocellular adenomas were reduced in the 125 and 250 ppm groups of female mice and there was no change in the incidence of hepatocellular carcinomas. The NOAEC for tumorigenicity in mice is 125 ppm, based on an increased incidence of hemangiosarcomas in males and squamous cell papillomas or carcinomas in females at 250 ppm. Proposed Mode of Action (MOA) for Forestomach and Liver Hemangiosarcomas in Female and Male Mice from EGBE Exposure The EU Risk Assessment Report (2006) evaluated the MOA for the forestomach and liver hemangiosarcomas in female and male mice and the relevancy of these tumors for human health risk assessment using the IPCS Human Relevance Framework. I. Postulated Mode of Action for the Induction of Tumours of the Forestomach in Female Mice The proposed mechanism of action for production of tumours in the forestomach is the local generation, as well as accumulation of cytoxic metabolite(s) that induce a sustained, compensatory cell proliferation, neoplasia arising out of this proliferating cell population. 6

74 The neoplasia was mainly papillomas, a single squamous cell carcinoma arising in the highest dose group of female mice. II. Postulated Mode of Action for the Induction of Hamangiosarcomas of the Liver in Male Mice The proposed mechanism of action for production of haemangiosarcomas of the liver is the deposition of haemosiderin in relevant cell-types, possibly including endothelial cells from which haemangiosarcomas arise, and the generation of cytotoxic reactive oxygen species that either induce genetic changes by this secondary mechanism or sustained cell proliferation within the endothelial target tissue, neoplasia arising out of this proliferating cell population. III. Summary EGBE is carcinogenic in male mice, where it causes a low incidence of haemangiosarcomas, and female mice, where is causes an increased incidence of forestomach tumours. It is not carcinogenic in rats. Genotoxicity is not an important toxicological property of this chemical and it is unlikely that the low, variable and uncertainly defined genotoxic activity can be the cause of the carcinogenic responses. Hypotheses have been proposed and supported by experiment data in an attempt to explain the carcinogenic responses In the case of forestomach tumors, the argument that they arise in a tissue subject to sustained abuse and consequent repair is clear. It is likely that this finding is in reality not sex specific but merely due to chance that the low level incidence in females rose above the level of statistical significance but it did not do so in males. In the case of the haemangiosarcomas, data from experiments with other chemicals show that mice are more susceptible to haemangiosarcoma development than are rats, and male mice are more sensitive than female mice, but the database in support of the hypothesis that life-long exposure of the liver to Kupffer cell pigmentation is a prerequisite for an increase in liver haemangiosarcoma rates in male mice is small, after exclusion of doubtful examples. With the rejection of genotoxicity as a possible mechanism, strong evidence for a potential source of reactive oxygen species within the liver, and a mode of action where each step has at least some supporting data, it is reasonable to presume that these may play a role in the neoplasia. With regard to human relevance, the mechanism proposed for the induction of haemangiosarcomas strongly suggests that EGBE is not likely to be a carcinogenic hazard under conditions of normal handling and use, because human erythrocytes are demonstrably more resistant to haemolysis than are rodent erythrocytes. The mechanism proposed for the induction of forestomach tumours would also point to a lack of human relevance under conditions of normal handling and use. As stated recently (IARC, 2003), while people do not possess forestomachs, they do have comparable squamous epithelial tissues in the oral cavity and the upper two-thirds of the oesophagus. Thus, in principle, carcinogens targeting the forestomach squamous epithelium in rodents are relevant for man. However, the relevance for man is probably low for agents that have no demonstrable genotoxicity and that are solely carcinogenic for the forestomach squamous 7

75 epithelium in rodents after oral administration. Consequently, for these agents, the mode of carcinogenic action could be specific to the experimental animals (IARC, 2003). EGBE satisfies only some of these conditions. On the other hand, there are proposed mechanisms that are supported by experimental evidence to show how this chemical, even when inhaled, can accumulate in the forestomach contents, where it can remain for many hours to cause damage directly or after its metabolism to BAA. In conclusion, given the species and sex specificity of the neoplastic responses and the current evidence supporting the hypothesis that the more likely mechanism of action is based on haematotoxicity, then EGBE is unlikely to be a human carcinogen. Reproductive Toxicity Male and female Swiss CD-1 mice were given in their drinking water 0 0.5, 1.0 or 2.0 % EGBE (equivalent to daily intakes of 0, 720, 1340 and 2050 mg/kg-day) during a continuous breeding phase (CBP) with a 7-day pre-mating period and a 98-day cohabitation period (Morrissey et al., 1988,1989; Heindel et al., 1990). There were significant adverse reproductive effects in the females at very high dose levels (>1,340 mg/kg) which also caused severe toxicity, including death. Under the conditions of the study, the NOAEL for reproductive toxicity of EGBE (fertility) was considered to be 720 mg/kg-day. For developmental toxicity, a NOAEL could not be derived. There was a very slight decrease in pup weight was observed at 720 mg/kg-day. No NOAEL or LOAEL can be determined for systemic parental toxicity because this kind of study is not designed to assess systemic toxicity although it is of note that there were effects (reduced fluid consumption) even at the lowest dose of 720 mg/kg-day. There were no macroscopic nor microscopic effects on the reproductive organs in the repeated dose toxicity studies at doses which does not result in severe general toxicity. Developmental Toxicity (Oral Studies Only) A developmental toxicity study was conducted by oral gavage using Fischer 344 rats with the purpose of determining whether the exposure of pregnant rats to EGBE during critical periods of cardiovascular development adversely affected the structure of the fetal heart and great vessels. Pregnant rats were dosed on GD 9-11 with 0, 30, 100 or 200 mg/kg EGBE, with some animals sacrificed on GD 12 and others sacrificed on GD 20. Other group of pregnant rats were dosed on GD with 0, 30, 100 or 300 mg/kg EGBE, with some animals sacrificed on GD 14 and the others sacrificed on GD 20. At doses of >100 mg/kg on GD 9-11 and GD 11-13, there was marked body weight reduction and/or weight gain, increased kidney and spleen weights, and severe hematotoxicity. The hematotoxicity was characterized by a large reduction in circulating red blood cells, hematocrit and hemoglobin, which occurred 24 hours post-treatment. The observed embryo/fetal effects included increased resorptions, non-live implants, and adversely affected implants per litter in the 200 mg/kg dose group of the GD 9 11 treated dams, 8

76 and decreased platelet count but no embryolethality in the 300 mg/kg dose group of the GD treated dams. EGBE treatment did not increase malformations, including cardiovascular malformations. Increased fetal lethality did occur in the 200 mg/kg dose group when given during GD 9-11, but there were no increased malformations. Increased platelet count was also seen in the fetuses of the 300 mg/kg dose group given during GD The maternal NOAEL for this study is 30 mg/kg-day. The developmental NOAEL is 100 mg/kg-day when EGBE is given on GD 9 11, and the NOAEL is 300 mg/kg-day when EGBE is given on GD (Sleet et al., 1991). Pregnant CD-mice were dosed by oral gavage with 0, 350, 650, 1,000, 1,500 or 2,000 mg/kg EGBE during GD 8 to 14. Maternal toxicity was evident in the dams at dosed of >650 mg/kg. There were hemolytic effects (>650 mg/kg) and mortality (>1,500 mg/kg). At 1,000 and 1,500 mg/kg, increased resorption rates and numerically reduced number of viable fetuses were observed at 1,000 and 1,500 mg/kg. Cleft palates were seen in 4/43 fetuses (in one litter) at 1,000 mg/kg/day and 1/25 at 1,500 mg/kg. The NOAELs for maternal and developmental toxicity are 350 and 650 mg/kg-day, respectively (Wier et al., 1987). A subsequent reproduction study was conducted which followed a Chernoff modification study design with treatment of the pregnant mice at 650 or 1,000 mg/kg-day between GD 8 and 14; the animals were allowed to give birth and the offspring were observed until PND 22. Hemolytic effects and reduction in body weight were seen in the dams at 1,000 mg. There were no adverse developmental effects including pup growth or survival. The NOAELs for maternal and developmental toxicity are 650 and 1,000 mg/kg/day, respectively. In another Chernoff assay, fifty mated CD-1 mice were dosed by oral gavage with 1,180 mg/kg-day EGBE (in corn oil) from GD 7 to 14, then allowed to litter and to rear pups to PND 3. Nineteen of the dams died (20%), maternal weight gain was reduced and there were only 24 viable litters (77 %) from the surviving dams compared with 97 % in the controls. There was no external malformations, pup survival to PND was unaffected, and there was no other evidence of developmental toxicity (Schuler et al., 1984). Key Study/Critical Effect for Screening Criteria An oral RfD was derived by U.S. EPA (2010) based on the findings of the NTP chronic inhalation studies, the rationale being the limited oral database and because the critical endpoint, hemosiderin pigmentation, was more pronounced in the chronic inhalation study (NTP, 2000) versus the available subchronic oral study (NTP, 1993). The following information on the NTP chronic studies was obtained from IRIS: In this chronic study, animals were exposed to EGBE 6 hours/day, 5 days/week at concentrations of 0, 31, 62.5, and 125 ppm (0, 150, 302, and 604 mg/m 3 ) for groups of 50 F344/N rats and 0, 62.5, 125, and 250 ppm (0, 302, 604, and 1,208 mg/m 3 ) for groups of 50 B6C3F1 mice. The researchers stated that the highest exposure was selected to 9

77 produce a 10-15% depression in hematologic indices. They reported that no effect on survival was observed in rats, but survival was statistically significantly decreased in male mice exposed to 125 or 250 ppm, compared with chamber controls (54, 52, and 78% respectively). Although statistics were not reported for mean body weights, the rats exposed to 31 and 62.5 ppm had similar mean body weights to the control rats. Mean body weights of the exposed mice were generally less than for controls, with females experiencing greater and earlier reductions. From week 17 to the end of the study, the mean body weights of 125 ppm female rats were generally less than those of controls. Non-neoplastic effects in rats included hyaline degeneration of the olfactory epithelium in males (13/48, 21/49, 23/49, 40/50) and females (13/50, 18/48, 28/50, 40/49) and Kupffer cell pigmentation in the livers of males (23/50, 30/50, 34/50, 42/50) and females (15/50, 19/50, 36/50, 47/50). The severity of the olfactory lesion was not affected by exposure. The Kupffer cell pigmentation is a result of hemosiderin accumulation and is a recognized secondary effect of the hemolytic activity of EGBE. Statistically significant effects observed in mice included forestomach ulcers and epithelial hyperplasia, hematopoietic cell proliferation and hemosiderin pigmentation in the spleen, Kupffer cell pigmentation in the livers, and bone marrow hyperplasia (males only). Hyaline degeneration of the olfactory epithelium (females only) was increased relative to chamber controls but was not statistically significant. As in the rats, the Kupffer cell pigmentation was considered a secondary effect of the hemolytic activity of EGBE. Bone marrow hyperplasia, hematopoietic cell proliferation, and hemosiderin pigmentation in the spleen were also attributed to the primary hemolytic effect; it was followed by regenerative hyperplasia of the hematopoietic tissue. The forestomach lesions did not appear to be related to the hemolytic effect of EGBE. Incidences of ulcer were significantly increased in all exposed female groups, as well as males exposed to 125 ppm. Incidences of epithelial hyperplasia, usually focal, were significantly increased in all exposed groups of males and females. The hyperplasia was often associated with ulceration, particularly in the females, and consisted of thickness of the stratified squamous epithelium and sometimes the keratinized layer of the forestomach. Ulceration consisted of a defect in the forestomach wall that penetrated the full thickness of the epithelium and frequently contained accumulations of inflammatory cells and debris. Using the same exposure levels described above, additional groups of rats (27/gender/exposure group) and mice (30/gender/exposure group) in the 2-year study were examined at 3, 6, and 12 months (8-10 animals/time point) for hematologic effects. Nine male and nine female rats were exposed to 31 ppm EGBE, specifically to evaluate hematology at 3 months and to receive a total evaluation at 6 months. Animals were continuously exposed, as described above, until their sacrifice at 3, 6, or 12 months. As in the 14-week study, inhalation of EGBE by both species resulted in the development of exposure-related hemolytic effects, inducing a responsive anemia. In rats, the anemia was persistent and did not progress or ameliorate in severity from 3 months to the final blood collection at 12 months. Statistically significant (p < 0.05) decreases in automated and manual hematocrit (Hct) values, hemoglobin (Hb) concentrations, and red blood cell (RBC) counts occurred at 3, 6, and 12 months in the 125 ppm female mice and the 250 ppm male and female mice. Statistically significant decreases in these same endpoints 10

78 were also observed in 62.5 ppm females at 6 months and in 125 ppm males at 6 and 12 months (decreases in Hct were observed only at 3 and 6 months). Mean cell volume (MCV) was increased in female mice at the highest duration (12 months) and exposure (250 ppm) levels. Reticulocyte counts were increased significantly in the 125 ppm females at 3 and 6 months and in the 125 ppm males at 6 months of exposure. U.S. EPA used a route to route extrapolation from the NTP (2000) study for the derivation for the RfD. The dose metric used for animal-to-human and route-to-route (inhalation-to-oral) extrapolation for the derivation of the RfD is the area under the curve (AUC) of BAA at 12 months in arterial blood. This dose metric was used for doseresponse modeling of chronic inhalation data to derive the point of departure (POD) of 133 µmol-hour/l, expressed as a BMDL based on animal data. The corresponding human BMDL was then back-calculated using the human PBPK model (Corley et al., 1994; Corley et al., 1997) to obtain an equivalent human oral drinking water dose (BMDLHED) of 1.4 mg/kg-day. A simplifying assumption was used that the entire dose of drinking water EGBE was consumed over a 12-hour period each day. U.S. EPA Oral Reference Dose (RfD) = 1.4/100 = 0.1 mg/kg/day Drinking water guidance value = 0.5 ppm References Corley R.A., Bormett G.A. and Ghanayem B.I. (1994). Physiologically-Based Pharmacokinetics of 2- Butoxyethanol and its Major Metabolite, Butoxyacetic Acid, in Rats and Humans. Toxicol. Appl. Pharmacol. 129: Corley R.A., Markham D.A., Banks C., Delorme P., Masterman A. and Houle J.M. (1997). PBPK and the dermal absorption of EGBE vapor by humans. Fundam. Appl. Toxicol. 39: EU (2006). European Union Risk Assessment Report on 2-Butoxyethanol (CAS No ), European Chemicals Bureau. Gollapudi, B.B., Barber E.D., Lawlor T.E. and Lewis S.A. (1996). Re-examination of the mutagenicity of ethylene glycol monobutyl ether to Samonella strain TA97a. Mutat.Res. 370: Heindel J.J., Gulati D.K., Russel V.S., Reel J.R., Lawton A.D. and Lamb J.C. (1990). Assessment of Ethylene Glycol Monobutyl and monophenol Ether reproductive toxicity using a continuous breeding protocol in Swiss CD-1 mice. Fundam. Appl. Toxicol. 15: Hoflack J.C., Lambolez L., Elias Z. and Vasseur P. (1995). Mutagenicity of ethylene glycol ethers and of their metabolites in Salmonella typhimurium his-. Mutat. Res., 341:

79 McKinney P.E., Palmer R.B., Blackwell W. and Benson B.E., (2000). Butoxyethanol ingestion with prolonged hyperchloremic metabolic acidosis treated with ethanol therapy. Clin. Toxicol. 38: Morrissey R.E., Lamb J.C., Schwetz B.A., Teague J.L. and Morris R.W. (1988). Association of sperm, vaginal cytology and reproductive organ weight data with results of continuous breeding reproduction studies in Swiss CD1 Mice. Fundam. Appl. Toxicol. 11: Morrissey R.E., Lamb J.C., Morris R.W., Chapin R.E., Gulati D.K. and Heindel J.J. (1989). Results and evaluations of 48 continuous breeding reproduction studies conducted in mice. Fundam. Appl. Toxicol. 13: NTP (1993). Toxicity studies on ethylene glycol ethers administered in drinking water. NIH Publication NTP Toxicity Report Series No. 26. NTP, Research Triangle Park, NC, USA. NTP (2000). Toxicology and Carcinogenesis Studies of 2-Butoxyethanol (CAS No ) in F344/N rats and B6C3F1 mice (Inhalation studies). NTP Technical Report Series No.484. NIH Publication No Schuler R.L., Hardin B.D., Niemeier R.W., Booth G., Hazelden K., Piccirillo V. and Smith K. (1984). Results of testing 15 glycol ethers in a short term in vivo reprotoxicity assay. Environ. Health Perspect. 57: Sleet R.B., Price C.J., Marr M.C., Morrissey R.M. and Schwetz B.A. (1991). Cardiovascular development (CVD) in F-344 rats following phase-specific exposure to butoxy ethanol. Teratol. 43: 466. U.S. EPA (2010). 2-Butoxyethanol, United States Environmental Protection Agency Integrated Risk Information System (IRIS). Wier P.J., Lewis S.C. and Traul K.A. (1987). A comparison of developmental toxicity evident at term to postnatal groth and survival using ethylene glycol monoethyl ether, ethylene glycol monobutyl ether and ethanol. Teratog. Carcinog. Mutagen. 7:

80 Toxicity Profile Ethanol (CAS No ) Ethanol has been reviewed in the OECD-SIDS program (OECD, 2004a,b). Acute Toxicity Ethanol has a low order of acute toxicity by all routes of exposure. The lowest wellconducted acute oral LD50 value is 8,300 mg/kg in mice, and an inhalation 1-hour LC50 value of >60,000 ppm (114,000 mg/m 3 ) in mice. Irritation Ethanol is a moderate eye irritant but it is not a skin irritant. Sensitization Ethanol is not a sensitizer. Repeated Dose Toxicity There are numerous repeat dose toxicity studies on ethanol by the oral route. Many of these studies were designed to increase our understanding of the risks associated with alcoholic beverages. Thus, these studies were carried out by the oral route and at high doses. The selected studies were considered appropriate for risk assessment purposes. In a 90-day study, male and female Sprague-Dawley rats were fed a diet containing 0, 1, 2, 3, 4, 5 or 10% ethanol. The 2% dose was calculated to be equivalent to 2,400 mg/kg/day. There were no deaths and no clinical signs of noted. Weight gain throughout the study was unaffected by treatment, but the final weights decreased with dose. Food and water consumption in the 10% group was reduced relative to controls. There was no treatment-related effect on serum liver enzymes and kidney clinical chemistry findings were minimal. In the liver, centrilobular steatosis increased in severity with dose as did the frequency and severity of Mallory bodies (hyaline) and acidophilic degeneration and necrosis. Most liver findings were absent or mild at 2% ethanol but became more significant at >3%. The NOAEL for this study has been established to at 2% (2,400 mg/kg/day). In an NTP 90-day study, F344/N rats and B6C3F1 mice were given in their drinking water 0 or 5% ethanol in drinking water. Based on the water consumption data in the 1

81 study and averaged body weights over the exposure period, this equated to doses of at least 4,000 mg/kg in rats and mg/kg in mice. Male rats showed minor changes to organ weights and hematology and clinical chemistry parameters (these were not considered to be adverse changes); female rats showed minor clinical chemistry changes and increased length of estrus cycle, as well as liver nodules. The male mice showed increased organ weights, fatty changes to the liver, and a decrease in sperm concentration. In the NTP two-year bioassay, B6C3F1 mice were given in their drinking 0, 2.5% and 5% ethanol Concentrations of 2.5% and 5% ethanol resulted in average daily consumption of approximately 100 and 180 mg ethanol for males and 80 and 155 mg for females. There was a marginal exposure-related increase in survival of the males, but no effect on the survival of the females. Water consumption was reduced with increasing concentrations of ethanol which was more marked in males than females. No further information was given on non-neoplastic effects. Genotoxicity The balance of evidence is that ethanol is not genotoxic. Negative results from a number of bacterial mutation assays appear to be reliable. Of the mammalian cell mutation assays a weak mutagenic effect in mouse lymphoma cells occurred only at very high ethanol concentrations. In vivo tests for chromosome aberrations in both rats and Chinese hamsters have given negative results. There is very little evidence to suggest that ethanol is genotoxic in somatic cells and it may have a very limited capacity to induce genetic changes in vivo but under very specific circumstances and at very high doses achievable in humans only by deliberate oral ingestion. Carcinogenicity Evidence of the carcinogenicity of ethanol is confined to epidemiological studies assessing the impact of alcoholic beverage consumption. These do not indicate any such hazard exists from potential exposure to ethanol in the work place or from the use of ethanol in consumer products. Reproductive/Developmental Toxicity The lowest reported NOAEL for fertility by the oral route was 2,000 mg/kg in rats, equivalent to a blood alcohol concentration of 1,320 mg/l, although this was based on a significant increase in the number of small pups rather than a direct effect on fertility; such direct effects are not seen until much higher doses. Many studies exist examining the developmental end point for ethanol. However, most use very high doses and few are individually robust enough to allow a NOAEL to be established. However, the collective weight of evidence is that the NOAEL for developmental effects in animals is high, 2

82 typically >6,400mg/kg, compared to maternally toxic effects at 3,600 mg/kg. The potential for reproductive and developmental toxicity exists in humans from deliberate over-consumption of ethanol. Blood ethanol concentrations resulting from ethanol exposure by any other route are unlikely to produce reproductive or developmental effects. Key Study/Critical Effect for Screening Criteria The lowest reported NOAEL is approximately 2,400 mg/kg/day from a 90-day dietary study with rats. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 2,400/1,000 = 2.4 mg/kg/day Drinking water guidance value = 8.4 ppm References OECD (2004a). IUCLID Data Set for Ethanol (CAS No ), UNEP Publications. OECD (2004b). Screening Information Dataset (SIDS) Initial Assessment Report for Ethanol (CAS No ), UNEP Publications. 3

83 Toxicity Profile Guar Gum (CAS No ) Guar gum (CAS No ) is the milled endosperm of the leguminous plant Cyanopsis tetragonolobus. Structurally, it is a galactomannan consisting of a main chain of D-mannose with a side chain of D-galactose at approximately every second mannose unit. The mannose units are β-(l-4) linked, and the single D-galactose units are joined to the main chain by α-(1-6) linkages. The estimated molecular weight of guar gum cranges from 200,000 to 300,000 daltons (Glickman, 1969). Guar gum is approved for use as a food additive by the U.S. Food and Drug Administration and is on the list of substances "generally recognized as safe" (CFR 1974). Acute Toxicity The oral LD50 is 8,100 mg/kg for mice and 9,400 mg/kg for rats (Bailey and Morgareidge, 1948). Irritation No data were found. Sensitization There are reports of respiratory sensitization in workers exposed occupationally to guar gum dusts (Maio, 1986). Repeat Dose Toxicity Studies F344 rats and B6C3F1 mice were given diets containing 0, 6,300, 12,500, 25,000, 50,000 or 100,000 ppm guar gum for 13 weeks (NTP, 1982). Mean body weights were decreased in male rats (100,000 ppm group) and in female mice (50,000 and 100,000 ppm). A dose-related decrease in feed consumption was observed for male and female rats; male and female mice were comparable or higher than that of controls. There were no compound-related clinical signs or histopathological effects. F344 rats and B6C3F1 mice were given diets containing 0, 25,000 ppm or 50,000 ppm guar gum for 103 weeks (NTP, 1982). Mean body weights of the high-dose females were 1

84 lower than those of the controls after week 20 for mice and week 40 for rats. No compound-related clinical signs or adverse effects on survival were observed. Feed consumption by dosed rats and mice of either sex was lower than that of controls. There were no non-neoplastic histopathological effects in either rats or mice that were treatment-related. Genotoxicity Guar gum was not mutagenic to Salmonella typhimurium TA 1530 or G-46 when tested without metabolic activation; however, it was mutagenic to Saccharomyces cerevisiae D- 3 (Green, 1977). Guar gum also was reported to cause chromosomal aberrations in human embryonic lung cells WI-38 (Green, 1977). No in vivo genotoxicity studies have been conducted on guar gum. Carcinogenicity F344 rats and B6C3F1 mice were given diets containing 0, 25,000 ppm or 50,000 ppm guar gum for 103 weeks (NTP, 1982). There were increased incidences of adenomas of the pituitary in male rats and pheochromocytomas of the adrenal in female rats that were statistically significant, but these differences were considered to be unrelated to guar gum administration. When pituitary adenomas or carcinomas and when pheochromocytomas or malignant pheochromocytomas are combined, the statistical differences disappear. Hepatocellular carcinomas occurred in treated male mice at incidences that were significantly lower than that in controls. The combined incidence of male mice with either hepatocellular adenomas or carcinomas was also significantly lower in the highdose group. It was concluded that under conditions of this bioassay, guar gum was not carcinogenic for F344 rats or B6C3F1 mice. Reproductive Toxicity No data were found. Developmental Toxicity Female rabbits were given daily (6 hours/day) dermal administration of 0, 2, 10 and 50 mg/kg guar gum during gestational days 6 through 18 (IRDC, 1988). Mortalities included 2 deaths at 50 mg/kg and 1 death at 10 mg/kg. A single animal was killed in extremis. A dose-related increase in dermal irritation (including erythema, edema, and desquamation) was observed in animals receiving 10 and 50 mg/kg. The number of early resorptions was significantly increased and the number of viable fetuses was correspondingly decreased at 50 mg/kg/day (p<0.05). The frequency of fetal malformations and variations in the treated groups was comparable to that of the control 2

85 group at all dose levels. The NOEL for this study is 2 mg/kg/day. Key Study/Critical Effect for Screening Criteria The key studies for the determination of a drinking water guidance value is the NTP twoyear chronic bioassays. The LOAELs are based on decreased mean body weights in female mice and rats fed 50,000 ppm guar gum in diet for 103 weeks. The NOAELs for these studies are 25,000 ppm guar gum. Rat: NOAEL (mg/kg/day) = 25,000 ppm * 0.05 = 1,250 mg/kg/day Mouse: NOAEL (mg/kg/day) = 25,000 ppm * 0.13 = 3,250 mg/kg/day Where 0.05 and 0.13 are the fraction of body weight that rats and mice, respectively, consume per day as food (U.S. EPA). The lowest NOAEL of 1,250 mg/kg/day for the rat will be used to derive a drinking water guidance value. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 1,250/100 = 12.5 mg/kg/day Drinking water guideline = 44 ppm References Bailey, D., and Morgareidge, K. (1976). Comparative acute oral toxicity of 12 food grade gums in the mouse, rat, hamster, and rabbit. Food and Drug Research Labs Papers No., 124; cited in NTP (1982). Glicksman M. (1969). Gum technology in the Food Industry, pp. 590, Academic Press, New York; cited in Yoon, S.-J., Chu, D.-C., and Juneja, L.R. (2008) Chemical and physical properties, safety and application of partially hydrolyzed guar gum as dietary fiber. J. Clin. Biochem. Nutr. 42: 1-7. Green, S. (1977). Present and future uses of mutagenicity tests for assessment of the safety of food additives. J. Environ. Pathol. Toxicol. 1: International Research and Development Corp (1988). Teratology Study of Guar Gum in Rabbits. TSCATS database, EPA Doc. No , Fiche No. OTS ; cited in NZ HSNO CCID. 3

86 Maio, J.L., Cartier, A., L Archevêque, J., Ghezzo, H., Soucy, F., Somers, J., and Dolovich, J. (1990). Prevalence of occupational asthma and immunologic sensitization to guar gum among employees at a carpet-manufacturing plant. J. Allergy Clin. Immunol. 86: NTP (1982). NTP Technical Report on the Carcinogenesis Bioassay of Guar Gum (CAS No ) in F344 Rats and B6C3F1 Mice (Feed Study), National Toxicology Program, Research Triangle Park, NC 4

87 Toxicity Profile Hydrochloric acid (CAS No ) Hydrochloric acid has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Hydrochloric acid (HCl) or hydrogen chloride is readily dissociated in water into hydrated protons and chloride ion. Acute Toxicity The oral LD50 value of hydrogen chloride is reported to be 238 to 277 mg/kg for female rats (Hoechst AG, 1966), and 900 mg/kg for rabbits (Loewy and Munzer, 1923). The lethal dose by dermal exposure is >5,010 mg/kg for rabbits (Monsanto, 1976). The LC50 values for HCl are reported to be mg/l/5min, mg/l/30min and mg/l/60min for rats; and 20.9 mg/l/5min, 3.9 mg/l/30min and 1.7 mg/l/60min for mice (Darmer et al., 1974; Hartzell et al., 1990; MacEwean et al., 1974). Irritation Concentrations >3.3% cause skin irritation, and concentrations >17% cause corrosion in animal studies (OECD, 2002a,b). An aqueous solution (4%) of hydrogen chloride was slightly irritating (Agner and Serup, 1988), and a 10% solution was determined to be Irritating to skin for the EU Dangerous Preparations Directive, in human volunteer experiments (York et al., 1996). 0.1 ml of 10% aqueous solution of HCl was highly irritating to the eyes of rabbits (Jacobs, 1992); 0.03 ml or more of 5% HCl was corrosive to the eyes of rabbits (Griffith et al., 1980); 0.1 ml of a 3.3% aqueous solution of HCL was slightly irritating the eyes of rabbits (Hoechst AG, 1966); and 0.1 ml of a 0.33% aqueous solution of HCl was not irritating to the eyes of rabbits. (Hoechst AG, 1966). Sensitization Hydrogen chloride is not a skin sensitizer to guinea pigs and humans (Gad et al., 1986). Repeated Dose Toxicity Rats were fed diets containing 280 to 1,250 mmol/kg hydrochloric acid (10.2 to 45.6 mg/kg) for 7-12 weeks. There was increased water intake in all treated groups. All 1

88 animals fed diet containing 937 mmol/kg and above for 9 weeks, and half of the animals fed diet containing 900 mmol/kg for 12 weeks died. Also at doses >937 mmol/kg, there was decreased body weight, food consumption, blood ph, femur length, rate of ash in bone (Upotn and L Estrange, 1977). In another study with rats, hydrochloric acid was administered via drinking water at ph 2-3 (study duration not provided). Decreased protein levels in urine and decreased urine volumes were observed in the treatment groups (Clausing and Gottschalk, 1989). Genotoxicity While consistent negative results have been obtained in the bacterial systems, positive results have been obtained in the non-bacterial systems (OECD, 2002a,b). The positive results were observed at high concentrations, but they were considered to be artifacts due to the low ph. Positive results were obtained in a sex-linked recessive lethal study with D. melanogaster; only one dose level was tested (Stumm-Tegethoff, 1966) There are no mammalian studies on in vivo mutagenicity with hydrogen chloride Carcinogenicity (Oral Studies only) A ten-month study has been conducted using mice (Dyer et al., 1946). Because the methods such as strain used, duration and dose of administration, presence of coadministration substance, were not appropriate, it cannot be used for the assessment of carcinogenicity of hydrochloric acid. Reproductive Toxicity No reliable studies were identified by the oral route. Developmental Toxicity No reliable studies were identified by the oral route. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for ph may apply to hydrochloric acid. References Agner, T., and Serup, J. (1988). Contact thermography for assessment of skin damage due to experimental irritants., Acta. Dermatol. Venerol., 68:

89 Clausing, P., and Gottschalk, M.Z. (1989). Effects of drinking water acidification, restriction of water supply and individual caging on parameter of toxicological studies in rats. Versuchstierkd., 32: Darmer, K.I. et al. (1974). Acute toxicity in rats and mice exposed to hydrogen chloride gas and aerosol. Am. Ind. Hyg. Assoc. J.,35: Dyer, H.M. et al. (1946). Effect of administration of hot water, acids, alkali, mecholyl chloride, or atropine sulfate upon the gastric mucosa of mice. J Natl. Cancer Inst. 7:67. Gad, S.C., Dunn, B.J., Dobbs, D.W., Reilly, C., and Walsh, R.D. (1986). Development and validation of an alternative dermal sensitisation test: the mouse ear swelling test (MEST)., Toxicol. Appl. Pharmacol. 84: Griffith, J.F. et al. (1980). Dose-response studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye. Toxicol. Appl. Pharmacol. 55,: Hartzell, G.E. et al. (1990). Toxicity of smoke containing hydrogen chloride. ACS Symp. Ser., 425: Hoechst AG (1966). Farbwerke Hoechst AG Report 150/66. Jacobs, G.A. (1992). OECD eye irritation tests on two acids. J. Am. Coll. Toxicol. 11: 734. Loewy, A., and Munzer, E. (1923). Beiträge zur von der experimentellen Säurevergiftung., Bioch. Z. 134, MacEwean, J.D. et al. (1974). Toxic Hazard Research Unit, Annual Technical Report, NTIS AD- A Monsanto (1976). Unpublished report YO ; cited in European Commission- European Chemical Bureau IUCLID (2000) OECD (2002a). IUCLID Data Set for Hydrogen chloride (CAS No ). OECD (2002b). Screening Information Dataset (SIDS) Initial Assessment Report for Hydrogen chloride (CAS No ), UNEP Publications. Stumm-Tegethoff, B.F.A. (1969). Formaldehyde-induced mutations in Drosophila melanogaster ndependence of the presence of acids. Theor. Appl. Genet. 39: Upotn, P.K., and L Estrange, J.L. (1977). Effects of chronic hydrochloric and lactic acid balance and bone composition of the rat. Quart. J. of Exp. Physiol. 62:

90 York, M.H., Griffiths, A.E., Whittle, E., and Basketter, D.A. (1996). Evaluation of a human patch test for the identification and classification of skin irritation potential. Contact Dermatitis 34:

91 Toxicity Profile 2-Propanol or Isopopranol (CAS No ) Acute Toxicity The acute oral LD50 has been reported as 4,700 mg/kg, 5,300 mg/kg, 5,500 mg/kg, and 5,8400 mg/kg in rats; 4,500 mg/kg in mice; and 5,030 mg/kg, 7,800 mg/kg, and 7,900 mg/kg in rabbits (31, 272, 274, 275, 144, 35 Kimura et al., 1971; WHO, 1990; Smyth and Carpenter, 1948; Lehman and Chase, 1994; Munch and Schwartze, 1925; Munch, 1972). The acute dermal LD50 in rabbits has been reported to be 12,900 mg/kg (Smyth and Carpenter, 1948). The acute inhalation 8-h LC50 in rats was 19,000 ppm in females and 22,500 ppm in males (Laham et al., 1980). Exposure of rats to 16,000 ppm for 8 h resulted in four deaths out of six animals (Smyth and Carpenter, 1948). Irritation Isopropanol applied to the intact or abraded skin of rabbits and guinea pigs produced negligible irritation (Nixon et al., 1975). Liquid isopropanol is moderately irritating to the eyes of rabbits (Griffith et al., 1980; WHO, 1990). Isopropanol produced little irritation when tested on the skin of six human subjects (Bevan, 2012). Sensitization There have been reports of isolated cases of dermal irritation and/or skin sensitization (Bevan, 2012). Except for three case reports, the positive reactions were observed on patch testing patients with contact dermatitis due to ethanol. These patients also had a positive reaction to ethanol. Repeated Dose Toxicity (Oral Studies only) In a drinking water study, rats ingested 0.5 to 10% of isopropanol for 27 weeks and showed decreased body weight gain but no gross or microscopic tissue abnormalities (Lehman and Chase, 1975). Increased formation of hyaline droplets in the proximal tubules was reported in male rats given 1 4% isopropanol in drinking water for 12 weeks (Pilegaard and Ladefoged, 1993). In another study, daily application of a 50% solution to the heads of rats for 187 days produced no apparent injury to the skin (Boughton, 1944). Isopropanol was administered in drinking water to three dogs for 7 months. The alcohol concentration was 4% from the end of the first month until the conclusion of the experiment. Tolerance to the alcohol developed as manifested by an increased degree of neuromuscular coordination at similar blood levels in habituated versus control animals 1

92 and by increased elimination of the alcohol. The only significant histopathological changes were noted in the kidneys of one dog that died (Lehman et al., 1945). Genotoxicity All genotoxicity assays conducted so far with isopropanol have been negative (OECD, 1997a,b; Bevan, 2012). Isopropanol was not mutagenic in the Salmonella microsomal assay using the spot test, in strains TA98, TA100, TA1535, and TA1537 with and without S9 from the livers of Aroclor-induced rats (Abbondandolo et al., 1980). Isopropanol was not mutagenic in Salmonella strains TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538 with and without metabolic activation, when tested using a plate-incorporation modification of this assay (Zeiger et al., 1992). Isopropanol was inactive in mutagenicity tests in Neurospora crassa (Brockman et al., 1984) and isopropanol did not enhance adenovirus (SA7) transformation using Syrian hamster embryo cells (Heidelberger et al., 1983). Isopropanol was inactive in a sister chromatid exchange assay with and without S9 metabolic activation (von der Hude et al., 1987). Isopropanol was inactive in an in vitro CHO/HGPRT gene mutation assay and in an in vivo bone marrow micronucleus assay in mice (Kapp, Jr. et al., 1993). Carcinogenicity No carcinogenicity studies have been conducted by the oral route. CD-1 mice were exposed by inhalation to 0, 500, 2500, or 5000 ppm of isopropanol vapor for 6 h/day, 5 days/week for 18 months. An additional group of mice (all exposure levels) were assigned to a recovery group which were exposed to isopropanol for 12 months and then retained until study termination at 18 months. There was no increased frequency of neoplastic lesions in any of the isopropanol-exposed animals. Nonneoplastic lesions were limited to the testes (males) and the kidney. In the testes, enlargement of the seminal vesicles occurred in the absence of associated inflammatory or degenerative changes. The kidney effects included tubular proteinosis and/or tubular dilatation. The incidence of testicular and kidney effects was not increased in the isopropanol-exposed recovery animals (Burleigh-Flayer et al., 1997). Fischer 344 rats were exposed to 0, 500, 2500, or 5000 ppm of isopropanol vapor for 6 h/day, 5 days/week for 24 months. The mortality rates for all male rats were 82, 83, 91, and 100% for the 0-, 500-, 2500-, and 5000-ppm groups, respectively. The corresponding values for the female rats were 54, 48, 55, and 69%. The main cause of death for the 5000-ppm rats (both sexes), as well as for much of the mortality of the 2500-ppm male rats, was chronic progressive nephropathy. Isopropanol exposure resulted in impaired kidney function, as indicated by various urine chemistry changes in male (2500- and 5000-ppm) and female (5000-ppm) rats. Animals in these groups also exhibited histopathological effects in the kidneys which appeared to be an exacerbated form of 2

93 chronic progressive nephropathy. The only neoplastic lesion noted was increased interstitial (Leydig) cell adenomas in male rats. The frequency of these tumors, although elevated above the control animals, was within the historical control range of the testing facility and within the range reported for control animals from the National Toxicology Program carcinogenicity studies (Burleigh-Flayer et al., 1997). The carcinogenic potential of isopropanol was evaluated via inhalation using three strains of mice. Male mice were exposed to 7.5 ppm of isopropanol for 3 to 7 h/day, 5 days/week for 5 to 8 months. Animals were killed at either 8 or 12 months. There was no significant increase in the number of lung tumors observed. Similarly, no increases in lung tumors were observed in the same strains of mice that received subcutaneous injections of isopropanol once weekly for 20 to 40 weeks (Weil et al., 1952). Reproductive Toxicity In a two-generation reproductive toxicity study, Sprague Dawley rats were dosed by oral gavage with 0, 100, 500, or 1,000 mg/kg isopropanol. There were seven parental deaths that were considered treatment-related: two high-dose F0 females, two F1 high-dose females, one mid-dose F0 female, and two low-dose F1 males. Lactation body weight gain was increased in the 500- and 1000-mg/kg females in both generations, and liver and kidney weights were increased in the 500- and 1,000-mg/kg groups in both sexes. Centrilobular hepatocyte hypertrophy was noted in some 1,000 mg/kg F1 males. There were some kidney effects in the 500- and 1000-mg/kg F0 males and in all treated F1 male rats. The kidney effects were characterized by an increased number of hyaline droplets in the convoluted proximal tubular cells, epithelial degeneration and hyperplasia, and proteinaceous casts. Increased mortality occurred in the high-dose F1 offspring during the early postnatal period; no other clinical signs of toxicity were observed in the offspring from either generation. Offspring body weight, however, in the 1,000-mg/kg group was reduced during the early postnatal period. There was significant mortality in the F1 weanlings (18/70) before the selection of the F1 adults. A statistically significant reduction was observed in the F1 male mating index of the 1,000-mg/kg group (73 versus 97% in the controls). There were no other treatment-related effects on reproduction, including fertility and gestational indices, or histopathology of the reproductive organs (Bevan et al., 1995). A benchmark dose level of 420 mg/kg/day (lower bound on dose associated with a 5% response rate for the decrease in the male mating index was calculated (Allen et al., 1998). In a one-generation reproductive/embryotoxicity study, male and female Wistar rats were given 0, 0.5%, 1.0% or 2.0% isopropanol in their drinking water. The calculated intakes for males were 383, 686 and 1107 mg/kg-day (pre-mating) and 347, 625 and 1030 mg/kg-day (18 weeks of treatment). The calculated intakes for females were 456, 835 and 1206 mg/kg-day (premating); 668, 1330 and 1902 mg/kg-day (gestation); and 1053, 1948 and 2768 mg/kg-day (postpartum). An immediate, statistically significant dosedependent decrease occurred in water intake in the male rats. Intake was reduced ~5-14% (1% group; premating period) and ~30% (2% group; days 7-11 to end of study). 3

94 Overall mean feed consumption was significantly lower in treated versus control animals. Male body weights (2% only) were reduced throughout the study. Water consumption was initially reduced in the 1% and 2% females, but the 2% group recovered to only ~70% of the control values (premating); it continued to be reduced during the gestation and lactation period. Mean maternal body weights were reduced (all treated groups) at the start of gestation, with partial recovery during the gestation period except for the 2% group. Overall weight gain during gestation in these groups were similar to the controls. Following parturition from PND 4 onward, the 2% dams had significantly lower body weights. There were no infertile males in any group, and no treatment-related effect on female fertility or on length of gestation. The number of pups/litter on GD 1 was reduced in the 2% group; because it was not replicated in the embryotoxicity portion, an increase in pup mortality during partuition or GD 0, followed by cannibalism of the dead pups by the dam was suggested. No macroscopic abnormalities were seen in females; nor was there any treatment-related histopathological changes seen in the reproductive tissue in the 2% parental animals. Absolute kidney weight and relative kidney, liver, and spleen weights were increased in the 2% F0 males; increased absolute liver and kidney weights and relative liver weights in the 2% F0 females. In the embryotoxicity portion, there was a statistically significant increase in the total number of preimplantation losses in the 2% animals. Whole body edema was seen in 40% of the fetuses in 3/8 litters in the 2% group. No macroscopic abnormalities of the viscera of these fetuses were detected, and the incidence of edema was not related to gender. In the one-generation portion, postnatal pup survival and in the average pup weight (by PND 7) were decreased in the 2% group. F1 generation animals of both sexes showed increased relative liver weights at all dose levels, and the 2% males had higher relative kidney weights. A slight but significant decrease in absolute brain weight and increase in relative empty cecum weights in both sexes of the 2% F1 generation group. No treatment-related gross abnormalities were observed in the F1 generation animals at necropsy (BIBRA, 1988). The effects of isopropanol (2.5% in drinking water) on the reproduction and growth of rats was assessed in a multigenerational study. No reproductive toxicity was observed (Lehman et al., 1945). Isopropanol was administerd as a 3% solution in drinking water to Wistar rats. Reduced parental body weight gain, food, and water consumption was observed in the treated animals compared with the controls. Fertility, litter size, and pup weights at postnatal days 4 and 21 were reduced in treated animals compared with the controls. In the second generation, the isopropanol concentration was reduced to 2%, and there were essentially no effects (Gallo et al., 1977). Developmental Toxicity Isopropanol was given at concentrations of 0, 0.5%, 1.25% or 2.5% in the drinking water to female Wistar rats on GD 6 to 16. The calculated intakes of isopropanol during GD 6-16 were 596, 1242 and 1605 mg/kg/day. There was an immediate reduction in water intake in the 2.5% dose group, and this was statistically significant throughout the 4

95 treatment period when compared to controls. A smaller reduction in water intake was also seen in the 1.25% females (statistically significant during GD 6-9), with no change in the 0.5% females. Palatability of the drinking water may have been the problem since water intake significantly increased the first day following the end of the treatment period for all dose groups. Feed consumption patterns paralleled the water consumption during and after treatment in the mid- and high-dose groups. Overall, mean body weights of the 2.5% females were lower than the controls from GD 7 to termination. Effects on weight gain in the 0.5% and 1.25% females were limited to a failure to gain weight during the first (0.5%) and second (1.25%) day of treatment. There were no treatment-related effects in postimplantation loss, mean number of implantation sites or live fetuses. There was a slight dose-dependent decrease in mean litter weight and a significant decrease in mean fetal weight in the 1.25% and 2.5% groups. A statistically significant increase in variations was observed, indicative of a lower degree of ossification in the treated animals There was a dose-dependent decrease in the number of fetuses with the 4 th sacral arch and a dose-dependent increase in the number of fetuses with less than 2 caudal arches. The sternum also showed reduced ossification because there were increased numbers of fetuses with small, absent, or incompletely ossified sternebrae (BIBRA, 1987). In a rat developmental study, female Sprague Dawley rats were dosed by oral gavage with either 0, 400, 800, or 1200 mg/kg of isopropanol during gestational days 6 to 15. Two dams (8%) died at 1200 mg/kg and one dam (4%) died at 800 mg/kg. At 1200 mg/kg, maternal body weights were reduced throughout gestation (GS 0-20; 89.9% of control value), associated with reduced gravid uterine weight. There were no other treatment-related effects on the dams. Fetal body weights per litter were also significantly reduced at the 800 and 1200 mg/kg dose levels, but there were no teratogenic effects. There were no adverse maternal or developmental effects at 400 mg/kg (Tyl et al., 1994). In a rabbit developmental study, female New Zealand white rabbits were dosed by oral gavage with either 0, 120, 240, or 480 mg/kg of isopropanol during gestational days 6 to 18. At 480 mg/kg, isopropanol was unexpectedly toxic to pregnant female rabbits, resulting in the deaths of four does (26%). Maternal body weights were significantly reduced during treatment (gestational days 6 18) and were associated with reduced maternal food consumption during this period. Profound clinical signs were noted at 480 mg/kg and included flushed and/or warm ears, cyanosis, lethargy, and labored respiration. No adverse maternal effects were noted at 120 or 240 mg/kg. There were no developmental or teratogenic effects at any dose tested (Tyl et al., 1994). Isopropanol was given by oral gavage to Sprague Dawley rats from gestational days 6 to 21 in doses of 0, 200, 700, or 1200 mg/kg. The dams were allowed to deliver, litters were culled on postnatal day (PND) 4, pups were weaned on PND 22, and their dams were killed. Weaned pups were assessed for day of testes descent or vaginal opening, motor activity, auditory startle, and active avoidance. The pups were killed on PND 68. Some of the pups were taken from each dose group and were perfused in situ for pathological examination of the central nervous system. There were no biologically significant findings in the behavioral tests, no changes in organ weights, and no pathological 5

96 findings of note. Thus, there was no evidence of developmental neurotoxicity from isopropanol exposure (Bates et al., 1994). Key Study/Critical Effect for Screening Criteria The benchmark dose level of 420 mg/kg/day from the two-generation reproductive toxicity study will be used to derive a drinking water guidance value for 2-propanol. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 420/100 = 4 mg/kg/day References Abbondandolo, A. et al. (1980). Mutat. Res. 79 : Allen B., Gentry R., Shipp A., and Van Landingham, C. (1998). Regul. Toxicol. Pharmacol. 28: Bates, H.K. et al. (1994). Fundam. Appl. Toxicol. 22: Bevan, C., Tyler, T.R., Gardiner, T.H., Kapp, R.W., Jr., Andrews, L., and Beyer, B.K. (1995). J. Appl. Toxicol. 15: Bevan, C. (2012). Monohydric Alcohols C1 to C6. In: Patty s Industrial Hygiene and Toxicology, 6th Edition, Volume 3, Chapter 55 (Bingham and B. Cohrssen, Eds.), pp , John Wiley and Sons, Inc., NY. BIBRA (1987). Unpublished report, The British Industrial Biological Research Association; reviewed in Faber, W.D. et al. (2008). Birth Defects Research (Part B) 83, (2008). BIBRA (1988). Unpublished report, The British Industrial Biological Research Association; reviewed in Faber, W.D. et al. (2008). Birth Defects Research (Part B) 83: Boughton, L.I. (1944). J. Am. Pharm. Assoc. 33: Brockman, H.E. et al. (1984). Mutat. Res. 133 : Burleigh-Flayer, H. et al. (1997). Fundam. Appl. Toxicol. 36: Gallo, M.A. et al. (1977). Toxicol. Appl. Pharmacol. 41:

97 Griffith, J.F. et al. (1980). Toxicol. Appl. Pharmacol. 55 : Heidelberger, C. et al. (1983). Mutat. Res. 114: Kapp Jr., R.W. et al. (1993). Environ. Mol. Mutagen. 22: Kimura, E.T. Ebert, D.M., and Dodge, P.W. (1971). Toxicol. Appl. Pharmacol. 19: Laham, S. et al. (1980). Drug Chem. Toxicol. 3: Lehman, A.J., and Chase, H.F. (1944). J. Lab. Clin. Med. 29: Lehman, A.J., Schwerma, H., and Rickards, E. (1945). J. Pharmacol. Exp. Ther. 85: Munch, J.C., and Schwartze, E.W. (1925). J. Lab. Clin. Med. 10: 985. Munch, J.C. (1972). Ind. Med. Surg. 41: Nixon, G.A., Tyson, C.A., and Wertz, W.C. (1975). Toxicol. Appl. Pharmacol. 31 : OECD (1997a). IUCLID Data Set for 2-Propanol (CAS No ), UNEP Publications. OECD (1997b). Screening Information Dataset (SIDS) Initial Assessment Report for 2- Propanol (CAS No ), UNEP Publications. Pilegaard, K., and Ladefoged, O. (1993). In Vivo 7: Smyth, H.F., and Carpenter, C.P. (1948). J. Ind. Hyg. Toxicol. 30: Tyl, R.W. et al. (1994). Fundam. Appl. Toxicol. 22: von der Hude, W. et al. (1987). Environ. Mutagen. 9: Weil, C.S., Smyth, H.F., and Nale, T.W. (1952). Arch. Ind. Hyg. Occup. Med. 5: WHO (1990). Environmental Health Criteria 103, World Health Organization, Geneva. Zeiger, E. et al. (1992). Environ. Mol. Mutagen. 19(Suppl. 21):

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99 Toxicity Profile Magnesium chloride (CAS No ) Acute Toxicity The oral LD50 of magnesium chloride hexahydrate in rats is >2,000 mg/kg. The dermal LD50 of magnesium chloride hexahydrate in rats is >2,000 mg/kg. Irritation Magnesium chloride hexahydrate was applied topically to the EPISKIN-SMätissue for 15 min followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. The test item showed no irritant effects. Single ocular instillation of magnesium chloride hexahydrate to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 6, 4 and 2 days postinstillation in animal no. 1, 2 and 3, respectively. Based on these findings, magnesium chloride hexahydrate is not an eye irritant. Sensitization Magnesium chloride hexahydrate was not a skin sensitizer when tested in guinea pigs. Repeated Dose Toxicity In an OECD 422 study, Wistar rats at dosages of 250, 500 and 1,000 mg/kg magnesium chloride hexahydrate revealed no major toxicological findings. The cause of death of animals during the conduct of the study could not be determined (a plausible cause may be gavage error or regurgitation). The NOAEL for this study is 1,000 mg/kg/day. Groups of 50 male and 50 female B6C3F1 mice were given magnesium hexahydrate at dose levels of 0, 0.5% and 2% in the diet for 96 weeks, after which all animals received the control diet for 8 weeks and were then necropsied. In the 2% females, a decrease in body weight was observed. However, survival rates did not differ between the treatment and control groups for males or females and clinical signs and urinary, hematological or serum clinical chemistry parameters showed no treatment-related effects. Therefore, this change was considered not to be of biological significance. The NOAEL for female and male mice was 2% in feed, which corresponded to 3,930 and 2,810 mg/kg/day, respectively. 1

100 Genotoxicity Magnesium chloride was not genotoxic in the in vitro mouse lymphoma assay and in the in vitro chromosomal aberration assay using human lymphocytes. Carcinogenicity Male and female B6C3F1 mice were given in their fed 0, 0.5% or 2% magnesium chloride hexahydrate for 96 weeks, after which all animals received the control diet for 8 weesk and were then necropsied. Tumors were mainly found in the skin/subcutis, liver and lymphatic system. With the exception of a significant decrease in the incidence of liver tumors among the 2% males, there were no differences in the tumor incidences between the treated and control animals. Reproductive/Developmental Toxicity In an OECD 422 study, Wistar rats at dosages of 0, 250, 500 or 1,000 mg/kg showed no treatment-related reproductive or developmental effects. The NOAEL for this study is 1,000 mg/kg/day. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for chloride may apply to magnesium chloride. Reference ECHA REACH database: 2

101 Toxicity Profile Magnesium nitrate (CAS No ) Acute Toxicity The acute oral LD50 in rats is >5,000 mg/kg. Irritation No studies are available. Sensitization Magnesium nitrate is not a skin sensitizer based on results of a Local Lymph Node Assay (LLNA). Repeat Dose Toxicity Studies No studies are available. Genotoxicity Magnesium nitrate hexahydrate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Carcinogenicity No data are available. Reproductive Toxicity No data are available. Developmental Toxicity No data are available. 1

102 Key Study/Critical Effect for Screening Criteria The Australian drinking water guidance value for nitrates may apply to magnesium nitrate. Reference ECHA REACH database: 2

103 Toxicity Profile Polypropylene glycol (CAS No ) Acute Toxicity Acute oral toxicity studies on PPGs of various molecular weights (300-3,900) have indicated LD50 values (rats) ranging from 500 to >40,000 mg/kg. In acute dermal toxicity studies, doses of PPG 1025 (20 ml/kg) and PPG 2025 (20 ml/kg) did not cause death to rabbits. Two of five rabbits dosed with 20 ml/kg PPG 425 and one of five dosed with 10 ml/kg PPG 425 died. Irritation Skin irritation was not noted after PPG 425, PPG 1025, or PPG 2025 was applied once to the skin of rabbits or when applied a total of eight times to the same area within 4 h. PPGs 425, 1025, and 2025 were classified as harmless agents in rabbits in another ocular irritation study; PPG 1200 induced slight, transient ocular irritation in an albino rabbit. Sensitization Neither skin irritation nor sensitization reactions were observed in 300 subjects who received continuous and repeated dermal applications of undiluted PPG Repeated Dose Toxicity PPG 2000 was administered to rats over a period of 100 days. Concentrations of 0.1, 0.3, 1.0, and 3.0% were administered in oral doses of 50 to 1,500 mg/kg/day. There were no adverse effects noted at concentrations of 0.1 to 1.0%. Slight decreases in growth were observed after the administration of 3% PPG In a 90-day study, PPG 2000 was administered orally to rats in doses ranging from 275 to 501 mg/kg/day. There was no evidence of adverse histopathologic, hematologic, or clinical chemistry effects in any of the animals tested. Body weight effects (not specified) were noted at the highest dose tested. Similar results were reported for dogs that received doses of PPG 2000 ranging from 526 to 810 mg/kg/day. PPG 1200 was fed to three groups of four pure bred beagle dogs for 90 days. Each group consisted of two male and two female dogs. The three groups ingested PPG 1200 at concentrations of 0.1, 0.3, and 1.0 % in the diet, respectively; six beagles in the control group were fed diets without PPG. At the highest no effect level (0.3%), average daily 1

104 consumption doses were 79 and 99 mg/kg for two male dogs and 90 and 123mg/kg for two female dogs. Data based on the following analyses did not indicate adverse effects at any of the concentrations tested: demeanor, feed consumption, hematologic parameters, clinical chemistry determinations, urinalysis, organ weights, organ/body weight and organ/brain weight ratios, or gross and microscopic examination of tissues and organs. Similar results were reported when three groups of 50 rats were dosed with 0.1, 0.3, and 1.0% PPG 1200, respectively, according to the same procedure. PPG 750 was administered to rats over a period of 100 days. Concentrations of 0.1 and 1% were administered at doses of 50 and 500 mg/kg/day. PPG 750 (0.1%) did not induce any adverse effects. However, in the group dosed with 1% PPG 750, there was a slight increase in liver and kidney weights; there were no histological changes. Neither of the doses resulted in a central nervous system stimulatory effect. Genotoxicity No data are available. Carcinogenicity No data are available. Reproductive/Developmental Toxicity No data are available. Key Study/Critical Effect for Screening Criteria Several rat subchronic toxicity studies indicate a NOAEL of 1% PPG in diet (500 mg/kg/day). In one study, it was reported that there was a slight increase in liver and kidney weights (no data are provided to determine if the change in organ weights were statistically significant). Nevertheless, these organ weight changes may not be considered adverse since there were no accompanying histopathologic changes. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 500/1,000 = 0.5 mg/kg/day Drinking water guidance value = 1.75 ppm 2

105 References Andersen F.A. (1994) Final report on the safety assessment of propylene glycol and polypropylene glycols. Int. J. Toxicol. 13:

106 Benzenesulfonic acid, 4-ethyenyl-, sodium salt, homopolymer (CAS No ) Acetic acid ethenyl ester, polymer with ethanol (CAS No ) Vinylidene chloride/methacrylate copolymer (CAS No ) Polyvinyl acetate, partially hydrolyzed (CAS No ) THIS IS NOT THE CORRECT CASRN All of the these substances are polymers. Mammalian toxicity data was located for only for acetic acid, ethenyl ester, polymer with ethanol (CAS No ) on a supplier s MSDS. However, all of the polymers listed above are expected to exhibit similar toxicity profiles. As polymers, they are not expected to be absorbed by the oral, dermal or inhalation routes of exposure due to their large molecular weights. They are also expected to be stable in biological media and resistant to metabolism. This polymers are expected to be essentially acutely non-toxic by the oral, dermal and inhalation routes of exposure. The oral LD50 in rats for CAS No was reported to be >11,000 mg/kg. Under certain conditions, dusts may be formed, which is to be considered as a nuisance dust. The polymers are expected to be non-irritating and not dermal sensitizers. No irritation or sensitization was observed in guinea pigs from CAS No These polymers are not expected to exhibit any systemic toxicity. Reference DuPont (2006). MSDS for ELVANOL 60-30, revised 27-Oct-2006.

107 Toxicity Profile Sodium carbonate (CAS No ) Sodium carbonate has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Sodium carbonate is a strong alkaline compound with a ph of 11.6 for a 0.1M aqueous solution. The pka of carbonate (CO3 2- ) is 10.33, which means that at a ph of both carbonate and bicarbonate are present in equal amounts. In water, sodium carbonate dissociates into sodium ion (Na + ) and carbonate (CO3 2- ). The carbonate ions will react with water, resulting in the formation of bicarbonate and hydroxide, until an equilibrium is established. Sodium carbonate is used in many countries (e.g. U.S. and EU) as a food additive. It is regarded as a Generally Recognised as Safe (GRAS) substance in food with no limitation other than current good manufacturing practice. Acute Toxicity An acute oral LD50 of sodium carbonate monohydrate in rats is 2,800 mg/kg, and the acute dermal LD50 is >2,000 mg/kg. These studies were done with sodium carbonate monohydrate but due to the relatively low water content of sodium carbonate monohydrate, the toxicity of sodium carbonate is not expected to be significantly different. The LC50s for guinea pigs, mice and rats were 800, 1,200 and 2,300 mg/m, respectively, when male animals were exposed for 2 hours to sodium combustion products containing mainly sodium carbonate. Irritation Skin irritation studies have been performed with solid sodium carbonate and a 50 % solution of sodium carbonate with both animals and human volunteers. Erythema and edema were not observed for the intact skin. Thus, sodium carbonate has little to no skin irritation potential. The available eye irritation tests show differing results. Studies using a dose of 0.1 ml sodium carbonate monohydrate and sodium carbonate (anhydrous) resulted in a classification of irritating and highly irritating, respectively. However, based on a study with a dose of 0.1 g sodium carbonate it was not classified as an ocular irritant. Overall, the results indicat that sodium carbonate is irritating to the eyes. 1

108 Sensitization No data are available. Repeated Dose Toxicity No animal data are available on repeated dose toxicity studies by the oral route. Carbonate would be neutralized in the stomach by the low ph of the gastric juice and would not be available for uptake into the body. Genotoxicity The available in vitro mutagenicity test with sodium carbonate was negative. Carcinogenicity No data are available. Reproductive/Developmental Toxicity Aqueous solutions of sodium carbonate were administered daily via oral intubation to pregnant mice at doses ranging from 3.4 to 340 mg/kg during days 6-15 of gestation. There was no effect on implantation or on the survival of dams and fetuses. There were no significant differences in soft and skeletal tissue anomalies between treated and control animals. Negative results were reported for rats and rabbits for daily doses from 2.45 to 245 mg/kg and 1.79 to 179 mg/kg, respectively. Key Study/Critical Effect for Screening Criteria The Australian drinking water screening value for sodium (180 ppm, aethestic) and ph may apply to sodium carbonate. References OECD (2002a). IUCLID Data Set for Sodium carbonate (CAS No ), UNEP Publications. OECD (2002b). Screening Information Dataset (SIDS) Initial Assessment Report for Sodium carbonate (CAS No ), UNEP Publications. 2

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110 Toxicity Profile Sodium hydroxide (CAS No ) Sodium hydroxide (NaOH) has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Sodium hydroxide (NaOH) is present in the environment as sodium (Na + ) and hydroxyl ions (OH - ). Sodium is a normal constituent of the blood and an excess is excreted in the urine. A significant amount of sodium is taken up via the food because the normal uptake of sodium via food is g per day according to Fodor et al. (1999). Exposure to NaOH could potentially increase the ph of the blood. However, the ph of the blood is regulated between narrow ranges to maintain homeostasis (ph of 7.4 to 7.5) by urinary excretion of bicarbonate and via exhalation of carbon dioxide. Acute Toxicity An oral LD50 of a 1-10% solution of NaOH in rabbits was reported to be 325 mg/kg (expressed as 100% NaOH). Mortality was also observed when 1% NaOH was dosed, but in this case, the applied volume was relatively high (24 ml per kg body weight). Irritation A NaOH solution of 8% can be considered corrosive based on animal data. Human data indicate that concentrations of 0.5 to 4% were irritating. In two different studies a concentration of 0.5% was irritating for only 55 and 61% of the volunteers, respectively. Thus, it is assumed that a concentration, which is slightly lower than 0.5%, is the nonirritating concentration. The available animal data on eye irritation showed that the non-irritant level was %, while the corrosive concentration was 1.2 % or >2%. Sensitization Male volunteers were exposed on the back to sodium hydroxide concentrations of % (induction). After 7 days the volunteers were challenged to a concentration of 0.125%. The irritant response correlated well with the concentration of NaOH, but an increased response was not observed when the previously patch tested sites were rechallenged. Based on this study sodium hydroxide has no skin sensitization potential. 1

111 Repeated Dose Toxicity No animal data are available on repeated dose toxicity studies by oral, dermal, inhalation or by other routes for NaOH. It is not useful to study the repeated dose toxicity of hydroxide via an oral study because at high concentrations the substance is corrosive or irritating, while at low concentrations the hydroxide will be neutralized in the stomach by gastric juice, which has a very low ph. Genotoxicity Both the in vitro and the in vivo genotoxicity tests are negative. Carcinogenicity No carcinogenicity studies on sodium hydroxide were identified. Reproductive Toxicity No valid studies were identified regarding toxicity to reproduction in animals after oral, dermal or inhalation exposure to NaOH. It is not useful to study the reproduction/developmental toxicity of hydroxide via an oral study because at high concentrations the substance is corrosive or irritating, while at low concentrations the hydroxide will be neutralized in the stomach by gastric juice, which has a very low ph. Developmental Toxicity No valid studies were identified regarding developmental toxicity in animals after oral, dermal or inhalation exposure to NaOH. It is not useful to study the reproduction/developmental toxicity of hydroxide via an oral study because at high concentrations the substance is corrosive or irritating, while at low concentrations the hydroxide will be neutralized in the stomach by gastric juice, which has a very low ph. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for ph may apply to sodium hydroxide. Reference 2

112 OECD (2002). IUCLID Data Set for Sodium hydroxide (CAS No ), UNEP Publications. OECD (2002). Screening Information Dataset (SIDS) Initial Assessment Report for Sodium hydroxide (CAS No ), UNEP Publications. 3

113 Toxicity Profile Talc (CAS No ) Talc refers to both mineral talc and industrial mineral products that are marketed under the name talc and contain proportions of mineral talc that range from about 35% to almost 100%. Industrial talc generally refers to products that contain abundant minerals other than talc; cosmetic talc now normally contains >98% talc but the content may have been lower in the past. Pharmaceutical talc contains >99% talc. Talcum powder is cosmetic-grade talc. Talc is a mineral composed of hydrated magnesium silicate with the chemical formula Mg3Si4O10(OH)2 or H2Mg3(SiO3)4. Talc is not soluble in water. The U.S. Food and Drug Administration (FDA) regulates talc and states that it is generally recognized as safe for use in color additives in foods, drugs and cosmetics, and in paper, paper products, cotton and cotton fabrics that come into contact with food. The Food Chemical Codex (2003) provides specifications for food-grade talc, including the statement that talc derived from deposits that are known to contain associated asbestos is not food grade. Under the voluntary guidelines initiated in 1976, the Cosmetic, Toiletry, and Fragrances Association stated that all cosmetic talc should contain at least 90% platy talc (hydrated magnesium silicate) that is free from detectable amounts (<0.5%) of fibrous, asbestos minerals. Toxicokinetics (Oral) The absorption and disposition of 3 H-labeled talc in rats, mice, and guinea pigs administered a single oral dose was studied by Phillips et al. (1978). The oral doses were 50 mg/kg for rats, 40 mg/kg for mice, and 25 mg/kg for guinea pigs. In rats, mice, and guinea pigs, more than 95% of the dose was excreted in the feces 3 to 4 days after dosing. Less than 2% of the radioactivity was recovered in the urine. This radioactivity probably reflected contamination of urine samples with feces. No radioactivity was found in the liver or kidneys of these animals. This information suggests that talc is not absorbed from the gastrointestinal tract following oral exposure. Acute Toxicity No data were located. 1

114 Repeated Dose Toxicity/Carcinogenicity Male and female Wistar rats were given in their diet 0 or 50 mg/kg of commercial talc [characteristics unspecified] for the life of the animals (average survival was 702 and 649 days, respectively). There was no significant difference in the talc-fed animals compared with control animals (Gibel et al., 1976). Male and female Wistar-derived rats were given in their diet 100 mg Italian talc (grade 00000; ready milled; mean particle size, 25 μm; containing 92% talc, 3% chlorite, 1% carbonate minerals and 0.5 1% quartz) per rat per day for 5 months (talc-containing diet was actually given for 101 days) and were then maintained on basal diet for life (average survival, 614 days). No differences in tumor incidence were noted between treated animals and control animals (eight/sex for an average survival of 641 days) (Wagner et al., 1977). In humans and experimental animals, the effects of talc are dependent on the route of exposure, and the dose and properties of the talc. Talc pneumoconiosis was somewhat more prevalent and severe among miners exposed to talc containing asbestiform minerals and/or asbestos than among those exposed to talc without such contaminants. However, the role of quartz and asbestos in the observed pneumoconiosis could not be ruled out. Among drug users, intravenous injection of talc present as a filler in the drugs resulted in microembolization in a variety of organs and alterations in pulmonary function. In animal studies, talc has been shown to cause granulomas and mild inflammation when inhaled. Observations of the effects that occurred in the lungs of rats exposed by inhalation to talc suggested that the operative mechanisms may be similar to those identified for carbon black, and talc is known to cause the release of cytokines, chemokines and growth factors from pleural mesothelial cells. IARC: There is inadequate evidence in humans for the carcinogenicity of inhaled talc not containing asbestos or asbestiform fibres. There is limited evidence in experimental animals for the carcinogenicity of talc not containing asbestos or asbestiform fibres. Inhaled talc not containing asbestos or asbestiform fibres is not classifiable as to its carcinogenicity (Group 3). Genotoxicity The IARC (1987) review of talc included unpublished results from a 1974 study conducted by Litton Bionetics that showed no mutagenic activity for talc in vitro or in vivo. Talc did not induce mutations in Salmonella typhimurium strains TA1530 or HisG46, or in the yeast, Saccharomyces cerevisiae. No chromosomal aberrations were observed in human fibroblasts treated with talc in vitro. In vivo tests conducted in rats gave negative results for induction of chromosomal aberrations in bone marrow cells and dominant lethal mutations in germinal cells. 2

115 Three samples of respirable talc failed to elicit significant unscheduled DNA synthesis (10, 20 and 50 μg/cm 2, 24 hours), sister chromatid exchange or aneuploidy (2, 5, 10 and 15 μg/cm 2, 48 hours) in rat pleural mesothelial cells, in contrast to various positive controls. The three samples, i.e. Spanish talc (No. 5725), Italian talc (No. 5726) and French talc (No. 7841), contained 90 95% talc; the remaining contents were chlorite and dolomite. Electron microscopy analysis revealed that talc particles were taken up by the rat pleural mesothelial cells, but no aneuploidy was observed in metaphases (Endo- Capron et al., 1993). Reproductive Toxicity No data are available. Developmental Toxicity No teratologic effects were observed in hamsters, rats, mice, or rabbits after oral administration of talc. The doses used were 1,600 mg/kg for rats and mice on days 6 through 15 of gestation; 1,200 mg/kg for hamsters on day 6 through 10 of gestation; and 900 mg/kg for rabbits on days 6 through 18 of gestation. Key Study/Critical Effect for Screening Criteria There are no adequate studies for which to derive a oral reference dose. Talc is poorly absorbed from the gastrointestinal tract, if at all, and the limited data available by the oral route indicate that talc is essentially non-toxic by the oral route of exposure. References Endo-Capron, S., Renier, A., Janson, X. et al. (1993). In vitro response of rat pleural mesothelial cells to talc samples in genotoxicity assays (sister chromatid exchanges and DNA repair). Toxicol. In Vitro 7: Gibel, W., LOhs, K., Horn, K.H., Wildner, G.P., and Hoffman, F. (1976) Experimental study on carcinogenic activity of asbestos filters (Ger.). Arch. Geschwulstforsch. 46: IARC (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 42, International Agency for the Research on Cancer, World Health Organization, Lyon, France; cited in NTP (1993). IARC (2010). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Carbon Black, Titanium Dioxide, and Talc, Volume 93, International Agency for the Research on Cancer, World Health Organization, Lyon, France. 3

116 NTP (1993). Toxicology and Carcinogenesis of Talc (CAS No ) in F344/N Rats and B6C3F1 Mice (Inhalation Studies), NTP TR 421, NIH Publication No , National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. Phillips, J.C., Young, P.J., Hardy, K., and Gangolli, S.D. (1978). Studies on the absorption and disposition of 'H-labelled talc in the rat, mouse, guinea-pig and rabbit. Food Cosmet. Toxicol. 16: Wagner, J.C., Berry, G., Cooke, T.J., Hill, R.J., Pooley, F.D., and Skidmore, J.W. (1977). Animal experiments with talc. In: Inhaled Particles (W.H. Walton and B. McGovern, Eds.), Vol. IV, Part 2, pp , Pergamon Press, Oxford. 4

117 Toxicity Profile Tetramethylammonium chloride (CAS No ) Surrogates: Didecyldimethylammonium chloride (CAS No ) Dodecyltrimethylammonium chloride (CAS No ) No data could be located on tetramethylammonium chloride. Toxicity data are available for didecyldimethylammonium chloride (DDAC) and dodecyltrimethyl-ammonium chloride (DTAC). Thus, these two substances have been used as surrogates for the alkylated quaternary chloride. Acute Toxicity The oral LD50 of 100% DDAC has been reported to be 84 mg/kg in rats and 268 mg/kg in mice. The oral LD50 of a 5-25% solution of DDAC in rats was reported to be 1,190 mg/kg (595 to 297 mg a.i./kg). The acute dermal LD50 of a 80% solution of DDAC in rats is 3,342 mg/kg (2674 mg a.i./kg). The acute oral LD50 of DTAC (24.7% aq. soln.) in rats is 490 mg/kg. The acute oral LD50 of DTAC (37.35% aq. soln.) in rats is 560 mg/kg. Irritation DDAC is a severe skin and eye irritant to rabbits. Sensitization DDAC is not a dermal sensitizer to guinea pigs or to humans. Repeatd Dose Toxicity Mice were given in their diet 0, 100, 300, 600, 1,000 or 3,000 ppm DDAC for days. For the lowest four dose groups, these doses correspond to mean intake levels of approximately 18, 52, 107, and 182 mg/kg for males, and 23, 68, 134, and 224 mg/kg for females. High mortality in the 3,000 ppm groups prohibited calculation of daily intakes. Treatment of mice with 3,000 ppm DDAC in the diet for several days resulted in virtually 1

118 100% mortality in both sexes, with only one male surviving to termination of the study. Death was attributed to treatment-related severe wasting and dehydration resulting from gastrointestinal effects. Treatment with 1,000 ppm DDAC produced a 5% decrease in body weight in males with associated decreases in body weight gain. Similar depressed body weight in females from this group was assumed to be related to DDAC exposure. No other changes were observed in males or females in the other dose groups. The NOEL for this study is 600 ppm (107 and 134 mg/kg/day for males and females, respectively). Rats were given in their diet 0, 100, 300, 600, 1,000 or 3,000 ppm DDAC for days. For the lowest four dose groups, these doses correspond to mean intake levels of approximately 6, 18, 37, and 61 mg/kg for males, and 8, 22, 44, and 74 mg/kg for females. High mortality in the 3,000 ppm group prohibited calculation of mean daily intakes. Dietary exposure to 3,000 ppm DDAC resulted in 80% mortality in both sexes. The three rats of each sex of this group that survived to the end of the study exhibited markedly reduced body weights, fluid- or gas-filled intestines at necropsy, and inflammation of the beginning of the large intestines (typhlitis). Pathological changes in clinical chemistry included decreased serum glucose and protein concentrations in both sexes, decreased albumin and globulin concentrations in females, and increased erythrocyte count and hemoglobin and hematocrit concentrations in males. Administration of 1,000 ppm of less of DDAC resulted in no treatment-related effects. The NOEL for this study is 61 and 74 mg/kg/day for males and females, respectively. Male and female beagle dogs were given DDAC in their diet at doses of 0, 5, 15 and 50 mg/kg for 90 days. There was marked decrease in body weight gain, food consumption and food efficiency in the 50 mg/kg dose group. Clinical chemistry, hematology, urinalysis, and pathological results did not reveal any treatment-related effects. The NOAEL for this study is 15 mg/kg/day. Beagle dogs were dosed orally with 0, 3, 10, or 30 mg/kg DDAC for 52 weeks. During the first four and a half weeks of the study, several dogs in the 30 mg/kg group showed potentially life threatening decreases in body weight and food consumption. The dose was therefore decreased to 20 mg/kg/day. In some cases, depressions in weight and food consumption were so severe that the dogs in this group were removed from treatment completely during study days 31-36, and then reinstated at the 20 mg/kg/day. Administration of 3 or 20 mg/kg/day was not associated with mortality, changes in organ weights, gross pathological findings, ophthalmologic changes, or microscopic changes in selected organs and tissues. The 20 mg/kg/day dose was associated with decreases in mean erythrocyte counts; hemoglobin and hematocrit values; and mean total cholesterol, total protein, and albumin values. The 10 and 20 mg/kg/day doses were associated with an increased incidence of emesis, salivation, and soft/mucoid/liquid feces compared to controls. The NOEL for systemic toxicity was considered to be 10 mg/kg/day. Sprague-Dawley rats were given in their diet 0, 300, 750, or 1,500 ppm DDAC for at least 104 weeks. These doses corresponded to approximate DDAC intakes of 13, 32, and 64 mg/kg/day for males, and 16, 41, and 83 mg/kg/day for females. Treatment-related 2

119 decreases in body weight and food consumption in both males and females were observed in the 1,500 ppm group. In addition, possible treatment-related microscopic changes including hyperplasia of bile ducts in female rats and changes in mesenteric lymph nodes in male and female rats related to blood in the sinuses were observed in the 1,500 ppm group. No treatment-related effects were seen in the type of incidence of clinical signs, survival, palpable masses, clinical pathology, organ weights, gross anatomic pathology, or ophthalmology. The NOEL for toxicity was considered to be 750 ppm (32 and 41 mg/kg/day for males and females, respectively). CD-1 mice were administered in their diet 0, 100, 500, or 1,000 ppm DDAC for at least 78 weeks. The approximate mean intake levels were 15, 76.3, and mg/kg/day for males and 18.6, 93.1, and mg/kg/day for females. Treatment-related findings included decreased body weights and body weight gains in both male and females from the 1,000 ppm groups. There were no treatment-related clinical signs of toxicity, increases in palpable masses, changes in food consumption, differences in organ weights or observations at necropsy or differences in histopathological findings. The NOEL for this study was considered to be 500 ppm (76.3 and 93.1 mg/kg/day for males and females, respectively). Genotoxicity Several studies evaluating the mutagenicity of DDAC have all given negative results. DDAC was not mutagenic to several strains of S. typhimurium in a bacterial reverse mutation assay with or without metabolic activation. DDAC was tested in the CHO/HGPRT forward mutation assay and was not mutagenic in the presence or absence of metabolic activation. DDAC did not induce chromosomal aberrations in CHO cells with or without metabolic activation. In the in vitro rat primary hepatocyte unscheduled DNA synthesis (UDS) assay, DDAC did not induce significant increases in UDS. No chromosomal damage was observed in the bone marrow of rats in an in vivo cyogenetics assay. DTAC (24.7% aq. soln.) was not mutagenic to bacteria in a reverse mutation assay with or without metabolic activation. DTAC (24.7% aq. soln.) was not mutagenic in a mouse lymphoma assay with or without metabolic activation. DTAC was inactive in a rat primary hepatocyte UDS assay. DTAC was not genotoxic when given by oral gavage in a rat bone marrow micronucleus assay. Carcinogenicity Sprague-Dawley rats received DDAC in their diet at concentrations of 0, 300, 750 or 1,500 ppm for at least 104 weeks. These doses corresponded to approximate mean DDAC intakes of 13, 32, and 64 mg/kg/day for males, and 16, 41, and 83 mg/kg/day for females. DDAC was not considered to be carcinogenic under the conditions of this study. 3

120 CD-1 mice were administered in the diet 0, 100, 500, or 1,000 ppm DDAC for at least 78 weeks. The approximate mean intake levels were 15, 76.3, and mg/kg/day for males, and 18.6, 93.1, and mg/kg/day for females. DDAC was not considered to be carcinogenic under the conditions of this study. Reproductive Toxicity A two-generation reproductive toxicity study has been conducted with DDAC. Sprague- Dawley rats were given in their diet 0, 300, 750 or 1,500 ppm for two generations. Results indicate that continuous exposure to DDAC in the diet for two generations resulted in no adverse reproductive effects. Parental toxicity was observed at 1,500 ppm (~112.6 mg/kg/day), limited to body weight reduction, weight gain depression, and decreased food consumption. Postnatal toxicity at 1,500 ppm was indicated by reduced pup body weights. Based on decreased body weight/weight gain and food consumption, the parental Toxicity NOAEL = 750 ppm (56 mg/kg/day); LOAEL = 1500 ppm (113 mg/kg/day). Based on decreased pup body weight/weight gain, the reproductive toxicity NOAEL = 750 ppm (56 mg/kg/day); LOAEL = 1500 ppm (113 mg/kg/day). Developmental Toxicity Female NZ rabbits were given by oral gavage 0, 1.0, 3.0 or 10 mg/kg/day of DDAC during GD Four of the 16 does in the highest group died prior to GD 13. One doe at 10 mg/kg and 2 does at 1.0 mg/kg delivered early and were removed from the study. Nonlethal indications of maternal toxicity were evident at 3 and 10 mg/kg, as evidenced by reduced weight gain and clinical signs during the treatment period. Developmental toxicity, including increased incidences of fetal mortality and reduced fetal body weight per litter, were observed only at the highest dose. No teratogenicity was observed at any dose level. The NOEL for maternal toxicity is 1 mg/kg/day, and the NOEL for developmental toxicity is 3 mg/kg/day. Female Sprague-Dawley rats were given by oral gavage 0, 1, 10 or 20 mg/kg DDAC during GD No females died, aborted, delivered early, or were removed early from the study. Maternal toxicity was indicated at 10 and 20 mg/kg by characteristic clinical signs of audible respiration. Reductions in body weight and food consumption were also observed at 20 mg/kg during the treatment period. No evidence of developmental toxicity including teratogenicity was observed at any dose level. The NOEL for maternal toxicity was 1 mg/kg/day. The NOEL for developmental toxicity is >20 mg/kg/day. 4

121 Key Study/Critical Effect for Screening Criteria The lowest NOAEL was 10 mg/kg/day for dogs in the one-year chronic toxicity study. The LOAEL of 20 mg/kg/day was based on increased incidence of clinical signs in males and females, and decreased total cholesterol levels in females. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 20/100 = 0.2 mg/kg/day Drinking water guidance value = 0.7 ppm References Henderson, N.D. (1992). A Review of the Environmental Impact and Toxic Effects of DDAC. Prepared for: BC Environment, Ministry of Environment, Lands and Parks, Victoria, British Columbia. Found as Appendix B of the Robust Summaries for the Fatty Nitrogen Derived (FND) Cationics Category in the U.S. HPV Chemical Challenge Program. U.S. EPA (2006). Reregistration Eligibility Decision for Aliphatic Alkyl Quaternaries (DDAC), EPA739-R , Office of Prevention, Pesticides and Toxic Substances (OPPTS), U.S. Environmental Protection Agency, August 2006 (docket #EPA-HQ-OPP ). U.S. HPVIS (FND Cationics Category): 5

122 APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES F.2 HALLIBURTON WATER AND GUAR BASED SYSTEMS

123 Toxicity Profile Acetic acid (CAS No ) Acetic acid dissociates in aqueous media to H + and the acetate anion (CH3CO2 - ). It is naturally occurring as the acid in apple cider vinegar and other fruit-derived products. It and several of its salts are commonly used as food additives (e.g., as flavor enhancers) and are listed as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA). ph: 2.5 at 50 g/l and 20 o C pka: 20 o C Acute Toxicity The oral LD50 in rats is 4,950 mg/kg, and the dermal LD50 in rabbits is 1,060 mg/kg. The 4-hour LC50 in rats is 11.4 mg/l. Irritation In the EU, acetic acid is classified as a skin and eye irritant at concentrations <25%. At >25%, it is classified as corrosive to the skin. Sensitization No data are available. Repeated Dose Toxicity In an 8-month study, rats dosed with 60 mg/kg acetic acid (three times per week) developed hyperplasia in the esophagus and forestomach. Genotoxicity Acetic acid was not mutagenic to Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 in an in vitro bacterial mutation assay with or without metabolic activation. Acetic acid (in media adjusted to a ph of 8.5) was also not genotoxic in an in vitro chromosomal aberration test using Chinese Hamster Ovary cells with or without metabolic activation. 1

124 Carcinogenicity No adequate carcinogenicity studies have been conducted on acetic acid. Reproductive Toxicity No studies could be located. Developmental Toxicity There were no effects on implantations or on maternal or fetal survival in mice, rats, or rabbits at doses up to 1,600 mg/kg. Key Study/Critical Effect for Screening Criteria There are no repeat dose toxicity studies that were considered adequate for human health risk assessment. The only study reported on the toxicity of acetic acid by oral gavage showed effects of irritation (probably due to the ph of acetic acid) at the site of contact in the gastrointestinal tract. No systemic effects were reported. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has maintained a group ADI of not limited for acetic acid and its potassium and sodium salts. The Australian drinking water guidance value for ph may apply to acetic acid. References ECHA REACH: JECFA: U.S. EPA HPVIS database: 2

125 Toxicity Profile Alkylated Quaternary chloride Surrogates: Didecyldimethylammonium chloride (CAS No ) Dodecyltrimethylammonium chloride (CAS No ) No data could be located on this alkylated quaternary chloride. Toxicity data are available for didecyldimethylammonium chloride (DDAC) and dodecyltrimethylammonium chloride (DTAC). Thus, these two substances have been used as surrogates for the alkylated quaternary chloride. Acute Toxicity The oral LD50 of 100% DDAC has been reported to be 84 mg/kg in rats and 268 mg/kg in mice. The oral LD50 of a 5-25% solution of DDAC in rats was reported to be 1,190 mg/kg (595 to 297 mg a.i./kg). The acute dermal LD50 of a 80% solution of DDAC in rats is 3,342 mg/kg (2674 mg a.i./kg). The acute oral LD50 of DTAC (24.7% aq. soln.) in rats is 490 mg/kg. The acute oral LD50 of DTAC (37.35% aq. soln.) in rats is 560 mg/kg. Irritation DDAC is a severe skin and eye irritant to rabbits. Sensitization DDAC is not a dermal sensitizer to guinea pigs or to humans. Repeatd Dose Toxicity Mice were given in their diet 0, 100, 300, 600, 1,000 or 3,000 ppm DDAC for days. For the lowest four dose groups, these doses correspond to mean intake levels of approximately 18, 52, 107, and 182 mg/kg for males, and 23, 68, 134, and 224 mg/kg for females. High mortality in the 3,000 ppm groups prohibited calculation of daily intakes. Treatment of mice with 3,000 ppm DDAC in the diet for several days resulted in virtually 100% mortality in both sexes, with only one male surviving to termination of the study. Death was attributed to treatment-related severe wasting and dehydration resulting from 1

126 gastrointestinal effects. Treatment with 1,000 ppm DDAC produced a 5% decrease in body weight in males with associated decreases in body weight gain. Similar depressed body weight in females from this group was assumed to be related to DDAC exposure. No other changes were observed in males or females in the other dose groups. The NOEL for this study is 600 ppm (107 and 134 mg/kg/day for males and females, respectively). Rats were given in their diet 0, 100, 300, 600, 1,000 or 3,000 ppm DDAC for days. For the lowest four dose groups, these doses correspond to mean intake levels of approximately 6, 18, 37, and 61 mg/kg for males, and 8, 22, 44, and 74 mg/kg for females. High mortality in the 3,000 ppm group prohibited calculation of mean daily intakes. Dietary exposure to 3,000 ppm DDAC resulted in 80% mortality in both sexes. The three rats of each sex of this group that survived to the end of the study exhibited markedly reduced body weights, fluid- or gas-filled intestines at necropsy, and inflammation of the beginning of the large intestines (typhlitis). Pathological changes in clinical chemistry included decreased serum glucose and protein concentrations in both sexes, decreased albumin and globulin concentrations in females, and increased erythrocyte count and hemoglobin and hematocrit concentrations in males. Administration of 1,000 ppm of less of DDAC resulted in no treatment-related effects. The NOEL for this study is 61 and 74 mg/kg/day for males and females, respectively. Male and female beagle dogs were given DDAC in their diet at doses of 0, 5, 15 and 50 mg/kg for 90 days. There was marked decrease in body weight gain, food consumption and food efficiency in the 50 mg/kg dose group. Clinical chemistry, hematology, urinalysis, and pathological results did not reveal any treatment-related effects. The NOAEL for this study is 15 mg/kg/day. Beagle dogs were dosed orally with 0, 3, 10, or 30 mg/kg DDAC for 52 weeks. During the first four and a half weeks of the study, several dogs in the 30 mg/kg group showed potentially life threatening decreases in body weight and food consumption. The dose was therefore decreased to 20 mg/kg/day. In some cases, depressions in weight and food consumption were so severe that the dogs in this group were removed from treatment completely during study days 31-36, and then reinstated at the 20 mg/kg/day. Administration of 3 or 20 mg/kg/day was not associated with mortality, changes in organ weights, gross pathological findings, ophthalmologic changes, or microscopic changes in selected organs and tissues. The 20 mg/kg/day dose was associated with decreases in mean erythrocyte counts; hemoglobin and hematocrit values; and mean total cholesterol, total protein, and albumin values. The 10 and 20 mg/kg/day doses were associated with an increased incidence of emesis, salivation, and soft/mucoid/liquid feces compared to controls. The NOEL for systemic toxicity was considered to be 10 mg/kg/day. Sprague-Dawley rats were given in their diet 0, 300, 750, or 1,500 ppm DDAC for at least 104 weeks. These doses corresponded to approximate DDAC intakes of 13, 32, and 64 mg/kg/day for males, and 16, 41, and 83 mg/kg/day for females. Treatment-related decreases in body weight and food consumption in both males and females were observed in the 1,500 ppm group. In addition, possible treatment-related microscopic 2

127 changes including hyperplasia of bile ducts in female rats and changes in mesenteric lymph nodes in male and female rats related to blood in the sinuses were observed in the 1,500 ppm group. No treatment-related effects were seen in the type of incidence of clinical signs, survival, palpable masses, clinical pathology, organ weights, gross anatomic pathology, or ophthalmology. The NOEL for toxicity was considered to be 750 ppm (32 and 41 mg/kg/day for males and females, respectively). CD-1 mice were administered in their diet 0, 100, 500, or 1,000 ppm DDAC for at least 78 weeks. The approximate mean intake levels were 15, 76.3, and mg/kg/day for males and 18.6, 93.1, and mg/kg/day for females. Treatment-related findings included decreased body weights and body weight gains in both male and females from the 1,000 ppm groups. There were no treatment-related clinical signs of toxicity, increases in palpable masses, changes in food consumption, differences in organ weights or observations at necropsy or differences in histopathological findings. The NOEL for this study was considered to be 500 ppm (76.3 and 93.1 mg/kg/day for males and females, respectively). Genotoxicity Several studies evaluating the mutagenicity of DDAC have all given negative results. DDAC was not mutagenic to several strains of S. typhimurium in a bacterial reverse mutation assay with or without metabolic activation. DDAC was tested in the CHO/HGPRT forward mutation assay and was not mutagenic in the presence or absence of metabolic activation. DDAC did not induce chromosomal aberrations in CHO cells with or without metabolic activation. In the in vitro rat primary hepatocyte unscheduled DNA synthesis (UDS) assay, DDAC did not induce significant increases in UDS. No chromosomal damage was observed in the bone marrow of rats in an in vivo cyogenetics assay. DTAC (24.7% aq. soln.) was not mutagenic to bacteria in a reverse mutation assay with or without metabolic activation. DTAC (24.7% aq. soln.) was not mutagenic in a mouse lymphoma assay with or without metabolic activation. DTAC was inactive in a rat primary hepatocyte UDS assay. DTAC was not genotoxic when given by oral gavage in a rat bone marrow micronucleus assay. Carcinogenicity Sprague-Dawley rats received DDAC in their diet at concentrations of 0, 300, 750 or 1,500 ppm for at least 104 weeks. These doses corresponded to approximate mean DDAC intakes of 13, 32, and 64 mg/kg/day for males, and 16, 41, and 83 mg/kg/day for females. DDAC was not considered to be carcinogenic under the conditions of this study. 3

128 CD-1 mice were administered in the diet 0, 100, 500, or 1,000 ppm DDAC for at least 78 weeks. The approximate mean intake levels were 15, 76.3, and mg/kg/day for males, and 18.6, 93.1, and mg/kg/day for females. DDAC was not considered to be carcinogenic under the conditions of this study. Reproductive Toxicity A two-generation reproductive toxicity study has been conducted with DDAC. Sprague- Dawley rats were given in their diet 0, 300, 750 or 1,500 ppm for two generations. Results indicate that continuous exposure to DDAC in the diet for two generations resulted in no adverse reproductive effects. Parental toxicity was observed at 1,500 ppm (~112.6 mg/kg/day), limited to body weight reduction, weight gain depression, and decreased food consumption. Postnatal toxicity at 1,500 ppm was indicated by reduced pup body weights. Based on decreased body weight/weight gain and food consumption, the parental Toxicity NOAEL = 750 ppm (56 mg/kg/day); LOAEL = 1500 ppm (113 mg/kg/day). Based on decreased pup body weight/weight gain, the reproductive toxicity NOAEL = 750 ppm (56 mg/kg/day); LOAEL = 1500 ppm (113 mg/kg/day). Developmental Toxicity Female NZ rabbits were given by oral gavage 0, 1.0, 3.0 or 10 mg/kg/day of DDAC during GD Four of the 16 does in the highest group died prior to GD 13. One doe at 10 mg/kg and 2 does at 1.0 mg/kg delivered early and were removed from the study. Nonlethal indications of maternal toxicity were evident at 3 and 10 mg/kg, as evidenced by reduced weight gain and clinical signs during the treatment period. Developmental toxicity, including increased incidences of fetal mortality and reduced fetal body weight per litter, were observed only at the highest dose. No teratogenicity was observed at any dose level. The NOEL for maternal toxicity is 1 mg/kg/day, and the NOEL for developmental toxicity is 3 mg/kg/day. Female Sprague-Dawley rats were given by oral gavage 0, 1, 10 or 20 mg/kg DDAC during GD No females died, aborted, delivered early, or were removed early from the study. Maternal toxicity was indicated at 10 and 20 mg/kg by characteristic clinical signs of audible respiration. Reductions in body weight and food consumption were also observed at 20 mg/kg during the treatment period. No evidence of developmental toxicity including teratogenicity was observed at any dose level. The NOEL for maternal toxicity was 1 mg/kg/day. The NOEL for developmental toxicity was >20 mg/kg/day. Key Study/Critical Effect for Screening Criteria The lowest NOAEL was 10 mg/kg/day for dogs in the one-year chronic toxicity study. The LOAEL of 20 mg/kg/day was based on increased incidence of clinical signs in males and females, and decreased total cholesterol levels in females. 4

129 Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 20/100 = 0.2 mg/kg/day Drinking water guidance value = 0.7 ppm References Henderson, N.D. (1992). A Review of the Environmental Impact and Toxic Effects of DDAC. Prepared for: BC Environment, Ministry of Environment, Lands and Parks, Victoria, British Columbia. Found as Appendix B of the Robust Summaries for the Fatty Nitrogen Derived (FND) Cationics Category in the U.S. HPV Chemical Challenge Program. U.S. EPA (2006). Reregistration Eligibility Decision for Aliphatic Alkyl Quaternaries (DDAC), EPA739-R , Office of Prevention, Pesticides and Toxic Substances (OPPTS), U.S. Environmental Protection Agency, August 2006 (docket #EPA-HQ-OPP ). U.S. HPVIS (FND Cationics Category), 5

130 Toxicity Profile Amorphous/Non-crystalline Silica (CAS No ) Silica Gel (CAS No ) Amorphous silica and silica gel have been reviewed in the OECD-SIDS program (OECD, 2004a,b). The CAS No is the general CAS No. for silicon dioxide which includes all forms of silicas (e.g. also crystalline and natural forms). Only the silica sub-classes, the synthetic amorphous silicas, are the subject of this assessment. Synthetic amorphous silicas (SAS) are in powder form which have a low water solubility: 70 mg/l Toxicokinetics After oral ingestion, there is no accumulation of SAS in body tissues. Upon cessation of exposure, rapid elimination occurs. Intestinal resorption appears to be insignificant in animals and humans. In a human study, the small apparent increases in the urine output of human volunteers were remarkably low as compared with the high dose of 2,500 mg SiO2 applied. After daily oral administration of 1,500 mg/kg SAS (FK 700) as aqueous suspension to rats for one month, there was no accumulation of SiO2 in the body: the average SiO2 content was: 1.5 μg in the liver, 6.4 μg in the kidney, and 5.3 μg in the spleen. The corresponding control values were 1.8, 7.2 and 7.8 μg SiO2, respectively. In a similar experiment in rats receiving 20 daily oral doses of 100 mg SAS (HDK V15) per animal (about 500 mg/kg) each, tissue values were slightly increased in liver and kidney: in liver 4.2 μg (control value 1.8 μg), in the spleen 5.5 μg (7.2 μg) and in the kidneys 14.2 μg (7.8 μg). In 12 human volunteers, no significant increased renal excretion of SiO2 was found following single oral ingestion of 2,500 mg (AEROSIL 175 and FK 700). Acute Toxicity The acute oral administration of various forms of SAS (aqueous suspension or gel) failed to produce signs of toxicity or deaths in treated animals with LD50 values greater than the 1

131 top doses applied, either by gavage: >3100 to >20000 mg/kg in mice and rats; or in the diet for 24 hours. Irritation Synthetic amorphous silicas are not irritating to the skin of rabbits exposed to 0.19 g (one case) or 0.5 g of dry or moistened test item under occlusive conditions for 4 or 24 hours. All products tested as a powder (0.1 g) have shown no or only weak and transient irritating effects on the conjunctivae of the eyes of rabbits with the iris and cornea not affected at all. Sensitization No experimental data are available on the synthetic amorphous silicas. Medical surveillance records on workers gave no evidence of skin sensitization over decades of practical experience. Repeated Dose Toxicity/Carcinogenicity Fischer rats and B6C3F1 mice were administered SAS in the diet at levels of 0, 1.25, 2.5, and 5% for 102 weeks. The animals were in good condition throughout and showed high survival. The tumor responses in all organs of SAS-fed rats and mice were not statistically significantly different from the controls. The NOAEL for rats and mice were 2,500 and 6,500 mg/kg/day, respectively. Genotoxicity There is no evidence for synthetic amorphous silica to induce mutations either in vitro or in vivo using standard genotoxicity tests. Reproductive Toxicity An early limited one-generation study on rats gave no evidence of adverse effects on reproduction performance at 500 mg/kg/day, the highest dose tested (NOAEL). But the reliability is poor due to the small group size of animals. Developmental Toxicity 2

132 SAS was examined for embryotoxic and developmental effects during the gestation phase in various animals species, rat, mouse, rabbit and hamster, at oral doses up to 1,600 mg/kg/day.. There were no significant signs of maternal or embryotoxic/developmental toxic effects in any species tested. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the frequencies occurring spontaneously in the control animals. Key Study/Critical Effect for Screening Criteria The lowest NOAEL from the two-year dietary study was 2,500 mg/kg/day for rats. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 2,500/100 = 2.5 mg/kg/day Drinking water guidance value = 0.09 ppm References OECD (2004a). IUCLID Data Set for Synthetic Amorphous Silica and Silicates: Silicon Dioxide (CAS No , , ; Silicic Acid, Aluminum Sodium Salt (CAS No ); Silicic Acid, Calcium Salt (CAS No ), UNEP Publications. OECD (2004b). Screening Information Dataset (SIDS) Initial Assessment Report for Synthetic Amorphous Silica and Silicates: Silicon Dioxide (CAS No , , ; Silicic Acid, Aluminum Sodium Salt (CAS No ); Silicic Acid, Calcium Salt (CAS No ), UNEP Publications. 3

133 Toxicity Profile 1,2-Benzothiazolin-3-one (CAS No ) Acute Toxicity The rat oral LD50 is 670 mg/kg in males and 784 mg/kg in females. The rabbit dermal LD50 is >2,000 mg/kg. Irritation 1,3-Benzothiazolin-2-one is a mild skin irritant and severe eye irritant to rabbits. Sensitization 1,3-Benzeothiazolin-3-one is a skin sensitizer to guinea pigs. Repeated Dose Toxicity Decreased body weight, increased incidence of forestomach hyperplasia, and nonglandular stomach lesions were observed in rats in a rat subchronic oral toxicity study. The NOAEL is 8.42 mg/kg/day. A dog subchronic oral toxicity study showed effects occurring at lower doses than in rats, and included alterations in blood chemistry (decreased plasma albumin, total protein, and alanine aminotransferase) and increased absolute liver weight. The NOAEL is 5 mg/kg/day. Genotoxicity All acceptable mutagenicity studies showed a negative mutagenic response for this chemical. Carcinogenicity No carcinogenicity studies are available. Reproductive Toxicity 1

134 In a two-generation reproductive toxicity study, parental toxicity was observed at 500 ppm and was characterized by lesions in the stomach. In pups, toxic effects were reported at 1,000 ppm and consisted of preputial separation in males and impaired growth and survival in both sexes. There was no evidence of increased susceptibility of the offspring. Developmental Toxicity Developmental toxicity studies were conducted in rats with maternal effects including decreased body weight gain, decreased food consumption, and clinical toxicity signs (audible breathing, haircoat staining of the anogenital region, dry brown material around the nasal area) as well as increased mortality. Developmental effects consisted of increases in skeletal abnormalities (extra sites of ossification of skull bones, unossified sternebra), but no external or visceral abnormalities. Key Study/Critical Effect for Screening Criteria The U.S. EPA Office of Prevention, Pesticides and Toxic Substances (OPPTS) chronic oral reference dose is based on increased incidence of emesis and clinical chemistry alterations at 20 mg/kg/day in a subchronic toxicity study in dogs, with a NOAEL of 5 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 5/100 = 0.05 mg/kg/day Drinking water guidance value = 0.2 ppm Reference U.S. Environmental Protection Agency, Reregistration Eligibility Decision for 1,2- Benzoisothiazolin-3-one (BIT), Office of Prevention, Pesticides and Toxic Substances, 739-R , September

135 Toxicity Profile C10-C12 Alcohol Ethoxylates (CAS No ) Alcohol ethoxylates (AE) are a class of non-ionic surfactants that have the basic structure Cx-yAEn. The subscript (x-y) following the C indicates the range of carbon chain units. AEs with carbon unit range between C8 to C18 are most commonly used in household detergent products. The hydrocarbon chain can be either linear or branched. AEs also contain an ethylene oxide (E) chain attached to the alcohol. The degree of ethylene oxide polymerization is indicated by the subscript (n) which indicates the average number of ethylene oxide units. In household products the average ethylene oxide chain length commonly ranges between 3 and 12 units. The toxicity profile of C10-C12 alcohol ethoxylates has been developed from a risk assessment report on alcohol ethoxylates ranging from C8 to C18 prepared by the household cleaning product and chemical industry (HERA, 2009). Acute Toxicity The acute oral LD50 of C9-11AE2.5 in rats was calculated to be >4,000 mg/kg and <10,000 mg/kg. The acute oral LD50 of C7-9AE6 in rats was determined to be <2,000 mg/kg. The acute oral LD50 of C9-11AE8 in fasted rats was found to be 1,200 mg/kg. The oral LD50 in rats for C12-13AE6.5 is 2,100 mg/kg. The oral LD50 in rats for C12-15AE7 is 1,700 mg/kg. For C14-15AE11, the acute oral LD50 values were reported to be 720 mg/kg (neat) and 1,800 mg/kg (given as 50% (m/v) solution in corn oil). The oral LD50 in rats for C12-15AE11 is >2,00 mg/kg for males and between 1,000 and 2,000 mg/kg for females. The oral LD50 in rats for C14-15AE13 is 1,100 and 1,000 mg/kg in two separate studies. The dermal LD50 of C7-9AE6 was >2 g/kg. The dermal LD50 values for AEs with an alkyl chain length of 9 11 carbon atoms in three different rat studies were determined to be >2,000 mg/kg and >4,000 mg/kg. An acute dermal LD50 value of >2,000 mg/kg was determined for C12-14AE3 and C12-14AE6 in two separate studies. The acute dermal LD50 of C12-15AE7 was determined to be >2,000 mg/kg. The acute rat 4 hr-lc50 of C9-11AE5 generated as a mist was determined to be >0.22 mg/l. Talmage (1994) reported that alcohol ethoxylates were not acutely toxic to rats at concentrations less than or equal to their saturated vapor concentrations in air. Acute toxic thresholds were reached only when animals were exposed to the undiluted test chemical in the form of a respirable mist or aerosol. Under these conditions, 1- or 4-hour inhalation LC50 values ranged from 1.5 to 20.7 mg/l. Some studies reported no mortalities (1-hours LC50 -study) occurred at concentrations as high as 52 mg/l. 1

136 Irritation Alcohol ethoxylates with varying alkyl chain lengths and ethoxylation degree were found to be slightly to severely irritating to skin in rabbits and rats. The degree of irritation was dependent on the type of patches used (open application versus full occlusion), the exposure time as well as the concentration of the test material. In humans, AEs are less irritating to skin than in animals. Neat applications of a range AEs in a 4-hr human patch test did not warrant these chemicals to be classified as skin irritants under EU legislation, while the same AEs would have been classified for skin irritation on the basis of animal data. Alcohol ethoxylates range from mildly to severely irritating to rabbit eyes. Generally, concentration of 0.1% were non-irritating, and concentrations of 1 to 10% ranged from slight to moderately irritating. No relationship could be established between the chemical structures of the tested AEs and their eye irritation responses. Sensitization Alcohol ethoxylates should not be considered as skin sensitizers. A substantial amount of skin sensitization studies in guinea pigs following either the Magnusson-Kligman maximization or the Buehler testing protocol are available to evaluate the skin sensitization potential of AEs. Although a mild skin sensitization reaction was observed in a study following the Magnusson-Kligman protocol, the weight of evidence clearly supports the assessment that AEs should not be considered as skin sensitizers. This is further supported by clinical and market data that demonstrate the absence skin sensitization responses to AEs when tested under the conditions of the HRIPT or when used in AE containing consumer products. Repeated Dose Toxicity C10AE5 was fed to rats at doses of 0, 125, 250 or 500 mg/kg for 90 days. There were not treatment-related clinical signs, body weight gain, food consumption or feed efficiency. There was a slight increase in absolute liver weights as well as a trend toward a dose-dependent increase in the liver weight/body weight ratio, with a statistically significant increase in the high dose group. However, the histological evaluation did not reveal any indication of hepatotoxicity and therefore the increase in liver weights was not interpreted to be a toxicological effect. It can be considered to be an adaptive response as a result of extensive metabolism of the test compound by the liver. There were no other gross or histopathological changes that were considered treatment-related. The NOAEL for this study can be considered to be 500 mg/kg/day. 2

137 Rats were given in their diet 0, 125, 250, 500, 1,000 and 3,000 ppm C9-11AE6 for 90 days. There were no signs of toxicity at any dose level. The NOAEL is 3,000 ppm (approximately 150 mg/kg/day). Rats were given in their diet 0. 04, 0.2 and 1.0% C9-11AE8 for 90 days. Lower body weight gain and decreased food consumption were noted in the 1% males and females and in the 0.2% females from week 1 through the end of the study. Further statistical analyses revealed a significant decrease in the mean body weight gain noted in the 1% females and the decreases in mean food consumption noted in the 1% males and females. The differences noted in the 0.2% females were not statistically significant. The investigators considered these observations to be the result of poor palatability of the test substance. There were no other treatment-related effects. The NOAEL for this study is 1.0% (about 400 mg/kg/day). Rats were fed C12-15AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% and 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the 0.25% and higher dose groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the 0.25% groups and higher. There were not treatment-related changes in hematology, urinary and clinical chemistry parameters. The histological examination of the liver at necropsy revealed hepatocytic enlargement at 0.125% and higher, suggesting an increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (102 mg/kg/day). Rats were fed C12-14AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% and 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the 0.25% and higher dose groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the 0.25% groups and higher. There were not treatment-related changes in hematology, urinary and clinical chemistry parameters. The histological examination of the liver at necropsy revealed hepatocellular hypertrophy at 0.125% and higher, suggesting an increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (110 mg/kg/day). Wistar rats were fed C14-15AE7 in the diet at concentrations of 0, 300, 1,000, 3,000, and 10,000 ppm for 90 days. All animals survived until their scheduled necropsy date. Significant treatment-related effects on body weight (i.e., reduced mean body weights in males at 10,000 ppm and in females at 3,000 ppm), food intake (i.e., reduced intake in both sexes at 10,000 ppm and at 3,000 ppm for females), organ weights (i.e., increased relative liver weight in both sexes at 3,000 and 10,000 ppm and in females also at 1,000 ppm; increased spleen weight in males at 10,000 ppm; clinical chemistry (i.e., confined to 10,000 ppm dose groups; significantly higher urea, chloride and potassium levels in males; significantly higher urea, chloride and cholesterol levels in females) and hematology (i.e., in both sexes at 10,000 ppm and in males also at 3,000 ppm increased 3

138 total leukocytes and lymphocytes; females at 10,000 ppm showed depression in numbers of neutrophils, mean cell volume and mean cell hemoglobin) were identified in one or both sexes fed with dietary concentrations of 3,000 and 10,000 ppm. There were no compound-related histopathological effects at any dose level. Minor, but statistically significant changes in liver weight, kidney weights and plasma urea concentration were recorded in female rats in the 1,000 ppm group were not of toxicological significance. The NOAEL for this study is 1,000 ppm (50 mg/kg/day). Rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1%, 0.5% and 1% for 90 days. There were no treatment-related changes in body weight, food intake, and organ weights including those of the reproductive system, clinical chemistry and hematology at any treatment level. The NOAEL is 1% in the diet, which corresponded to 700 and 785 mg/kg/day for males and females, respectively. Rats were fed C12-13AE6.5 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Rats were fed C14-15AE7 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Charles River rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1, 0.5 and 1% for two years. There was a dose-related decrease in body weights in the 0.5 and 1% females and in the 1% males, the likely cause being poor palatability of the diet. At study termination, elevated organ-to-body weight ratios were noted for the liver, kidney, heart and thyroid/parathyroid glands in the 1% dietary group. The only significant histopathological finding prevalent in all dose groups was a dose-related increase in incidence of focal myocarditis at 12 months but not at study termination at 2 years. No other treatment-related histopathology was noted. The NOAEL was established at the 0.5% dietary level, which corresponds to about 162 and 190 mg/kg/day for males and females, respectively. Genotoxicity In all available in vitro and in vivo genotoxicity assays, there was no indication of genetic toxicity of broad range of structurally different alcohol ethoxylates. 4

139 Carcinogenicity Alcohol ethoxylates as a class are not carcinogenic by the oral route based of available oral long term toxicity/carcinogenicity studies on AEs. Charles River rats were given in their diet 0, 0.1, 0.5 or 1% C14-15AE7 for two years. There was no treatment-related changes in general behavior and appearance. The survival rate of the test animals was comparable if not better than the controls. Body weights of 0.5% females and 1% males and females had significantly lower weight gains than the control. There were no treatment-related effects on organ weights and tumor incidence. Sprague-Dawley rats were fed C14-15AE7 at 0.1, 0.5 and 1% in the diet for two years. A treatment-related body weight depression was observed in females at the two highest treatment levels and in males at the 1% dose level, probably due to the poor palatability of the diet. There was no evidence for any carcinogenic activity. Sprague-Dawley rats were fed C12-13AE6.5 in the diet at doses up to 1% (500 mg/kg/day). Reduced food consumption was noted at the higher dose levels (i.e., 0.5 and 1% for females and 1% for males), resulting in a lower body weight gain compared to the control group. No treatment-related histopathology was found and no increase in tumor incidence was observed. Reproductive Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% ( about 0, 25, 50 and 250 mg/kg/day). No treatment related effects in the parents or pups on general behaviour, appearance or survival were observed. Fertility of treated groups was comparable with the controls. The only observation was related to a reduced weight gain of parental rats and pups relative to the control at the 0.5% dose level. The NOAEL for reproduction was therefore set at the highest dose level which was 0.5% dietary level (250 mg/kg/day). In a two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% (about 0, 25, 50 and 250 mg/kg). The control group and the six treated groups comprised of 25 male and 25 female animals. Three of the groups received the compound continuously during the study. In the other three groups the females received the compound only during the 6th through the 15th day of gestation and the males were untreated. No treatment-related changes in behavior or appearance were observed in the parental rats or pups throughout the study. Female rats from the 0.5% continuous treatment group gained slightly less body weight compared to control females. No other consistent differences in body weight were observed. Food consumption was similar for control and treated rats. No compound related differences were seen between control and treated rats with respect to fertility, gestation or viability indices. The average 21-day body weights for pups at the 0.5% continuous treatment group were significantly lower as compared to the average pup 5

140 body weights in the controls. No other compound-related changes in body weight were observed. None of the deaths of parental rats during the study was considered to be compound-related. Examination of organ weight values revealed that compound-related effects were limited to increased group mean relative liver weights of male and female F1 from the 0.5% continuous feeding group at the 91-day sacrifice, and increases in group mean relative liver weights of males from the 0.5% continuous feeding group of the F2 generation at the 60-day and caesarean section sacrifices. No compound-related histopathological lesions were observed in any of the tissues examined from rats for the F0 and F1 generations. The NOAEL for reproductive toxicity is at least 0.5% in the diet (250 mg/kg/day). Developmental Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% ( about 0, 25, 50 and 250 mg/kg/day). General behavior, appearance and survival were not affected by treatment. At the 0.5% dose level, adults and pups gained less weight than the control rats. In the 0.5% dose group, there was a statistical increase in embryo lethality and soft tissue anomalies and at the 0.1% there was a statistical decrease in mean fetal liver weight. Neither of these effects was considered to be treatment-related by the authors as they showed no dose response characteristics. The NOAEL for maternal toxicity is 50 mg/kg/day. The NOAEL for developmental and teratogenicity is 0.1% (50 mg/kg/day). Pregnant rabbits were given by oral gavage 0, 50, 100 or 200 mg/kg C12AE6 from GD 2 to16. Nine control rabbits and 31 treated rabbits died during the study. Surviving rabbits at the 200 mg/kg dose level generally showed slight losses of body weight. At 100 and 200 mg/kg, ataxia and a slight decrease in body weight was observed in the pregnant animals. In seven treated and two control rabbits early deliveries were recorded. There were no treatment-related effects on corpora lutea, implantations, number of live fetuses and spontaneous abortions. The NOAEL for maternal toxicity is 50 mg/kg/day; the NOAEL for developmental toxicity is 200 mg/kg/day. In two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 and 0.5% (about 0, 25, 50 and 250 mg/kg). On the 13th day of the gestation period a representative number of female rats from each treatment group of the FC generation (i.e., pups from the 3rd mating of the F0 and F1 parental generation) were sacrificed. Laparotomies were performed and the uterus was examined for uterine abnormalities, normal implantation and resorption sites. Remaining females were sacrificed on the 21st day of gestation. Various maternal and fetal parameters showed occasional values that were significantly different from the corresponding controls. However these were not considered related to the material tested as none occurred at the high feeding level and no dose response for these parameters was apparent. With respect to body weight gains, parental female rats and pups of the high dose group did not gain as much body weight as the control rats. Examination of organ weight values reveal compound related effects were limited to increased group mean liver weighs of male and female P1 generation from the 0.5% continuous feeding group at the 6

141 91 day sacrifice and increase in group mean relative liver weights of males of the 0.5% continuous feeding group of the P2 generation at the 60 day section sacrifices. T he NOAEL for maternal and developmental toxicity was established at the 0.1% in the diet (50 mg/kg/day). Key Study/Critical Effect for Screening Criteria AEs of different structures with regard to the length of the alkyl chain and the degree of ethoxylation have been evaluated in a number of 90-day and two-year oral toxicity studies. The lowest NOAEL of AEs for systemic toxicity was established at 50 mg/kg/day in two chronic (two-year) dietary studies on C12-13AE6.5 and C14-15AE7. Effects observed at the LOAEL were related to significantly elevated organ-to-body weight ratios for liver, kidney and heart, although there were no adverse histopathological changes. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 50/100 = 0.5 mg/kg/day Drinking water guidance value = 1.8 ppm References Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Alcohol Ethoxylates (2009), Talmage, S.S. (1994). Environmental and Human Safety of Major Surfactants Alcohol Ethoxylates and Alkylphenol Ethoxylates, pp. 35, The Soap and Detergent Association, Lewis Publishers, Boca Raton, Florida. 7

142 Toxicity Profile Calcium chloride (CAS No ) Calcium chloride has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Calcium chloride is easily dissociated into calcium and chloride ions in water. The absorption, the distribution and the excretion of the ions in animals are regulated separately. Once calcium chloride is taken up, the effect of the substance on animals should be attributed to the effect of calcium, the effect of chloride, or both. Calcium and chloride ions are essential constituents of all animals. Absorption, transport, distribution, excretion, and the homeostatic regulation of calcium and chloride ions in animals are well established, as are the mechanisms of action. Calcium is essential for the formation and maintenance of bones and teeth, and for the regulation of various physiological functions such as neural transmission and muscle contraction. Chloride is also essential for the regulation of acid-base balance of the body and intracellular osmotic pressure and acid-base buffering. Acute Toxicity The oral LD50 values were 2,045 (male) and 1,940 mg/kg (female) in mice, 3,798 (male) and 4,179 mg/kg (female) in rats, and 500-1,000 mg/kg in male rabbits. Acute oral toxicity of the substance is attributed to the severe irritating property of the original compound or its high-concentration solutions to the gastrointestinal tract, causing perforation and ulceration of the contact area of the tract. The dermal LD50 value in male/female rabbits was >5,000 mg/kg. Irritation Calcium chloride is non to slightly irritating to skin of rabbits; it is, however, severely irritating to eyes of rabbits. Irritating effect of calcium chloride has been observed in human skin injuries caused by the incidental contact with the substance or its highconcentration solutions. Sensitization There are no reliable data available for sensitization of calcium chloride. 1

143 Repeated Dose Toxicity Rats (40-day old) were fed 20 mg/g calcium chloride in the diet for 12 months. There were no treatment-related effects on mortality, weight gain, or daily food consumption. In addition, no neoplastic lesions were observed in gastrointestinal tract, urinary tract, liver, heart, brain or spleen of the animals. From the food consumption (22 g diet/day), the daily intake of calcium chloride was estimated to be 440 mg. Considering that 1 mg/g diet is equivalent to 100 and 50 mg/kg/day for young and old rats, respectively, the dose used in this study (20 mg calcium chloride/g diet) corresponds to 1,000 to 2,000 mg/kg/day. Calcium and chloride are both essential nutrients for humans as well as other animal species. As for healthy humans, the tolerable upper intake level for calcium is set at 2,500 mg per day (equivalent to 6.9 g CaCl2 per day) and the reference nutrient intake for chloride at 2,500 mg/day (equivalent to 3.9 g CaCl2 per day) (U.S Institute of Medicine Dietary Reference Intakes). The estimated intake of calcium chloride as food additives ( mg/day) is considerably smaller than these values. Consistent with this, the establishment of the ADI for calcium chloride has not been deemed necessary by JECFA. The substance has also been considered a GRAS substance by the U.S. FDA. It is thus very unlikely that calcium chloride taken orally as food additives adversely affects human health. Genotoxicity In vitro studies for genetic toxicity with sufficient reliability have been reported. Genetic toxicity of calcium chloride was negative in the bacterial mutation tests and the mammalian chromosome aberration test. Carcinogenicity No carcinogenicity studies on calcium chloride were located. Reproductive Toxicity No reproductive toxicity studies on calcium chloride were located. Developmental Toxicity A developmental toxicity study showed no toxic effects on dams or fetuses at doses up to 189 mg/kg/day (mice), 176 mg/kg/day (rats) and 169 mg/kg/day (rabbits). 2

144 Key Study/Critical Effect for Screening Criteria The drinking water guidelines for chloride and hardness (as calcium carbonate) may apply to calcium chloride. References OECD (2002). IUCLID Data Set for Calcium chloride (CASRN ), UNEP Publications. OECD (2002). Screening Information Dataset (SIDS) Initial Assessment Report for Calcium chloride (CASRN ), UNEP Publications. 3

145 Toxicity Profile Cocoamidopropyl betaine (CAS No ) Cocoamidopropyl betaine has been reviewed in the OECD-SIDS program (OECD, 2006). Acute Toxicity The acute toxicity of cocoamidopropyl betaine ( % aqueous solution) in rats is low. The oral LD50 is >4,900 mg/kg, and the dermal LD50 is >2,000 mg/kg. Irritation A 30% aqueous solution of cocoamidopropyl betaine was only very slightly irritating to the skin of rabbits. In human studies, up to 3% solutions were weakly irritating. A 5-10% solution of cocamidopropyl betaine produced mild to moderate irritation to the eyes of rabbits; solutions containing 15% were irritating to highly irritating; and a 30% aqueous solution was irritating with irreversible damage. Sensitization The sensitizing potential of cocoamidopropyl betaine in humans is low. Commercial cocoamidopropyl betaine may, however, contain impurities identified as sensitizers (amidoamine and/or 3-dimethylaminopropylamine) which may explain positive results in human patch tests. There is no evidence for a photosensitizing potential. Repeated Dose Toxicity Rats were given a 30% aqueous solution of cocoamidopropyl betaine by oral gavage at doses of 0, 250, 500 or 1,000 mg/kg/day (corresponding to about 0, 75, 150, and 300 mg active substance/kg/day, respectively) 5 days/week for 28 days (Henkel KgA, 1991). The only treatment-related findings were forestomach lesions at the highest dose level, probably as a result of the irritant effect of the test substance. The NOAEL for this study is 500 mg/kg/day (150 mg cocoamidopropyl betaine/kg/day). Rats were given a 30% aqueous solution of cocoamidopropyl betaine by oral gavage at doses of 0, 250, 500 or 1,000 mg/kg/day (corresponding to about 0, 75, 150, and 300 mg active substance/kg/day, respectively) five days/week for 90 days (Th. Goldschmidt AG, 1991). The only treatment-related findings were forestomach lesions at the 500 and 1,000 mg/kg dose levels, probably as a result of the irritant effect of the test substance. The NOAEL for this study is 250 mg/kg day (75 mg cocoamidopropyl betaine/kg/day). 1

146 Genotoxicity There is no evidence for a genotoxic potential of cocoamidopropyl betaine. A 30% aqueous solution of cocoamidopropyl betaine was not genotoxic in vitro in either a bacteria mutation test or in a mouse lymphoma test. A limited intraperitoneal mouse micronucleus test with 27 % active cocamidopropyl betaine was negative. Carcinogenicity No valid carcinogenicity studies have been conducted. Reproductive Toxicity No reproductive toxicity studies have been conducted on cocoamidopropyl betaine. However, there was no evidence of an adverse effect on the reproductive organs in rats given oral doses of up to 1,000 mg/kg/day of a 30% aqueous solution of cocoamidopropyl betaine (300 mg active substance/kg/day) for 90 days (Th. Goldschmidt AG, 1991). Developmental Toxicity A 28.9% aqueous solution of cocoamidopropyl betaine was tested in a rat developmental toxicity at doses of 330, 990, and 3,300 mg/kg/day (corresponding to 95, 286, and 950 mg/kg/day, respectively) (CESIO, 2004). There were dose-related maternal toxic effects (reduced body weights and stomach ulcers) at 990 mg/kg/day and above. Embryotoxic effects (increased numbers of resorptions, decreased number of viable fetuses, decreased fetal body weight) were found only at the maternal toxic dose level of 3,300 mg/kg/day. The NOAEL for maternal toxicity was 330 mg/kg/day (corresponding to 95 mg active substance/kg-day) and the NOAEL for developmental toxicity was 990 mg/kg/day (corresponding to 286 mg active substance/kg/day). Key Study/Critical Effect for Screening Criteria The key study is a 90-day rat oral gavage study in which forestomach lesions were seen at dose levels of 500 and 1,000 mg/kg of a 30% aqueous solution of cocoamidopropyl betaine. The NOAEL for this study is 250 mg/kg/day (75 mg active substance/kg/ay). NOAELadjusted = 75 * 5/7 = 53.6 mg/kg/day 2

147 Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 0.05 mg/kg/day Drinking water guideline: 0.19 ppm References CESIO (2004). Prenatal development toxicity study in rats with cocamidopropyl betaine by oral administration - according to OECD guideline DRAFT. Essen, LPT Study No /03, Henkel KGaA (1991). Dehyton K; 28-Tage-Test mit wiederholter oraler Verabreichung an Ratten. TED ; Juli 1991, Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Cocoamidopropyl betaine (CAPB) (CAS No.: , , ), 2005; OECD (2006). SIDS Initial Assessment Profile for Alkylamidopropyl betaines. Th. Goldschmidt AG (1991). Tego-Betain. 90 day oral (gavage) subchronic toxicity study in the rat. Essen, Th. Goldschmidt AG, , ,

148 Toxicity Profile Crystalline Silica, Cristobalite (CAS No ) Crystalline Silica, Quartz (CAS No ) Silica is an off-white granule that occurs naturally in various crystalline and amorphous or other non-crystalline forms. Crystalline silica is characterized by silicon dioxide (SiO2) molecules oriented in fixed, periodic patterns to form stable crystals. The primary crystalline form of silica is quartz. Other crystalline forms of silica include cristobalite, tripoli and tridymite. Particle size is a key determinate of silica toxicity, since toxicity is restricted to particles that are small enough to be deposited into the target regions of the respiratory tract. Oral Exposure No oral studies were located; however, crystalline silica is not expected to exhibit toxicity by the oral route. Although absorption studies were not found for crystalline silica, kinetic studies on amorphous silica show no absorption from the gastrointestinal tract. Dermal Exposure No dermal studies were located; however, crystalline silica is not expected to exhibit toxicity by the dermal route. Inhalation Exposure See attached OECD-SIDS Initial Targeted Assessment Profile on Quartz and Cristobalite, SIAM 32, April 2011.

149 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

150 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

151 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

152 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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153 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

154 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

155 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

156 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

157 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

158 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

159 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

160 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

161 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

162 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 4 of 10

163 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

164 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

165 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

166 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

167 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

168 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

169 Toxicity Profile Endo-1,4-beta-Mannanase (CAS No ) No data could be located on endo-1,4-beta-mannanase. There is information, however, on α-amylases. α-amylase catalyzes the endo-hydrolysis of 1,4-α-D-glycosidic linkages in polysaccharides like starches, glycogen, and oligosaccharides containing three or more 1,4-α-linked glucose units. Given the similarity of the substrates, amylases and endo-1,4- beta-mannanase would be expected to have similar physico-chemical properties relevant to evaluating their mammalian toxicity potential. The data on α-amylases have come from detergent amylases, the typical ones being Bacillus, which has an neutral isoelectric point and a broad ph optimum between ph 5 and 9. The enzyme is characterized by a molecular weight of 58 kd (HERA, 2005). Acute Toxicity No deaths were observed when male and female rats were given an aqueous suspension of 0, 4,000 or 10,000 mg/kg of α-amylase ( salt free batch PPY 1316, enzyme derived from B. subtilis) by oral gavage. The actual enzyme content of this batch was 239 mg aep/g. In another study, male and female rats were given an aqueous suspension of 0 or 5,000 mg/kg α-amylase preparation derived from B. licheniformis, the actual enzyme content of the preparation was mg aep/g. There were no deaths. Rats were exposed by inhalation to either 1.6 mg/l of a production α-amylase (from B. subtilis) batch ADTA , a mixture of two batches prepared by the standard production process (45.9% of particles <4.7 μm) or 1.08 mg/l of a salt-free α-amylase (from B. subtilis) batch PPY 1316, prepared from production batch ADTA by removal of NaCl (33.3% of particles <4.7 μm), for 4 hours. An air-exposed control group was also included. The actual amount of enzyme protein in the test aerosols was mg aep/l (production batch) and mg aep/l (salt-free batch). There were no deaths occurred. In another study, rats were exposed to 1.6 mg/l α-amylase (from B. subtilis) preparation (highest concentration attainable) derived from a genetically modified strain of B. subtilis for 4 hours. Total organic solids comprised 83.3% of the test substance (active and inactive enzyme as well as other organic material). There were no deaths (HERA, 2005). Irritation α-amylase (from B. subtilis) has a low potential for irritation to the skin and eyes of rabbits (HERA, 2005). In a human repeat insult patch test (HRIPT), although skin irritation did not appear after a single application, irritation was reported in human 1

170 volunteers receiving nine topical applications of 1, 2.5, 5 or 10% α-amylase (from B. subtilis) in distilled water. The magnitude of responses increased with increasing concentration such that the use of the 10% concentration was discontinued and was replaced for the rest of the study by a 0.5 % α-amylase. The irritation was thought to be due to residual protease activity present in the amylase preparation (HERA, 2005). Sensitization α-amylase (from B. subtilis) was not a skin sensitizer to guinea pigs in two different studies (HERA, 2005). In a human repeat insult patch test (HRIPT) reported above, there were no significant reactions indicative of skin sensitization in the challenge phase (HERA, 2005). Repeated Dose Toxicity Rats and dogs have been given amylase enzymes orally for up to 90 days. These studies were not reported in any detail and the actual amount of enzyme in the formulations tested in these studies is unclear. No findings of toxicological significance were observed in either species exposed to any of the α-amylase formulations tested other than slight reductions in food consumption at high dietary doses (>5% of the diet) or irritation of the stomach of rats dosed by oral gavage with >3,000 mg/kg/day (HERA, 2005). Genotoxicity α-amylase (from B. licheniformis and from B. subtilis) was not mutagenic to S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 in bacterial reverse mutation assays in the presence or absence of metabolic activation (HERA, 2005). α-amylase (from B. licheniformis) did not induce chromosome aberrations in human lymphocytes, which were obtained from one donor only (HERA, 2005). It was also inactive when tested in an in vivo bone marrow chromosomal aberrations study, in which rats were dosed orally up to 5,000 mg/kg (HERA, 2005). Carcinogenicity No data were found. Reproductive/Developmental Toxicity Two α-amylases, one derived from B. stearothermophilus and one derived from a genetically modified strain of B. subtilis have been evaluated for effects on fertility in one-generation studies in rats. The diets containing 0, 36 or 72 units of α-amylase/g 2

171 food. No treatment-related effects on fertility or other findings of toxicological significance were observed for either enzyme. Key Study/Critical Effect for Screening Criteria Oral studies (dietary and/or oral gavage) up to 90 days on α-amylase have been conducted in rats and dogs. These studies have been poorly reported. Slight reductions in food consumption was reported at >5% in the diet (2,500 mg/kg/day in rats) and irritation of the stomach of rats dosed with >3,000 mg/kg/day. These studies are considered unreliable because of the lack of reported key information needed for risk assessment purposes. There is, however, a 13-week rat dietary study conducted on a cellulase enzyme (HERA, 2005). The cellulase enzymes cleave the ß-1,4-glycosidic bonds in cellulose. For the purposes of risk assessment, the structure of amylases would be expected to be relatively similar to that of celluloses, given that amylases and cellulases are both enzyme families that hydrolyze polysaccharides, although differing in their substrates. Thus, the toxicity data on cellulases will be used as read-across to amylases for the repeat dose toxicity endpoint. In the 13-week dietary study on a cellulase enzyme, there was reduced body weight gain in rats given 3,000 mg/kg/day. No other adverse effects were observed. The NOAEL for this study is 600 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 600/1000 = 0.6 mg/kg/day Drinking water guidance value = 2.1 ppm Reference Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: α-amylase, CAS No ; Cellulase (β-(1,4)-glucanase), CAS No ; Lipase, ,

172 Toxicity Profile Ethanol (CAS No ) Ethanol has been reviewed in the OECD-SIDS program (OECD, 2004a,b). Acute Toxicity Ethanol has a low order of acute toxicity by all routes of exposure. The lowest wellconducted acute oral LD50 value is 8,300 mg/kg in mice, and an inhalation 1-hour LC50 value of >60,000 ppm (114,000 mg/m 3 ) in mice. Irritation Ethanol is a moderate eye irritant but it is not a skin irritant. Sensitization Ethanol is not a sensitizer. Repeated Dose Toxicity There are numerous repeat dose toxicity studies on ethanol by the oral route. Many of these studies were designed to increase our understanding of the risks associated with alcoholic beverages. Thus, these studies were carried out by the oral route and at high doses. The selected studies were considered appropriate for risk assessment purposes. In a 90-day study, male and female Sprague-Dawley rats were fed a diet containing 0, 1, 2, 3, 4, 5 or 10% ethanol. The 2% dose was calculated to be equivalent to 2,400 mg/kg/day. There were no deaths and no clinical signs of noted. Weight gain throughout the study was unaffected by treatment, but the final weights decreased with dose. Food and water consumption in the 10% group was reduced relative to controls. There was no treatment-related effect on serum liver enzymes and kidney clinical chemistry findings were minimal. In the liver, centrilobular steatosis increased in severity with dose as did the frequency and severity of Mallory bodies (hyaline) and acidophilic degeneration and necrosis. Most liver findings were absent or mild at 2% ethanol but became more significant at >3%. The NOAEL for this study has been established to at 2% (2,400 mg/kg/day). In an NTP 90-day study, F344/N rats and B6C3F1 mice were given in their drinking water 0 or 5% ethanol in drinking water. Based on the water consumption data in the 1

173 study and averaged body weights over the exposure period, this equated to doses of at least 4,000 mg/kg in rats and mg/kg in mice. Male rats showed minor changes to organ weights and hematology and clinical chemistry parameters (these were not considered to be adverse changes); female rats showed minor clinical chemistry changes and increased length of estrus cycle, as well as liver nodules. The male mice showed increased organ weights, fatty changes to the liver, and a decrease in sperm concentration. In the NTP two-year bioassay, B6C3F1 mice were given in their drinking 0, 2.5% and 5% ethanol Concentrations of 2.5% and 5% ethanol resulted in average daily consumption of approximately 100 and 180 mg ethanol for males and 80 and 155 mg for females. There was a marginal exposure-related increase in survival of the males, but no effect on the survival of the females. Water consumption was reduced with increasing concentrations of ethanol which was more marked in males than females. No further information was given on non-neoplastic effects. Genotoxicity The balance of evidence is that ethanol is not genotoxic. Negative results from a number of bacterial mutation assays appear to be reliable. Of the mammalian cell mutation assays a weak mutagenic effect in mouse lymphoma cells occurred only at very high ethanol concentrations. In vivo tests for chromosome aberrations in both rats and Chinese hamsters have given negative results. There is very little evidence to suggest that ethanol is genotoxic in somatic cells and it may have a very limited capacity to induce genetic changes in vivo but under very specific circumstances and at very high doses achievable in humans only by deliberate oral ingestion. Carcinogenicity Evidence of the carcinogenicity of ethanol is confined to epidemiological studies assessing the impact of alcoholic beverage consumption. These do not indicate any such hazard exists from potential exposure to ethanol in the work place or from the use of ethanol in consumer products. Reproductive/Developmental Toxicity The lowest reported NOAEL for fertility by the oral route was 2,000 mg/kg in rats, equivalent to a blood alcohol concentration of 1,320 mg/l, although this was based on a significant increase in the number of small pups rather than a direct effect on fertility; such direct effects are not seen until much higher doses. Many studies exist examining the developmental end point for ethanol. However, most use very high doses and few are individually robust enough to allow a NOAEL to be established. However, the collective weight of evidence is that the NOAEL for developmental effects in animals is high, 2

174 typically >6,400mg/kg, compared to maternally toxic effects at 3,600 mg/kg. The potential for reproductive and developmental toxicity exists in humans from deliberate over-consumption of ethanol. Blood ethanol concentrations resulting from ethanol exposure by any other route are unlikely to produce reproductive or developmental effects. Key Study/Critical Effect for Screening Criteria The lowest reported NOAEL is approximately 2,400 mg/kg/day from a 90-day dietary study with rats. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 2,400/1,000 = 2.4 mg/kg/day Drinking water guidance value = 8.4 ppm References OECD (2004a). IUCLID Data Set for Ethanol (CAS No ), UNEP Publications. OECD (2004b). Screening Information Dataset (SIDS) Initial Assessment Report for Ethanol (CAS No ), UNEP Publications. 3

175 Toxicity Profile Ethoxylated Fatty Acid Ester II This substance is a hydrophilic, non-ionic surfactant used in pharmaceutical preparations, and cosmetics as an emulsifier, dispersant, or stabilizer, and as a food additive. It is considered a mixture and thus a UVCB substance (substance of unknown or variable composition, complex reaction products or biological materials). Acute Toxicity Reported oral LD50 values for rats range from approximately 33,800 mg/kg to approximately 54,500 mg/kg, while the oral LD50 for mice is greater than 25,000 mg/kg. Clinical experience with humans who have ingested large quantities of this substance indicates that it is essentially nontoxic by the oral route, with the estimated lethal dose being 15,000 mg/kg. Irritation In 24-hour rabbit skin-patch tests, this substance was found to produce minimal or no irritation. Daily application of 100% solutions of this substance to the backs of rabbits for 30 days produced histologic changes in the skin, including mild to moderate inflammation, acanthosis, and necrosis. Application of 1% or 2.5% aqueous solutions of this substance to the backs of mice for up to 9 days produced no gross or microscopic lesions. Studies conducted in humans did not result in skin irritation. Sensitization Repeated intradermal injections of this substance into guinea pigs did not produce skin sensitization, and studies conducted in humans did not result in sensitization. Repeated Dose Toxicity In a multi-generation study, doses of 5%, l0%, and 20% of test material were administered in the feed to male and female rats. Composition of the diet was altered for different dose levels to compensate for the decrease in caloric value caused by addition of the test material. Diarrhea was seen in animals fed 10% or 20% test substance, and was presumably due to the osmotic effect of the large amounts of polyoxyethylene moiety in the intestinal tract. There was some reduction in growth rate, postnatal survival of pups, lactation and breeding efficiency, and longevity in rats fed the diet containing 20% test material. Hematologic examination showed no abnormalities and no treatment-related histologic lesions were seen upon examination of kidneys, livers, and gross lesions. No 1

176 significant findings were reported in a three-generation study using groups of 30 rats fed 2% test substance. Degeneration of the heart, liver, and kidney were reported in groups of 12 Charles Foster rats administered a daily dose of 1.5 ml of a solution of 1%, 2%, or 3% test substance for 3 months. Similar effects were not reported in other studies using higher doses for longer periods, so the significance of the findings of this single study is debatable. Daily administration of 1g of this substance to each of two monkeys for 17 months produced no compound-related gross or microscopic lesions. Evidence indicates that the long-term ingestion of this substance by humans does not result in toxicity. Long-term consumption (one or more years) of 4.5 to 6 g of this substance per day by human patients for the treatment of lipid malabsorption syndromes has been reported to cause no adverse effects. The consumption of doses as high as 15 g/day for several months caused no clinical indications of toxicity. Groups of 10 F344/N rats and 10 B6C3F1 mice of each sex received diets containing 0, 3,100, 6,200, 12,500, 25,000 or 50,000 ppm test material. All animals survived to the end of the studies. The final mean body weights of dosed rats and mice were similar to those of the controls. No clinical findings, changes in absolute or relative organ weights, or gross or microscopic lesions in rats or mice were related to test material administration. The NOAEL is 50,000 ppm. Genotoxicity Studies This substance is not genotoxic is a variety of in vitro and in vivo assays. Carcinogenicity studies In one study, 28 mice were fed diets containing 10 mg of this substance daily for 51 week, no neoplasms occurred as the result of treatment. Groups of 60 F344/N rats and 60 B6C3F1 mice of each sex received diets containing 0, 25,000, or 50,000 ppm test material for up to 103 weeks. Interim evaluations were performed on 7 to 10 rats and mice from each dose group at 15 months. There were no significant changes in absolute or relative organ weights. Incidences of hyperplasia and inflammation of the forestomach were increased in female mice that received 50,000 ppm. No other chemical-related lesions occurred in rats or male mice evaluated at 15 months. The mean body weights in male and female rats and male mice administered test material were similar to those of the controls throughout the studies. The final mean body weight of female mice receiving 50,000 ppm was 11% lower than that of the controls. No clinical findings were associated with administration of test material. The survival of dosed male rats was lower than that of the controls (0 ppm, 29/50; 25,000 ppm, 18/50; 50,000 ppm, 18/50); the survival of dosed female rats and male and female mice was 2

177 similar to that of the respective controls (female rats: 23/50, 25/50, 25/50; male mice: 33/49, 34/50, 32/50; female mice: 30/50, 28/50, 26/50). The incidence of adrenal medulla pheochromocytoma was marginally increased in highdose male rats (21/50, 19/50, 29/50). The slight increase in incidence of pheochromocytoma in the high-dose male rat group was judged to be of questionable significance based on historical control data. The incidence of hyperplasia of the adrenal medulla was increased in low-dose male rats but not in high-dose male rats (11/50, 22/50, 12/50). The marginal increased incidence of pheochromocytoma in combination with the increased incidence of hyperplasia were considered to be an equivocal finding. No chemical-related increases in the incidences of neoplasms occurred in male or female mice. The incidences of squamous hyperplasia and inflammation of the forestomach were significantly increased in high-dose male and female mice; forestomach ulcers were significantly increased in high-dose females. Developmental Toxicity Timed-mated Sprague-Dawley rats were exposed to 0, 500 or 5,000 mg/kg/day of test material on gestational days (GD) 6 through 15. All treated females survived to scheduled necropsy and pregnancies per group were confirmed. No dose-related signs of toxicity were observed for individual animals during the in-life phase of the study or at scheduled necropsy. Average maternal body weight (GD 0, 3, 6, 9, 12, 15, 18, or 20) did not differ among treatment groups, nor was there a treatment-related change in maternal weight gain during treatment or gestation (absolute or corrected). There were no treatment-related effects upon the following maternal organ weights: gravid weight (absolute), kidney weight (absolute or relative), and heart weight (absolute or relative). Relative maternal liver weight (% body weight on GD 20 or % corrected body weight) was elevated in both treated groups and absolute liver weight was elevated at 500 mg/kg/day. Maternal food intake was comparable across groups during the pre- and posttreatment periods, but was decreased by 14% during the first 3 days of treatment at 5,000 mg/kg/day relative to the vehicle control group. Maternal relative water intake was comparable among treatment groups throughout gestation. No differences among groups were noted for the number of corpora lutea per dam, the number of implantation sites per dam or the percent preimplantation loss per litter. No adverse effects were noted on the growth, viability or morphological development of the conceptuses. In conclusion, the maternal LOAEL was 500 mg/kg/day (based upon an increase in maternal relative liver weight). No definitive adverse effects of test material upon prenatal development were noted in this study. Thus, the developmental NOAEL was greater than 5,000 mg/kg/day. Key Study/Critical Effect for Screening Criteria Compound-related lesions of the forestomach occurred in all dosed mouse groups in a two-year feeding study. These included squamous hyperplasia of the epithelium, 3

178 inflammation, and, in high-dose females, ulcers. It was suggested that this substance,as a surfactant, produced forestomach injury by localized irritation to the epithelial cells. LOAEL = 25,000 ppm in mice (forestomach lesions) LOAEL (mg/kg/day) = 25,000 ppm * 0.13 = 3,250 mg/kg/day Where 0.13 is the fraction of body weight that is consumed per day as food for the mouse (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (LOAEL to NOAEL) Oral RfD = 3,250/1,000 = 3.25 mg/kg/day Drinking water guidance value = 11.4 ppm 4

179 Toxicity Profile Ethoxylated Sorbitan Monooelate Polysorbate 80 (CAS No ) Ethoxylated sorbitan monooleate or polysorbate 80 is a hydrophilic, non-ionic surfactant used in pharmaceutical preparations, and cosmetics as an emulsifier, dispersant, or stabilizer, and as a food additive. It is a mixture of oleic esters of sorbitan and its anhydrides copolymerized with approximately 20 mol of ethylene oxide (Sweetman, 2002). Thus, polysorbate 80 is considered a UVCB substance (substance of unknown or variable composition, complex reaction products or biological materials). The information below and the references can be found in NTP (1992a). Metabolism The metabolism of radiolabeled polysorbates in rats has been studied. After oral administration, the ester linkage of the polysorbate molecule is hydrolyzed by pancreatic lipase and the released fatty acid is absorbed and metabolized; essentially all of the fatty acid in polysorbate 80 fed as 10% of the diet is hydrolyzed and absorbed. The sorbitolpolyoxyethylene moiety is poorly absorbed and most is excreted in the feces. In metabolism studies conducted with rats fed polysorbate 80 containing radiolabeled sorbitol, 91% of the radioactivity was found in the feces, 2.1% was found in the urine, and 1.6% was found in the carcass. None was found in the liver, kidney, adrenal glands, spleen, brain, or fat. Following intravenous administration of polysorbates to rats, the ester linkage of the polysorbates is hydrolyzed by blood lipases. Intravenous studies using radiolabeled polysorbate 20 in rats demonstrated that the fatty acid portion was metabolized and excreted mostly as expired carbon dioxide, with lesser amounts appearing in the urine and feces; the sorbitol-polyoxyethylene moiety was not catabolized and was excreted mainly in the urine, although a small amount was found in the feces, indicating some biliary excretion; trace amounts were detected in the carcass and liver. Metabolism of polysorbates in humans appears to be similar to that in rats. Analysis of urine and feces from human volunteers who consumed 4.5 g of polysorbate 80 daily showed that 95% of the polyoxyethylene portion was excreted in the feces and 5% in urine; apparently, none was retained in the body. Acute Toxicity Reported oral LD50 values for rats range from approximately 33,800 mg/kg to approximately 54,500 mg/kg, while the oral LD50 for mice is greater than 25,000 mg/kg. Clinical experience with humans who have ingested large quantities of polysorbate 80 indicates that the compound is essentially nontoxic by the oral route, with the estimated lethal dose being 15,000 mg/kg. 1

180 Irritation In 24-hour rabbit skin-patch tests, polysorbate 80 was found to produce minimal or no irritation. Daily application of 100% solutions of polysorbate 80 to the backs of rabbits for 30 days produced histologic changes in the skin, including mild to moderate inflammation, acanthosis, and necrosis. Application of 1% or 2.5% aqueous solutions of polysorbate 80 to the backs of mice for up to 9 days produced no gross or microscopic lesions. Studies conducted in humans did not result in skin irritation. Sensitization Repeated intradermal injections of polysorbate 80 into guinea pigs did not produce skin sensitization, and studies conducted in humans did not result in sensitization. Repeated Dose Toxicity In a multi-generation study, doses of 5%, l0%, and 20% polysorbate 80 were administered in the feed to male and female rats. Composition of the diet was altered for different dose levels to compensate for the decrease in caloric value caused by addition of polysorbate 80. Diarrhea was seen in animals fed 10% or 20% polysorbate 80, and was presumably due to the osmotic effect of the large amounts of unabsorbed sorbitolpolyoxyethylene moiety in the intestinal tract. There was some reduction in growth rate, postnatal survival of pups, lactation and breeding efficiency, and longevity in rats fed the diet containing 20% polysorbate 80. Hematologic examination showed no abnormalities and no treatment-related histologic lesions were seen upon examination of kidneys, livers, and gross lesions. No significant findings were reported in a three-generation study using groups of 30 rats fed 2% polysorbate 80. Degeneration of the heart, liver, and kidney were reported in groups of 12 Charles Foster rats administered a daily dose of 1.5 ml of a solution of 1%, 2%, or 3% polysorbate 80 for 3 months. Similar effects were not reported in other studies using higher doses for longer periods, so the significance of the findings of this single study is debatable. Daily administration of 1g polysorbate 80 to each of two monkeys for 17 months produced no compound-related gross or microscopic lesions. Evidence indicates that the long-term ingestion of polysorbate 80 by humans does not result in toxicity. Long-term consumption (one or more years) of 4.5 to 6 g polysorbate 80 per day by human patients for the treatment of lipid malabsorption syndromes has been reported to cause no adverse effects. The consumption of doses as high as 15 g/day for several months caused no clinical indications of toxicity. Groups of 10 F344/N rats and 10 B6C3F1 mice of each sex received diets containing 0, 3,100, 6,200, 12,500, 25,000 or 50,000 ppm polysorbate 80. All animals survived to the 2

181 end of the studies. The final mean body weights of dosed rats and mice were similar to those of the controls. No clinical findings, changes in absolute or relative organ weights, or gross or microscopic lesions in rats or mice were related to polysorbate 80 administration. The NOAEL is 50,000 ppm (NTP, 1992a). Genotoxicity Studies Polysorbate 80 is not genotoxic is a variety of in vitro and in vivo assays. Carcinogenicity studies In one study, 28 mice were fed diets containing 10 mg polysorbate 80 daily for 51 week, no neoplasms occurred as the result of treatment. Groups of 60 F344/N rats and 60 B6C3F mice of each sex received diets containing 0, 25,000, or 50,000 ppm polysorbate 80 for up to 103 weeks. Interim evaluations were performed on 7 to 10 rats and mice from each dose group at 15 months. There were no significant changes in absolute or relative organ weights. Incidences of hyperplasia and inflammation of the forestomach were increased in female mice that received 50,000 ppm. No other chemical-related lesions occurred in rats or male mice evaluated at 15 months. The mean body weights in male and female rats and male mice administered polysorbate 80 were similar to those of the controls throughout the studies. The final mean body weight of female mice receiving 50,000 ppm was 11% lower than that of the controls. No clinical findings were associated with administration of polysorbate 80. The survival of dosed male rats was lower than that of the controls (0 ppm, 29/50; 25,000 ppm, 18/50; 50,000 ppm, 18/50); the survival of dosed female rats and male and female mice was similar to that of the respective controls (female rats: 23/50, 25/50, 25/50; male mice: 33/49, 34/50, 32/50; female mice: 30/50, 28/50, 26/50). The incidence of adrenal medulla pheochromocytoma was marginally increased in highdose male rats (21/50, 19/50, 29/50). The slight increase in incidence of pheochromocytoma in the high-dose male rat group was judged to be of questionable significance based on historical control data. The incidence of hyperplasia of the adrenal medulla was increased in low-dose male rats but not in high-dose male rats (11/50, 22/50, 12/50). The marginal increased incidence of pheochromocytoma in combination with the increased incidence of hyperplasia were considered to be an equivocal finding. No chemical-related increases in the incidences of neoplasms occurred in male or female mice. The incidences of squamous hyperplasia and inflammation of the forestomach were significantly increased in high-dose male and female mice; forestomach ulcers were significantly increased in high-dose females (NTP, 1992a). 3

182 Developmental Toxicity Timed-mated Sprague-Dawley-derived (CD rats were exposed to 0, 500 or 5,000 mg/kg/day of polysorbate 80 on gestational days (GD) 6 through 15. All treated females survived to scheduled necropsy and pregnancies per group were confirmed. No dose-related signs of toxicity were observed for individual animals during the in-life phase of the study or at scheduled necropsy. Average maternal body weight (GD 0, 3, 6, 9, 12, 15, 18, or 20) did not differ among treatment groups, nor was there a treatment related change in maternal weight gain during treatment or gestation (absolute or corrected). There were no treatment-related effects upon the following maternal organ weights: gravid weight (absolute), kidney weight (absolute or relative), and heart weight (absolute or relative). Relative maternal liver weight (% body weight on gd 20 or % corrected body weight) was elevated in both treated groups and absolute liver weight was elevated at 500 mg/kg/day. Maternal food intake was comparable across groups during the pre- and post-treatment periods, but was decreased by 14% during the first 3 days of treatment at 5000 mg/kg/day relative to the vehicle control group. Maternal relative water intake was comparable among treatment groups throughout gestation. No differences among groups were noted for the number of corpora lutea per dam, the number of implantation sites per dam or the percent preimplantation loss per litter. No adverse effects were noted on the growth, viability or morphological development of the conceptuses. In conclusion, the maternal LOAEL was 500 mg/kg/day (based upon an increase in maternal relative liver weight). No definitive adverse effects of polysorbate 80 upon prenatal development were noted in this study. Thus, the developmental NOAEL was greater than 5,000 mg/kg/day (NTP 1992b). Key Study/Critical Effect for Screening Criteria Compound-related lesions of the forestomach occurred in all dosed mouse groups in a two-year feeding study. These included squamous hyperplasia of the epithelium, inflammation, and, in high-dose females, ulcers. It was suggested that polysorbate 80 as a surfactant produced forestomach injury by localized irritation to the epithelial cells. LOAEL = 25,000 ppm in mice (forestomach lesions) LOAEL (mg/kg/day) = 25,000 ppm * 0.13 = 3,250 mg/kg/day Where 0.13 is the fraction of body weight that is consumed per day as food for the mouse (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (LOAEL to NOAEL) Oral RfD = 3,250/1,000 = 3.25 mg/kg/day 4

183 Drinking water guidance value = 11.4 ppm References NTP (1992a). Toxicology and Carcinogenesis Studies of Polysorbate 80 (CAS No ) in F344/N rats and B6C3F1 Mice (Feed Studies), NTP TR 415, NIH Publication No , National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. NTP (1992b). Developmental Toxicology of Polyoxyethylene Sorbitan Monooleate (CAS # ) in Sprague-Dawley CD Rats, NTP Study: TER91009, National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. Sweetman, S.C. (2002). Martindale: the Complete Drug Reference, London, Pharmaceutical Press; cited in Carlsson et al. (2006). 5

184 Toxicity Profile Fatty Acid Ester This fatty acid ester is a non-ionic surfactant-active agent that typically finds use as an emulsifier, stabilizer and thickener in foods, cosmetics, medical products, and lubricants, and other applications. It is approved by FDA as an indirect food additive. Acute Toxicity The acute oral LD50 of this substance in rats was reported to be > 39,800 mg/kg. Irritation No data were located. Sensitization No data were located. Repeated Dose Toxicity/Carcinogenicity Oral feeding toxicity studies have been carried out in rats for 16 weeks with this substance at dietary concentrations of 0, 2.5, 5 and 10%. The LOAEL was 2.5% dietary concentration (~1800 mg/kg/day) based on increased kidney weight findings that were considered significant in both male and female rats. This substance fed to rats at 5% concentrations in the diet for 2 years showed no adverse effects on growth, hematology, clinical chemistry, survival, organ size or histopathology. The NOAEL was reported to be 5% in the diet. Genotoxicity No genotoxicity studies could be located for this substance; however, there are two studies conducted on a structurally similar substance. The surrogate was not mutagenic to Salmonella typhimurium TA98 and TA100 in a Ames mutation assay in the presence or absence of metabolic activation. It did not cause any chromosomal aberrations in the Syrian golden hamster embryo cell assay and did not show clastogenic activity. 1

185 Reproductive/Developmental Toxicity No studies could be located for this substance; however, there is one study conducted in a structurally similar substance. The surrogate was administered in the diet at dose levels of 0, 5, 10, and 20% over a period of two years and over four generations of rats. No effects were observed on growth, food efficiency, reproduction, lactation, metabolism, behavior, mortality, or in the gross and histopathological examination. At the 20% dose level in the diet, newborn mortality was not increased but there were slight effects on growth and impairment of lactation. Key Study/Critical Effect for Screening Criteria No treatment-related effects were noted in a two-year carcinogenicity study in which rat were fed this fatty acid ester in the diet at concentrations up to 5% (50,000 ppm). NOAEL (mg/kg/day) = 50,000 ppm * 0.05 = 2,500 mg/kg/day Where 0.05 is the fraction of body weight that is consumed per day as food for the rat (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 2,500/100 = 25 mg/kg/day Drinking water guidance value = 87.5 ppm 2

186 Toxicity Profile Guar Gum (CAS No ) Guar gum (CAS No ) is the milled endosperm of the leguminous plant Cyanopsis tetragonolobus. Structurally, it is a galactomannan consisting of a main chain of D-mannose with a side chain of D-galactose at approximately every second mannose unit. The mannose units are β-(l-4) linked, and the single D-galactose units are joined to the main chain by α-(1-6) linkages. The estimated molecular weight of guar gum cranges from 200,000 to 300,000 daltons (Glickman, 1969). Guar gum is approved for use as a food additive by the U.S. Food and Drug Administration and is on the list of substances "generally recognized as safe" (CFR 1974). Acute Toxicity The oral LD50 is 8,100 mg/kg for mice and 9,400 mg/kg for rats (Bailey and Morgareidge, 1948). Irritation No data were found. Sensitization There are reports of respiratory sensitization in workers exposed occupationally to guar gum dusts (Maio, 1986). Repeat Dose Toxicity Studies F344 rats and B6C3F1 mice were given diets containing 0, 6,300, 12,500, 25,000, 50,000 or 100,000 ppm guar gum for 13 weeks (NTP, 1982). Mean body weights were decreased in male rats (100,000 ppm group) and in female mice (50,000 and 100,000 ppm). A dose-related decrease in feed consumption was observed for male and female rats; male and female mice were comparable or higher than that of controls. There were no compound-related clinical signs or histopathological effects. F344 rats and B6C3F1 mice were given diets containing 0, 25,000 ppm or 50,000 ppm guar gum for 103 weeks (NTP, 1982). Mean body weights of the high-dose females were 1

187 lower than those of the controls after week 20 for mice and week 40 for rats. No compound-related clinical signs or adverse effects on survival were observed. Feed consumption by dosed rats and mice of either sex was lower than that of controls. There were no non-neoplastic histopathological effects in either rats or mice that were treatment-related. Genotoxicity Guar gum was not mutagenic to Salmonella typhimurium TA 1530 or G-46 when tested without metabolic activation; however, it was mutagenic to Saccharomyces cerevisiae D- 3 (Green, 1977). Guar gum also was reported to cause chromosomal aberrations in human embryonic lung cells WI-38 (Green, 1977). No in vivo genotoxicity studies have been conducted on guar gum. Carcinogenicity F344 rats and B6C3F1 mice were given diets containing 0, 25,000 ppm or 50,000 ppm guar gum for 103 weeks (NTP, 1982). There were increased incidences of adenomas of the pituitary in male rats and pheochromocytomas of the adrenal in female rats that were statistically significant, but these differences were considered to be unrelated to guar gum administration. When pituitary adenomas or carcinomas and when pheochromocytomas or malignant pheochromocytomas are combined, the statistical differences disappear. Hepatocellular carcinomas occurred in treated male mice at incidences that were significantly lower than that in controls. The combined incidence of male mice with either hepatocellular adenomas or carcinomas was also significantly lower in the highdose group. It was concluded that under conditions of this bioassay, guar gum was not carcinogenic for F344 rats or B6C3F1 mice. Reproductive Toxicity No data were found. Developmental Toxicity Female rabbits were given daily (6 hours/day) dermal administration of 0, 2, 10 and 50 mg/kg guar gum during gestational days 6 through 18 (IRDC, 1988). Mortalities included 2 deaths at 50 mg/kg and 1 death at 10 mg/kg. A single animal was killed in extremis. A dose-related increase in dermal irritation (including erythema, edema, and desquamation) was observed in animals receiving 10 and 50 mg/kg. The number of early resorptions was significantly increased and the number of viable fetuses was correspondingly decreased at 50 mg/kg/day (p<0.05). The frequency of fetal malformations and variations in the treated groups was comparable to that of the control 2

188 group at all dose levels. The NOEL for this study is 2 mg/kg/day. Key Study/Critical Effect for Screening Criteria The key studies for the determination of a drinking water guidance value is the NTP twoyear chronic bioassays. The LOAELs are based on decreased mean body weights in female mice and rats fed 50,000 ppm guar gum in diet for 103 weeks. The NOAELs for these studies are 25,000 ppm guar gum. Rat: NOAEL (mg/kg/day) = 25,000 ppm * 0.05 = 1,250 mg/kg/day Mouse: NOAEL (mg/kg/day) = 25,000 ppm * 0.13 = 3,250 mg/kg/day Where 0.05 and 0.13 are the fraction of body weight that rats and mice, respectively, consume per day as food (U.S. EPA). The lowest NOAEL of 1,250 mg/kg/day for the rat will be used to derive a drinking water guidance value. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 1,250/100 = 12.5 mg/kg/day Drinking water guideline = 44 ppm References Bailey, D., and Morgareidge, K. (1976). Comparative acute oral toxicity of 12 food grade gums in the mouse, rat, hamster, and rabbit. Food and Drug Research Labs Papers No., 124; cited in NTP (1982). Glicksman M. (1969). Gum technology in the Food Industry, pp. 590, Academic Press, New York; cited in Yoon, S.-J., Chu, D.-C., and Juneja, L.R. (2008) Chemical and physical properties, safety and application of partially hydrolyzed guar gum as dietary fiber. J. Clin. Biochem. Nutr. 42: 1-7. Green, S. (1977). Present and future uses of mutagenicity tests for assessment of the safety of food additives. J. Environ. Pathol. Toxicol. 1: International Research and Development Corp (1988). Teratology Study of Guar Gum in Rabbits. TSCATS database, EPA Doc. No , Fiche No. OTS ; cited in NZ HSNO CCID. 3

189 Maio, J.L., Cartier, A., L Archevêque, J., Ghezzo, H., Soucy, F., Somers, J., and Dolovich, J. (1990). Prevalence of occupational asthma and immunologic sensitization to guar gum among employees at a carpet-manufacturing plant. J. Allergy Clin. Immunol. 86: NTP (1982). NTP Technical Report on the Carcinogenesis Bioassay of Guar Gum (CAS No ) in F344 Rats and B6C3F1 Mice (Feed Study), National Toxicology Program, Research Triangle Park, NC 4

190 Toxicity Profile Hydrocarbons, Terpene Processing By-products (CAS No ) Terpenes and Terpenoids, Sweet Orange Oil (CAS No ) CAS No is a by-product of the manufacturing process in which high-quality gum turpentine oil or alpha pinene is synthesized into terpineol. It is largely composed of alpha terpinene, dl-limonene, cineol, gamma terpinene, terpinolene and other terpene hydrocarbons. The composition of CAS No in the HPV Test Plan for Monoterpene Hydrocarbon Category is: 22-34% limonene, 22-33% terpinolene, 5-10% mycene, 18% limonene isomers, 10% other terpene hydrocarbons. CAS No is composed of 91-95% d-limonene, 1-3% beta-myrcene, and 1-2% alpha-pinene. Acute Toxicity The oral and dermal LD50 values for limonene, myrcene, terpinolene, dihydromyrcene and sweet orange peel oil indicate a low order of both oral and dermal toxicity. All rabbit and rat dermal, and mouse and rat oral LD50 values are >4,000 mg/kg with the majority of values being >5,000 mg/kg. Irritation No data were located. Sensitization No data were located. Repeated Dose Toxicity/Carcinogenicity Selected repeat dose toxicity studies were included in this toxicity profile. For summaries on all conducted studies, see the U.S. HPVIS. Groups of ten Sprague Dawley rats of each sex were administered 0, 240, 600 or 1,500 mg/kg sweet orange oil in 1% methyl cellulose by gavage daily for 30 days. Observations included survival, clinical observations, body weights, food consumption, clinical 1

191 pathology, gross pathology, organ weights and histopathology. No treatment related effects were reported for survival, clinical observations, body weights or food consumption. Decreases in glucose levels related to treatment were reported in the middose females and high-dose males and females. Increases in serum albumin and total serum protein were observed in all treated females and the high-dose males. Histopathology revealed treatment related lesions in the nonglandular stomach of the high-dose males and females and in the kidney of all treated male groups. Kidney weights were also increased in all of the treated male groups and in the high-dose female group. Liver weight increases related to treatment were reported for the high-dose females and all treated male groups. The authors concluded that the no-observed-effectlevel (NOEL) under conditions of this study was less than 240 mg/kg/day for both male and female rats. The authors noted that the kidney changes observed in the male rat at all dose levels were expected given the known interaction between limonene and alpha-2- microglobulin. Limonene is the principal constituent of orange oil. In a NTP 13-week study, male and female F344/N rats were dosed by oral gavage 0, 150, 300, 600, 1,200 or 2,400 mg/kg d-limonene in corn oil for 5 days/week. At 2,400 mg/kg, 9/10 female rats and 5/10 male rats (5/10) died within the first week of the study. The final mean body weights of male rats receiving the three highest doses (600, 1,200 or 2,400 mg/kg) were reported to be 6%, 12%, or 23% lower than that of the controls, respectively. Rough hair coats, lethargy, and excessive lacrimation were observed for all animals in the 1,200 and 2,400 mg/kg dose groups. Kidney effects were seen in all treated male rats, with a dose-related increase in severity. The kidney effects were characterized by degeneration of epithelium in the convoluted tubules, granular casts with tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets were observed in the epithelium of the proximal convoluted tubules in all groups of male rats including vehicle controls. In a NTP 13-week study, male and female B6C3F1 mice were dosed by oral gavage with 0, 125, 250, 500, 1,000 or 2,000 mg/kg d-limonene for five days/week. At 2,000 mg/kg, 1/10 males and 2/10 females died before the end of the study; 1/10 females in the 500 mg/kg group also died before study termination. Several other animals also died as a result of gavage error. Mean body weights were 10% lower than control for male mice and 2% lower than control for female mice for the 1,000 and 2,000 mg/kg groups. Clinical signs of rough hair coats and decreased activity were reported for the two highest dose levels. Male rats were dosed by oral gavage with 0, 2, 5, 10, 30 or 75 mg/kg d-limonene 5 days/week for 13 weeks. Linear regression analyses indicated increased relative kidney and liver weights at 30 and 75 mg/kg. Histological examination showed changes in the kidney characterized by hyaline droplet formation, granular casts and multiple cortical changes. Exacerbation of hyaline droplet formation was reported at the earliest necropsy eight days after administration at the 10 mg/kg dose level. In a two-year NTP bioassay, male and female F344/N rats were dosed with 0, 75 or 150 mg/kg or 0, 300 or 600 mg/kg d-limonene for five days/week. Mean body weights for 2

192 the 150 mg/kg male rats were generally 4-7% lower than vehicle controls from week 2 to study termination. Mean body weights of the 600 mg/kg females were generally 4-7% lower than vehicle controls from week 28 to study termination. No treatment-related clinical signs were reported for the duration of the study. Survival of the 150 mg/kg male group was significantly greater than that of the vehicle alone after week 81. Survival of the 600 mg/kg female group was significantly lower than that of the vehicle controls after week 39. In the male rat kidneys, a dose-related increase was observed in the incidence of mineralization and epithelial hyperplasia. A dose-related increase in the severity of spontaneous nephropathy was reported in the treated male rats. Increased incidences in tubular cell hyperplasia and neoplasia were also reported in dosed male rats. Tubular cell adenoma incidence in 150 mg/kg male rats and tubular cell adenoma or tubular cell carcinomas (combined) in dosed male rats were significantly greater than vehicle controls. There was no evidence of carcinogenic activity of d-limonene for female rats receiving 300 or 600 mg/kg. The NOAEL for male rats (excluding the kidney effects) is 150 mg/kg/day (see below on the relevance of the male rat kidney effects for human risk assessment). The NOAEL for female rats is 300 mg/kg/day based on low survival in the 600 mg/kg dose group. In a NTP two-year bioassay, male and female B6C3F1 mice were dosed by oral gavage with 0, 250, or 500 mg/kg or 0, 500 or 1,000 mg/kg d-limonene, respectively, five days a week. Mean body weights for the 1,000 mg/kg female mice administered 1,000 mg/kg were generally 5-15% lower than vehicle controls from week 28 to study termination. No treatment related clinical signs were reported for the duration of the study. Survival of the 250 mg/kg male group was significantly lower than that of the vehicle controls by study termination. In the 500 mg/kg males, livers exhibited presence of cells with abnormal numbers of nuclei and cytomegaly. There was no evidence of carcinogenic activity of d-limonene for male or female B6C3F1 mice at the dose levels tested. The NOAEL for male mice is 250 mg/kg/day based on liver effects at 500 mg/kg/day. Although there was reduced survival in the 250 mg/kg male mice, NTP did not considered this to be dose-related. The NOAEL for female mice is considered to be 500 mg/kg/day based on decreased survival and lower body weights (5-15%) in the 1,000 mg/kg dose group. The kidney changes seen in male rats administered limonene have been well characterized, and are known to be specific to the male rat and of no significance in human risk assessment. Genotoxicity Given the structural similarity between the members of this chemical category, the substances in this category exhibit low genotoxic potential in vitro. Mutagenicity/genotoxicity testing has been performed on three members of this chemical category, including a complete battery of in vitro genotoxicity tests using limonene. No evidence of mutagenicity was observed when limonene was incubated with Salmonella 3

193 typhimurium strains TA98, TA100, TA102, TA1535, TA1537, and TA1538 with and without S-9 metabolic activation at concentrations up to and including 150,000 μg/plate. Limonene did not induce chromosomal aberrations when incubated with Chinese hamster ovary cells at a concentration of μg/ml, nor did it induce sister chromatid exchanges in Chinese hamster ovary cells at concentrations of μg/ml. In a mouse lymphoma forward mutation assay, limonene was negative in L5178Y cells with and without S-9 metabolic activation up to a maximum concentration of 100 μg/ml. When incubated with Syrian hamster embryo cells up to 100 μg/ml or 3 mm, limonene did not induce statistically significant cell transformation. The effects of limonene on gap junction intercellular communications were also tested at concentrations up to 1 mm in Syrian hamster embryo cells, and showed no effects. In an in vitro chromosome aberration test with human lymphocytes, myrcene did not induce chromosomal aberrations at concentrations up to 1,000 μg/ml with and without metabolic activation. When incubated with Chinese hamster ovary cells in a V79- HPRT Gene Mutation Assay, myrcene was not mutagenic with or without metabolic activation. In sister chromatid exchange (SCE) tests with human lymphocytes, myrcene did not induce sister chromatid exchanges at concentrations up to 1,000 μg/ml with or without metabolic activation. Additionally, there was no evidence of genotoxicity when myrcene was incubated with V79 and hepatic tumor (HPT) Chinese hamster cells at concentrations up to 500 μg/ml in SCE assays. In fact, myrcene reduced the SCE inducing effect of S-9 mix activated cyclophosphamide in human lymphocytes and Chinese hamster ovary (CHO) cells, and aflatoxin B1 in V79 and HTC Chinese hamster cells in a dose dependent manner. No evidence of mutagenicity was observed when sweet orange peel oil was incubated with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with and without S-9 metabolic activation at concentrations up to 5000 μg/plate. In a mouse lymphoma forward mutation assay, sweet orange peel oil was positive in L5178Y cells with and without S-9 metabolic activation up to a maximum concentration of 75 μg/ml but only at highly toxic concentrations. The authors noted that that positive results in this assay may be associated with changes in physiologic culture conditions (ph and osmolality). Negative results were obtained with sweet orange peel oil in the Rec DNA repair assay using Bacillus subtilis strains H17 and M45. In eighteen separate in vitro tests on the mutagenicity and genotoxicity of limonene, myrcene, and sweet orange peel oil; the majority was negative, with the exception of the mouse lymphoma assay using sweet orange peel oil. This result is questionable given the culture conditions present and the negative results of the mouse lymphoma assay using limonene since limonene is the majority (greater than 90%) constituent of sweet orange peel oil. Two in vivo genotoxicity assays are available for two substances in this chemical category. In an in vivo mammalian spot test, no evidence of mutagenicity was reported when mouse embryos were treated in utero with 215 mg/kg/day limonene on days of gestation. 4

194 In an in vivo cytogenetic bone marrow assay, beta-myrcene (100, 500 or 1,000 mg/kg) was orally administered via gavage to up to male and female Wistar rats. A dose- related increase in the mitotic index in bone marrow cells was reported for rats administered the test substance. The authors commented that this might be an interaction between betamyrcene, which is known to induce CYP-450 enzymes, and colchicine, which arrests cell division at metaphase. beta-myrcene may have increased the bioavailability of colchicine leading to the increase in mitotic index observed in the experiment. No significant increases in chromosomal aberrations were reported in the treated animals at either 24 or 48 hours. The authors concluded that given the results, beta-myrcene was not clastogenic to the rat when orally administered at dose levels up to 1,000 mg/kg. Based on the results of this in vivo genotoxicity assay and the numerous in vitro genotoxicity assays, it is unlikely that any of these materials would exhibit a significant genotoxic potential in vivo. Reproductive Toxicity Selected repeat dose toxicity studies were included in this toxicity profile. For summaries on all conducted studies, see the U.S. HPVIS. beta-myrcene was administered via gavage to female Wistar rats at dose levels of 250, 500, 1,000 and 1,500 mg/kg of beta-myrcene from the 15th day of gestation until weaning of the offspring which was day 21 postnatal. Fertility was impaired in female offspring exposed to 1,000 or 1,500 mg/kg beta-myrcene. In a one-generation reproductive toxicity study, male and female Wistar rats were dosed by oral gavage with 0, 100, 300 or 500 mg/kg beta-myrcene. The exposure period was 91 days prior to and during the mating period for the males and 21 days prior to and during the mating period for females, pregnancy, and lactation until 21 days post parturition. Neither deaths nor signs of toxicity were reported in male rats at any dose level. No statistically significant differences in body weight gain were reported between control and test animals. A slight increase in liver and kidney weights was reported for treated male (highest dose only) and female rats. No morphological alterations of the liver or testis tissue were revealed upon microscopic examination. No effects were reported on the number of spermatids in the testis or on the number of spermatozoa in the cauda epididymis. No adverse effects on body weight gain and no other signs of toxicity were observed in treated female rats during the pre-mating or mating periods. No treatment related effects were reported on fertility as measured by the mating index and pregnancy index upon comparison to controls. At the highest dose level, a slight increase in the resorption rate and a parallel decrease in the ratio of live fetuses per implantation site were reported. Increases in the occurrence of fetal skeleton abnormalities between control and treated groups were reported at the 500 mg/kg level. No adverse effects were reported on duration of pregnancy, labor, pup mortality, and maternal or offspring weight changes. Slight delays in incisor eruption (300 mg/kg) and eye opening (100, 300 mg/kg) were reported but were not dose-related. The authors attributed the increase in skeletal 5

195 abnormalities at the highest dose level tested to known strain-specific anomalies including increases of dislocated sternums, and lumbar extra ribs. The authors concluded that the NOAEL for toxic effects on fertility and general reproductive performance via the oral route was 300 mg /kg for beta-myrcene. Female rats were dosed by oral gavage with 0, 375, 750 or 1,500 mg/kg sweet orange peel for seven days prior to and through cohabitation, gestation, delivery and a four day lactation period. No deaths occurred at any dose level. Statistically significant numbers of rats from all three dose groups experienced excess salvation during the pre-mating and gestation periods, and during the lactation period for high-dose animals. The dosed rats had decreased weight gains compared to the control rats during the seven day premating period. Absolute and relative maternal food consumption was significantly decreased for the 750 and 1,500 mg/kg dose groups during the seven day premating period. No treatment related effect on mating performance or fertility was reported at any dose level. A significant increase in stillbirths and pup deaths was reported for the highest dose group compared to the control group (see next section for developmental effects). Given the results of three reproductive toxicity assays using sweet orange peel oil predominantly composed of d-limonene and beta-myrcene, it may be concluded that the substances within this chemical category exhibit low reproductive toxicity potential. Developmental Toxicity Pregnant female Wistar rats were dosed by oral gavage with 0, 591 or 2,869 mg/kg d- limonene on days 9-15 of gestation. At the highest dose level, increases in maternal mortality and decreases in maternal and fetal body weights were reported. Additionally at the highest dose level, delayed ossification of fetal metacarpal bones and proximal phalanx and decreased weights of the thymus, spleen, and ovaries were reported. The NOAEL for both maternal and offspring toxicity was reported to be 591 mg/kg. Pregnant Japanese white rabbits were administered 0, 250, 500 or 1,000 mg/kg d- limonene on days 6 to 18 of gestation. Increased maternal mortality was reported at the highest dose level. Significant decreases in maternal body weight gain and food consumption were temporarily observed at the 500 and 1,000 mg/kg dose levels. No treatment related effects were reported for the offspring. The NOAEL for maternal toxicity was reported to be 250 mg/kg/day. The NOAEL for offspring toxicity was reported to be greater than 1,000 mg/kg/day. Pregnant ICR mice were administered 0, 591 or 2,363 mg/kg d-limonene on days 7 to 12 of gestation. Significant decreases in body weight gain were reported for pregnant ICR mice administered the highest dose level of d-limonene. In the offspring, increased incidence of fused ribs compared to that of the controls, delayed ossification of some bones and decreased body weight gain were reported at the highest dose level tested. The NOAEL for both maternal and offspring toxicity was reported to be 591 mg/kg/day. 6

196 Pregnant Wistar rats were administered 0, 250, 500 or 1,200 mg/kg beta-myrcene on gestational days The vehicle was corn oil. Decreased maternal weight gain was reported at the 1,200 mg/kg dose. Increased fetal skeletal malformations were reported at the 1,200 mg/kg dose level. The NOAEL for both maternal and offspring toxicity was reported to be 500 mg/kg/day. beta-myrcene was administered via gavage to female Wistar rats at dose levels of 250, 500, 1,000 or 1,500 mg/kg from pregnancy day 15 until weaning of the offspring, which was day 21 postnatal. Mortality, weight gain and post-natal development were evaluated. Reproductive capacity was assessed in the exposed offspring upon reaching maturity (120 days). No adverse effects were noted in the offspring at the lowest dose level tested. Decreased body weight, increased perinatal mortality, and delayed developmental landmarks were noted at the 500, 1000 and 1500 mg/kg dose levels. Fertility was impaired in female offspring exposed to the two highest doses of beta-myrcene. The NOAEL for peri- and post-natal development was set at 250 mg/kg/day. Female rats were dosed by oral gavage with 0, 375, 750 or 1,500 mg/kg sweet orange peel for seven days prior to and through cohabitation, gestation, delivery and a four day lactation period. No deaths occurred at any dose level. Statistically significant numbers of rats from all three dose groups experienced excess salvation during the premating and gestation periods, and during the lactation period for high-dose animals. The dosed rats had decreased weight gains compared to the control rats during the seven-day premating period. Absolute and relative maternal food consumption was significantly decreased for the 750 and 1500 mg/kg/day dose groups during the seven day premating period. No treatment related effects were reported on maternal body weight, changes in body weight, and absolute and relative feed consumption during the lactation period. No treatment related effect on mating performance or fertility was reported at any dose level. A significant increase in stillbirths and pup deaths was reported for the highest dose group compared to the control group. The treatment with sweet orange oil had no effect on the incidence of malformations or gross lesions in the pups. The NOAEL for this study was reported to be less than 375 mg/kg/day for maternal toxicity and 750 mg/kg/day for offspring development. Given the results of six developmental toxicity assays using limonene, sweet orange oil and beta-myrcene, it may be concluded that the substances within this chemical category exhibit low developmental toxicity potential. Key Study/Critical Effect for Screening Criteria The toxicity data on d-limonene will be used as read-across to CAS No and The kidney changes seen in male rats administered limonene have been well characterized, and are known to be specific to the male rat and of no significance in human risk assessment. Thus, the male rat kidney effects will not be considered in the determining the critical effect for a drinking water guideline value. 7

197 In the NTP two-year rat bioassay, the NOAELs for male and female rats were 150 and 300 mg/kg/day, respectively. In the NTP two-year mouse bioassay, the NOAELs for male and female mice were 250 and 500 mg/kg/day, respectively. The lowest NOAEL is 150 mg/kg/day for male rats in the NTP 2-year bioassay based on liver effects. Adjusting for 5 days/week to 7 days/week, results in a NOAEL for continuous exposure of 107 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 107/100 = 1.1 mg/kg/day Drinking water guidance value = 3.8 ppm Reference U.S. EPA HPVIS database: 8

198 Toxicity Profile Hydrochloric acid (CAS No ) Hydrochloric acid has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Hydrochloric acid (HCl) or hydrogen chloride is readily dissociated in water into hydrated protons and chloride ion. Acute Toxicity The oral LD50 value of hydrogen chloride is reported to be 238 to 277 mg/kg for female rats (Hoechst AG, 1966), and 900 mg/kg for rabbits (Loewy and Munzer, 1923). The lethal dose by dermal exposure is >5,010 mg/kg for rabbits (Monsanto, 1976). The LC50 values for HCl are reported to be mg/l/5min, mg/l/30min and mg/l/60min for rats; and 20.9 mg/l/5min, 3.9 mg/l/30min and 1.7 mg/l/60min for mice (Darmer et al., 1974; Hartzell et al., 1990; MacEwean et al., 1974). Irritation Concentrations >3.3% cause skin irritation, and concentrations >17% cause corrosion in animal studies (OECD, 2002a,b). An aqueous solution (4%) of hydrogen chloride was slightly irritating (Agner and Serup, 1988), and a 10% solution was determined to be Irritating to skin for the EU Dangerous Preparations Directive, in human volunteer experiments (York et al., 1996). 0.1 ml of 10% aqueous solution of HCl was highly irritating to the eyes of rabbits (Jacobs, 1992); 0.03 ml or more of 5% HCl was corrosive to the eyes of rabbits (Griffith et al., 1980); 0.1 ml of a 3.3% aqueous solution of HCL was slightly irritating the eyes of rabbits (Hoechst AG, 1966); and 0.1 ml of a 0.33% aqueous solution of HCl was not irritating to the eyes of rabbits. (Hoechst AG, 1966). Sensitization Hydrogen chloride is not a skin sensitizer to guinea pigs and humans (Gad et al., 1986). Repeated Dose Toxicity Rats were fed diets containing 280 to 1,250 mmol/kg hydrochloric acid (10.2 to 45.6 mg/kg) for 7-12 weeks. There was increased water intake in all treated groups. All 1

199 animals fed diet containing 937 mmol/kg and above for 9 weeks, and half of the animals fed diet containing 900 mmol/kg for 12 weeks died. Also at doses >937 mmol/kg, there was decreased body weight, food consumption, blood ph, femur length, rate of ash in bone (Upotn and L Estrange, 1977). In another study with rats, hydrochloric acid was administered via drinking water at ph 2-3 (study duration not provided). Decreased protein levels in urine and decreased urine volumes were observed in the treatment groups (Clausing and Gottschalk, 1989). Genotoxicity While consistent negative results have been obtained in the bacterial systems, positive results have been obtained in the non-bacterial systems (OECD, 2002a,b). The positive results were observed at high concentrations, but they were considered to be artifacts due to the low ph. Positive results were obtained in a sex-linked recessive lethal study with D. melanogaster; only one dose level was tested (Stumm-Tegethoff, 1966) There are no mammalian studies on in vivo mutagenicity with hydrogen chloride Carcinogenicity (Oral Studies only) A ten-month study has been conducted using mice (Dyer et al., 1946). Because the methods such as strain used, duration and dose of administration, presence of coadministration substance, were not appropriate, it cannot be used for the assessment of carcinogenicity of hydrochloric acid. Reproductive Toxicity No reliable studies were identified by the oral route. Developmental Toxicity No reliable studies were identified by the oral route. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for ph may apply to hydrochloric acid. References Agner, T., and Serup, J. (1988). Contact thermography for assessment of skin damage due to experimental irritants., Acta. Dermatol. Venerol., 68:

200 Clausing, P., and Gottschalk, M.Z. (1989). Effects of drinking water acidification, restriction of water supply and individual caging on parameter of toxicological studies in rats. Versuchstierkd., 32: Darmer, K.I. et al. (1974). Acute toxicity in rats and mice exposed to hydrogen chloride gas and aerosol. Am. Ind. Hyg. Assoc. J.,35: Dyer, H.M. et al. (1946). Effect of administration of hot water, acids, alkali, mecholyl chloride, or atropine sulfate upon the gastric mucosa of mice. J Natl. Cancer Inst. 7:67. Gad, S.C., Dunn, B.J., Dobbs, D.W., Reilly, C., and Walsh, R.D. (1986). Development and validation of an alternative dermal sensitisation test: the mouse ear swelling test (MEST)., Toxicol. Appl. Pharmacol. 84: Griffith, J.F. et al. (1980). Dose-response studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye. Toxicol. Appl. Pharmacol. 55,: Hartzell, G.E. et al. (1990). Toxicity of smoke containing hydrogen chloride. ACS Symp. Ser., 425: Hoechst AG (1966). Farbwerke Hoechst AG Report 150/66. Jacobs, G.A. (1992). OECD eye irritation tests on two acids. J. Am. Coll. Toxicol. 11: 734. Loewy, A., and Munzer, E. (1923). Beiträge zur von der experimentellen Säurevergiftung., Bioch. Z. 134, MacEwean, J.D. et al. (1974). Toxic Hazard Research Unit, Annual Technical Report, NTIS AD- A Monsanto (1976). Unpublished report YO ; cited in European Commission- European Chemical Bureau IUCLID (2000) OECD (2002a). IUCLID Data Set for Hydrogen chloride (CAS No ). OECD (2002b). Screening Information Dataset (SIDS) Initial Assessment Report for Hydrogen chloride (CAS No ), UNEP Publications. Stumm-Tegethoff, B.F.A. (1969). Formaldehyde-induced mutations in Drosophila melanogaster ndependence of the presence of acids. Theor. Appl. Genet. 39: Upotn, P.K., and L Estrange, J.L. (1977). Effects of chronic hydrochloric and lactic acid balance and bone composition of the rat. Quart. J. of Exp. Physiol. 62:

201 York, M.H., Griffiths, A.E., Whittle, E., and Basketter, D.A. (1996). Evaluation of a human patch test for the identification and classification of skin irritation potential. Contact Dermatitis 34:

202 Toxicity Profile Monoethanolamine borate (CAS No ) The toxicity profile includes toxicity data on boric acid, reaction products with ethanolamine and triethanolamine (CAS No ), which is structurally similar to monoethanolamine borate. Acute Toxicity The acute oral LD50 in rats is >2,000 mg/kg, and the acute dermal LD50 of CAS in rats is >2,000 mg/kg. Irritation Monoethanolamine borate was slightly irritating and was not considered to be a skin irritant when tested on the skin of rabbits. Monoethanolamine (MEA) Polyborate 1:1 and MEA Polyborate 1:3 were tested for eye irritancy using the Bovine Corneal Opacity and Permeability test (BCOP test). Minimal to no ocular irritation was observed in both studies. Sensitization CAS No was not a skin sensitizer to guinea pigs using the Buehler method. Repeated Dose Toxicity Groups of male and female rats received CAS No by oral gavage at dose levels of 0, 62.5, 250 or 1,000 mg/kg body weight per day for a period of 28 days. No deaths occurred throughout the study. Behavior, state of health, body weight development and food consumption, were not affected by administration of the test substance. No treatment-related clinical signs were observed in all animals. No changes of the oral mucosa or impairment of dental growth was observed in all study groups. There were no substance related adverse findings either in hematological examinations, clinical chemistry or urine analyses throughout the study period. As a non-adverse finding, the test substance induced slightly lower total bilirubin, cholesterol and triglycerides in high dose group rats (1,000 mg/kg body weight) and possibly marginally lower hemoglobin and hematocrit content when compared to the controls at the end of the study period. These changes were at the lower range of the historical control data for this rat strain and age and hence, not considered to be of toxicological significance. The test 1

203 substance did not influence any of the neurotoxicological parameters. No test article related changes in organ weights were observed. Necropsy at terminal sacrifice revealed no gross pathology findings in the animals of all experimental groups. All microscopic findings sporadically observed in different organs of single animals were interpreted as not compound related. The NOAEL for this study is 1,000 mg/kg/day. Genotoxicity In an in vitro chromosomal aberration test using cultured peripheral human lymphocytes, MEA Polyborate 1:1 showed a clastogenic response in experiments where the ph of the treated cultures was above the ph of the solvent control. The clastogenicity was only observed in the first cytogenetic assay. Due to fact that the adjustment of the ph of the cultures treated with MEA Polyborate 1:1 did not show a clastogenic response in the third cytogenetic assay, it has to be assumed that the high ph is responsible for the detected clastogenic activity. In an in vitro chromosomal aberration test using cultured peripheral human lymphocytes, MEA Polyborate 1:3 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. CAS No did not induce gene mutations in an in vitro HPRT-test with V79 Chinese hamster lung fibroblast cells, either in the presence or in the absence of metabolic activation. Carcinogenicity No data are available. Reproductive Toxicity No data are available. Developmental Toxicity No data are available. Key Study/Critical Effect for Screening Criteria A 28-day oral toxicity study conducted was conducted in rats with CAS No (Boric acid, reaction products with ethanolamine and triethanolamine). The NOAEL for this study is 1,000 mg/kg/day. Data from CAS No is being used as readacross to monoethanolamine borate. 2

204 Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subacute to chronic) Oral RfD = 1000/1000 = 1 mg/kg/day Drinking water guideline: 3.5 ppm Reference ECHA REACH database: 3

205 Toxicity Profile PEG Oleate Ester [Poly(oxy-1,2-ethanediyl),.alpha.-hydro-.omega.-hydroxy-, (9Z)-9-octadecenoate] (CAS No ) Ethoxylated fatty acids are also called PEG fatty acid esters because the substance is a PEG chain linked to a fatty acid via an ester linkage. Data were located on the PEG stearate, PEG laurate, and PEG castor oil; these substances will be used as a surrogates for this substance. Acute Toxicity The acute oral LD50 of PEG stearates (50% preparations) were >10,000 mg/kg in rats, and the acute dermal LD50 of PEG stearate (15% preparation) was >10,000 ml/kg in rabbits. Irritation PEG stearates (100%) were mildly irritating the skin of rabbits, and non-irritating to human skin. PEG stearates (100%) was minimally irritating to the eyes of rabbits. PEG castor oils (100%) was considered non-irritating to the eyes of rabbits. Sensitization Clinical studies on the PEG stearates indicate that these ingredients are neither irritants nor sensitizers at concentrations of 25%. Repeated Dose Toxicity PEG-8 stearate at 5% in the diet did not produce any treatment-related effects in rabbits after 19 weeks, or any treatment-related effects at 4% after 4 months. In a two-year dietary study, PEG-8 stearate at 4% produced no treatment-related effects over three generations. Likewise, PEG-100 at 2% did not produce any treatment-related effects over three generations in a two-year dietary study. In a two-year dietary study, male and female rats (12/sex/dose) were given 0, 2, 5, 10, and 25% PEG-20 laurate in feed, At 25%, growth reduction occurred in the male rats, three rats had gastric mucosa hyperplasia, hepatic cysts in five rats, and cecal enlargement in 17 animals. At 10%, there were hepatic cysts in four rats and cecal 1

206 enlargement in four animals. At 5%, there was one hepatic cyst and cecal enlargement in three animals. At 2%, there was one hepatic cyst, and cecal enlargement in two animals (Fitzhugh et al., 1960). Genotoxicity There is no indication of a genotoxic effect of PEG fatty acids. Carcinogenicity No carcinogenicity studies could be located for PEG fatty acids. Reproductive/Developmental Toxicity Multi-generation studies with PEG- 40 stearates showed no adverse effects on reproduction and development. There was no evidence of developmental toxicity seen in mice and rat feeding studies with PEG-35 castor oil. Key Study/Critical Effect for Screening Criteria Liver cysts and cecal enlargement were seen in all tested rats given PEG-20 laurate in feed for two years. It cannot determined whether the lowest concentration tested (2%) is a LOAEL or a NOAEL. Thus, for the purposes of this risk assessment, the 2% concentration in this study will be considered a LOAEL. NOAEL (mg/kg/day) = 20,000 ppm * 0.05 = 1,000 mg/kg/day Where 0.05 is the fraction of body weight that is consumed per day as food for the rat (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (LOAEL to NOAEL) Oral RfD = 1,000/1,000 = 1 mg/kg/day Drinking water guidance value = 3.5 ppm References Fitzhugh, O.G., Schouboe, P.J., and Nelson, A.A. (1960). Oral toxicities of lauric acid and certain lauric acid derivatives. Toxicol. Appl. Pharmacol. 2:

207 Fruijtier-Pölloth, C. (2005). Safety assessment on polyethylene glycols (PEGs) and their derivatives as used in cosmetics. Toxicol. 214:

208 Toxicity Profile Sodium hydroxide (CAS No ) Sodium hydroxide (NaOH) has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Sodium hydroxide (NaOH) is present in the environment as sodium (Na + ) and hydroxyl ions (OH - ). Sodium is a normal constituent of the blood and an excess is excreted in the urine. A significant amount of sodium is taken up via the food because the normal uptake of sodium via food is g per day according to Fodor et al. (1999). Exposure to NaOH could potentially increase the ph of the blood. However, the ph of the blood is regulated between narrow ranges to maintain homeostasis (ph of 7.4 to 7.5) by urinary excretion of bicarbonate and via exhalation of carbon dioxide. Acute Toxicity An oral LD50 of a 1-10% solution of NaOH in rabbits was reported to be 325 mg/kg (expressed as 100% NaOH). Mortality was also observed when 1% NaOH was dosed, but in this case, the applied volume was relatively high (24 ml per kg body weight). Irritation A NaOH solution of 8% can be considered corrosive based on animal data. Human data indicate that concentrations of 0.5 to 4% were irritating. In two different studies a concentration of 0.5% was irritating for only 55 and 61% of the volunteers, respectively. Thus, it is assumed that a concentration, which is slightly lower than 0.5%, is the nonirritating concentration. The available animal data on eye irritation showed that the non-irritant level was %, while the corrosive concentration was 1.2 % or >2%. Sensitization Male volunteers were exposed on the back to sodium hydroxide concentrations of % (induction). After 7 days the volunteers were challenged to a concentration of 0.125%. The irritant response correlated well with the concentration of NaOH, but an increased response was not observed when the previously patch tested sites were rechallenged. Based on this study sodium hydroxide has no skin sensitization potential. 1

209 Repeated Dose Toxicity No animal data are available on repeated dose toxicity studies by oral, dermal, inhalation or by other routes for NaOH. It is not useful to study the repeated dose toxicity of hydroxide via an oral study because at high concentrations the substance is corrosive or irritating, while at low concentrations the hydroxide will be neutralized in the stomach by gastric juice, which has a very low ph. Genotoxicity Both the in vitro and the in vivo genotoxicity tests are negative. Carcinogenicity No carcinogenicity studies on sodium hydroxide were identified. Reproductive Toxicity No valid studies were identified regarding toxicity to reproduction in animals after oral, dermal or inhalation exposure to NaOH. It is not useful to study the reproduction/developmental toxicity of hydroxide via an oral study because at high concentrations the substance is corrosive or irritating, while at low concentrations the hydroxide will be neutralized in the stomach by gastric juice, which has a very low ph. Developmental Toxicity No valid studies were identified regarding developmental toxicity in animals after oral, dermal or inhalation exposure to NaOH. It is not useful to study the reproduction/developmental toxicity of hydroxide via an oral study because at high concentrations the substance is corrosive or irritating, while at low concentrations the hydroxide will be neutralized in the stomach by gastric juice, which has a very low ph. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for ph may apply to sodium hydroxide. Reference 2

210 OECD (2002). IUCLID Data Set for Sodium hydroxide (CAS No ), UNEP Publications. OECD (2002). Screening Information Dataset (SIDS) Initial Assessment Report for Sodium hydroxide (CAS No ), UNEP Publications. 3

211 Toxicity Profile Sodium sulfate (CAS No ) Sodium sulfate has been reviewed in the OECD-SIDS program (OECD, 2005a,b). Acute Toxicity The acute toxicity (LD50) of sodium sulfate has not been reliably established. Human data indicate a very low acute toxicity of sodium sulfate. High oral doses of sodium sulfate, from 300 mg/kg up to 20 grams for an adult, are well tolerated, except from (intentionally) causing severe diarrhea. There are no data on acute dermal toxicity. Irritation Sodium sulfate is not irritating to the skin and slightly irritating to the eyes. Respiratory irritation has never been reported. Sensitization Sodium sulfate is unlikely to be a skin or respiratory sensitizer based on wide practical experience with sodium sulfate, in combination with the natural occurrence of sulfate in the body. Repeated Dose Toxicity Valid oral repeated dose toxicity studies with 21, 28 and 35 day studies in hens and pigs are available. Toxicity was confined to changes in bodyweight, water and feed intake and diarrhea These changes occurred only at very high doses of sodium sulfate. The available data do not allow for the derivation of a NOAEL. Based on available consumer data, a daily dose of around 25 mg/kg/day is well tolerated by humans. Genotoxicity Sodium sulfate was not mutagenic in an Ames test. Carcinogenicity There is no valid oral carcinogenicity study. Limited data from experimental studies 1

212 support the notion that a substance that is abundantly present in and essential to the body is unlikely to be carcinogenic. Reproductive/Developmental Toxicity Limited data of poor validity did not provide an indication of toxicity to reproduction. Key Study/Critical Effect for Screening Criteria Exposure to sodium sulfate occurs via drinking water and through naturally occurring amounts in foodstuffs. In drinking water (wells) concentrations up to 2 g/l were measured in the U.S. The taste threshold for sodium sulfate is mg/l. The maximum acceptable concentration for drinking water is mg/l sulfate and is based on taste. No data on the sulfate content of foodstuffs were found; however, according to WHO, sulfates are used as additives in the food industry and the estimated average daily intake of sulfate in food in the U.S. is 453 mg/person, based on data on food consumption and reported usage of sulfates as additives. An Acceptable Daily Intake for sodium sulfate has not been established. The Australian drinking water guideline values for sodium and sulfate may apply to sodium sulfate. References OECD (2005). IUCLID Data Set for Sodium sulfate (CAS No ), UNEP Publications. OECD (2005). Screening Information Dataset (SIDS) Initial Assessment Report for Sodium sulfate (CAS No ), UNEP Publications. 2

213 Toxicity Profile Sodium sulfite CAS No ) Sodium sulfite has been reviewed in the OECD-SIDS program (OECD, 2008a,b). Sodium sulfite is completely dissociated in aqueous media into 2 Na + and sulfite anion (SO3 2- ). Sodium sulfite is a salt of sulfurous acid (H2SO3). In aqueous solutions, sulfurous acid dissociates, the dissociation constants are pka1 of 1.8 and pka2 of 7.0 at 25 C. At neutral ph, a mixture of 50% sulfite (SO3 2- ) and 50% bisulfite (HSO3 2- ) is present. At concentrations above 1M, bisulfite anions will dimerize with the elimination of water to form metabisulfite (S2O5 2- ). At low concentrations, metabisulfite will hydrolyze to form bisulfite. In surface waters, sulfite is oxidized to sulfate either catalytically by air oxygen or by microbial action. The presence of cations like iron, copper or manganese in the environment accelerates the oxidation rate significantly. Sodium sulfite is rapidly absorbed from the gastro-intestinal tract. Sulfate is the main metabolite formed by the action of sulfite oxidase in many tissues. Tissue accumulation of sulfite-derived S is highest in stomach, skin and hair, intestine and kidney. Excretion is rapid, mainly in the urine. Acute Toxicity The oral LD50 values are 3,560 mg/kg in rats and mg/kg in mice. No dermal LD50 values or acute LC50 values are available. Irritation Sodium sulfite was not irritating to the skin or eyes of rabbits. Sensitization No dermal sensitization studies in animals are available. In humans, some cases of sensitization from topical contact with sodium sulfite have been described. Only 1.4% of a population of 1762 eczema patients showed a positive reaction to sodium sulfite in patch tests. 1

214 Repeated Dose Toxicity In a 90-day feeding study, a NOAEL for male rats of 2% in diet corresponding to 1,670 mg/kg/day was found. The LOAEL was 4% in diet based on decreased body weight gain and increased relative weights of testis and brain. In female rats, the highest tested dose of 4% in diet was the NOAEL corresponding to 3,070 mg/kg/day. A two-year rat feeding of sodium metabisulfite (Na2S2O5). The NOAEL was 0.5%, which is equivalent to a dose of 144 mg/kg/day calculated as sodium sulfite. The LOAEL for local effects (forestomach and glandular stomach hyperplasia or inflammation) corresponded to 1.0% in diet, which is equivalent to 300 mg/kg/day calculated as sodium sulfite. The NOAEL for systemic effects corresponded to the highest dose of 2% in diet (equivalent to 625 mg/kg/day calculated as sodium sulfite). Genotoxicity Sodium sulfite is not genotoxic based on the available studies that have been conducted in vitro and in vivo. In vitro, sodium sulfite gave no indication of mutagenic or clastogenic activity up to cytotoxic concentrations both in the absence and presence of metabolic activation systems when tested in Ames tests/salmonella typhimurium reversion assays, in gene mutation tests with Saccharomyces cerevisiae and in mammalian V79 cells (without metabolic activation), in a DNA damage and repair assay with Escherichia coli, as well as in a chromosomal aberration test with CHL cells (without metabolic activation). There are no in vivo tests with application of pure sodium sulfite. Several genotoxicity studies in vivo with sulfites other than sodium sulfite were negative. The negative findings in whole animals are regarded as consistent with the high reactivity of sulfite e.g. with proteins and its rapid inactivation in mammals due to metabolism. However, in contrast to the consistent negative findings in earlier studies, recent investigations of a single working group demonstrated dose-dependent increases of micronuclei in bone marrow and DNA damage in tissues of mice after intraperitoneal injection of a mixture of sodium sulfite and sodium bisulfite. No parallel investigations were performed with the pure compounds as controls. Carcinogenicity There are no carcinogenicity studies available with administration of sodium sulfite. Male and female Wistar rats exposed to sodium metabisulfite (Na2S2O5) given in the diet with 0.125, 0.25, 0.5, 1.0 and 2.0% and supplemented with thiamine due to its breakdown by sulfite in the diet) for 104 weeks. The number of lymphoreticular pulmonary tumors in males decreased with increasing levels of sulfite. The incidence of thyroid and 2

215 pituitary tumors in control males was exceptionally low, whereas those noted in the various test groups represented numbers normally found in the strain of Wistar rats used. All other neoplasms occurred in a sporadic manner with no apparent relationship between number, location or type of tumors and the treatment. Male and female mice were given 0, 1 or 2% potassium metabisulfite in drinking water for 104 weeks. There was no increased incidence of tumors in the treated mice compared to controls. Reproductive Toxicity There are no reproductive toxicity studies with sodium sulfite investigating effects on male or female fertility. In a three-generation study with sodium metabisulfite (Na2S2, there was no suggestive evidence of reproductive toxicity or impairment of fertility in rats that received up to 2% in the diet. No effects on gonads were seen histologically. This NOAEL for reproductive toxicity would be equivalent to a dose of 625 mg/kg bw/day calculated as sodium sulfite, which is higher than the NOAEL for chronic toxicity of 0.5% in the diet (based on local irritation of the stomach), being equivalent to a dose of 144 mg/kg bw/day calculated as sodium sulfite. In a long-term (see repeat dose toxicity section for results) and three-generation study, rats were treated with 0, 0.125, 0.25, 0.5, 1.0 and 2.0% of sodium metabisulfite (corresponding to 0, 32, 70, 144, 300 and 625 mg/kg/day as Na2SO3 equivalents) in a supplemented diet with thiamine, since sulfites are known to break down thiamine in the diet. F0- and F1-generation were exposed for 104 weeks, and the F2-generation for 30 weeks. The F0-generation was mated at week 21 of treatment. Half of the animals were mated again at week 34. Animals from the first litter were selected at weaning to become the F1a-generation being mated at weeks 12 and 30 to produce F2a- and F2b-generations. Animals from the F2a litters were mated to produce the F3a- and F3b-generations by pairing on weeks 14 and 22. Body weight was not reduced in any treatment group in the F0-generation. There was a marginal reduction in body weight in both sexes of the 2% group in the F1- and F2- generations. Results in successive generations showed no substantial treatment-related effects in terms of fertility, the number of animals/litter or the birth weight or mortality of the young. During lactation the body weight of the young in the 2% group was generally lower than the controls and the lower-dosed groups. This reduced body weight was probably not a true substance-related effect because it was transient and could be due to a higher initial body weight in the control groups. A reduction in the number of F2ageneration offspring (F3a pups) was observed in the 0.5, 1.0 and 2.0% dose groups, but it was not dose-dependent and did not occur in the F2b-generation offspring (F3b pups). No pronounced effects were observed on reproductive performance in any generation and no effects on gonads were seen histologically. The NOAEL for reproduction toxicity was 3

216 the highest dose of 2% in the diet (625 mg/kg/day calculated as sodium sulfite ). The NOAEL for chronic toxicity of the F0-generation corresponding to 0.5% in the diet (144 mg/kg/day calculated as sodium sulfite). The LOAEL for local effects (irritation of the stomach) corresponded to 1.0% in diet (300 mg/kg/day calculated as sodium sulfite). The NOAEL for systemic effects corresponded to the highest dose of 2% (625 mg/kg/day calculated as sodium sulfite ). Developmental Toxicity Pregnant female Wistar rats were given in their diet 0, 0.32, 0.63, 1.25, 2.5 or 5% sodium sulfite heptahydrate (ca. 0, 150, 300, 550, 1,050 or 1,650 mg/kg as sodium sulfite) on gestational days Satellite groups of 4 rats received 0, 0.32 or 5% in the diet on the same days and were allowed to deliver and rear their litters to weaning. In dams, statistically significant decreased food consumption was seen at 150, 300 or 1650 mg sodium sulfite/kg and body weight gains were also significantly decreased at 1,650 mg/kg during the exposure period. In fetuses of all dosed animals, there was a decrease in fetal body weights without clear dose-response relationship. In the satellite groups there was no evidence of toxicity or growth retardation in dams or offspring during a 21-day lactation period. The observed fetal growth retardation was without clear dose-response relationship and there were also no adverse findings concerning this endpoint in the satellite groups allowed to deliver and rear their litters to weaning. Therefore, the relevance of this isolated effect is questionable and is not considered as adverse finding. There was no indication for teratogenic effects at all tested doses. The NOAEL for maternal toxicity is 1,050 mg/kg/day and the NOAEL for developmental effects is 1,650 mg/kg/day. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline values for sodium and sulfate may apply to sodium sulfate. Reference OECD (2008). IUCLID Data Set for Sodium sulfite (CAS No ). OECD (2008). Screening Information Dataset (SIDS) Initial Assessment Report for Sodium sulfite (CAS No ). 4

217 Toxicity Profile Sodium thiosulfate (CASRN ) Thiosulfate is a metastable, moderately reducing oxyanion of sulfur. Chemical reactions (redox reactions) of thiosulfate generate chemical species of sulfur that differ in their oxidation state. The thiosulfate anion (S2O3 2- ) is only stable in neutral or alkaline media. It is unstable in acid media. In aqueous media, thiosulfate irreversibly disproportionates to sulfide and sulfate. The chemistry of sulfur in soils (and in water) is complex because of its many oxidation states. The terminal, thermodynamically-favored reaction product of thiosulfate is sulfate, except in highly reduced soils. Acute Toxicity The acute oral LD50 of an aqueous solution of calcium thiosulfate is >2,000 mg/kg in female rats. For ammonium thiosulfate, the acute oral rat LD50 is 4,054 mg/kg in males, 3,500 mg/kg, and 3,824 mg/kg in combined sexes. The acute oral LD50 of potassium thiosulfate solution was estimated to be >5,000 mg/kg (for pure test item > 2,500 mg/kg) in the rat. Irritation No data were located. Sensitization Ammonium thiosulfate at concentrations of 10%, 25% and 50% (w/w) in aqua ad iniectabilia did not reveal any sensitising properties in the local lymph node assay. Repeated Dose Toxicity No data on sodium thiosulfate were located. Genotoxicity Ammonium thiosulfate was not mutagenic in the bacterial reverse mutation assay using S. typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli strain WP2 uvra in the presence and absence of metabolic activation system. Ammonium thiosulfate was negative in an in vitro chromosomal aberration assay using CHO cells. Ammonium thiosulfate was also negative in a mouse lymphoma cells. 1

218 Carcinogenicity No data on sodium thiosulfate were located. Reproductive Toxicity No data on sodium thiosulfate were located. Developmental Toxicity The administration of up to 550 mg/kg sodium thiosulfate to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. Thus, the NOAEL for maternal and developmental toxicity can be expected above the highest dose of 550 mg/kg. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline values for sodium and sulfate may apply to sodium thiosulfate. References ECHA REACH database: U.S. EPA (2007). Reregistration Elibility Decision (RED) for Ammonium Thiosulfate, Office of Prevention, Pesticides an Toxic Substances, EPA 738-R , December 20,

219 Toxicity Profile Sorbitan Monooelate (CAS No ) Sorbitan fatty acid esters are non-ionic surfactant-active agents that typically find use as emulsifiers, stabilizers and thickeners in foods, cosmetics, medical products, lubricants and other applications (Andersen, 2002; Eisenhard, 1999). Sorbitan monooleate is approved by FDA as an indirect food additive. The information in this toxicity profile was obtained from the U.S. EPA HPV Test Plan for Sorbitan Esters Category of the Aliphatic Esters Chemicals, November 26, Metabolism Metabolism of the sorbitan esters in animals has been reported to occur initially via enzymatic hydrolysis, leading to sorbitan and the corresponding natural fatty acids. Oral gavage studies in rats with radiolabelled sorbitan monostearate, which is structurally similar to sorbitan monooleate, have demonstrated that about 90% of the sorbitan monostearate dose was absorbed and hydrolyzed to stearic acid and sorbitan (Elder, 1985; Wick, 1953). The resulting sorbitan and fatty acid metabolites, in turn would be expected to be metabolized further (via fatty acid beta-oxidation or carbohydrate metabolic pathways) to either smaller and more polar water-soluble metabolites, which can be excreted in the urine or as carbon dioxide exhaled in the lungs. Acute Toxicity The acute oral LD50 for sorbitan monooleate in rats was reported to be > 39,800 mg/kg (Elder, 1985). Irritation No data were located. Sensitization No data were located. Repeated Dose Toxicity/Carcinogenicity Oral feeding toxicity studies have been carried out in rats for 16 weeks with sorbitan 1

220 monooleate at dietary concentrations of 0, 2.5, 5 and 10% (Ingram et al., 1978). The LOAEL was 2.5% dietary concentration (~1800 mg/kg/day) based on increased kidney weight findings that were considered significant in both male and female rats. Sorbitan monooleate fed to rats at 5% concentrations in the diet for 2 years showed no adverse effects on growth, hematology, clinical chemistry, survival, organ size or histopathology (ACI, 1970). The NOAEL was reported to be 5% in the diet for sorbitan monooleate in this study. Genotoxicity No genotoxicity studies could be located for sorbitan monooleate; however, there are two studies conducted on the structurally similar substance sorbitan monostearate. Sorbitan monostearate was not mutagenic to Salmonella typhimurium TA98 and TA100 in a Ames mutation assay in the presence or absence of metabolic activation (Inoue et al., 1980). Sorbitan monostearate did not cause any chromosomal aberrations in the Syrian golden hamster embryo cell assay and did not show clastogenic activity Inoue et al., 1980). Reproductive/Developmental Toxicity No studies could be located for sorbitan monooleate; however, there is one study conducted in the structurally similar substance sorbitan monostearate. Sorbitan monostearate was administered in the diet at dose levels of 0, 5, 10, and 20% over a period of two years and over four generations of rats (Oser et al., 1956a,b; Oser et al., 1957a,b). No effects were observed on growth, food efficiency, reproduction, lactation, metabolism, behavior, mortality, or in the gross and histopathological examination. At the 20% dose level in the diet, newborn mortality was not increased but there were slight effects on growth and impairment of lactation. Key Study/Critical Effect for Screening Criteria No treatment-related effects were noted in a two-year carcinogenicity study in which rat were fed sorbitan monooleate in the diet at concentrations up to 5% (50,000 ppm). NOAEL (mg/kg/day) = 50,000 ppm * 0.05 = 2,500 mg/kg/day Where 0.05 is the fraction of body weight that is consumed per day as food for the rat (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) 2

221 Oral RfD = 2,500/100 = 25 mg/kg/day Drinking water guidance value = 87.5 ppm References Andersen, F.A. (2002). Final report on the safety assessment of sorbitan caprylate, sorbitan cocoate, sorbitan diisostearate, sorbitan dioleate, sorbitan distearate, sorbitan isostearate, sorbitan olivate, sorbitan sesquiisostearate, sorbitan sesquistearate, and sorbitan triisostearate. Int. J. Toxicol. 21 (Suppl. 1): Atlas Chemical Industries (ACI) (1970). Summaries of toxicological data. Lifespan feeding studies on sorbitan monolaurate and sorbitan monooleate. Food Cosmet. Toxicol. 8: (1970). Summary of data from Toxicology Report on Span 20 and Span 80 (ACI Studies). Eisenhard, W.C, (1999). Esterification in Fatty Acids in Industry: Process, Properties, Derivatives, Applications. Johnson RW, Fritz E (eds.), Marcel Dekker, New York, pp Elder, R.L. (1985). Final report on the safety assessment of sorbitan stearate, sorbitan laurate, sorbitan sesquioleate, sorbitan oleate, sorbitan tristearate, sorbitan palmitate, and sorbitan trioleate, J. Amer. Coll. Toxicol. 4(3): Ingram, A.J., Butterworth, K.R., Gaunt, I.F., Grasso, P., and Gangoli, S.D. (1978). Shortterm toxicity study of sorbitan monooleate (Span 80) in rats. Food Cosmet. Toxicol. 16: Inoue, K., Sunakawa, T., and Takayama, S. (1980). Studies of in vitro cell transformation and mutagenicity by surfactants and other compounds. Food Cosmet. Toxicol. 18: Oser, B.L., and Oser, M. (1956a). Nutritional studies on the diets containing high levels of partial ester emulsifiers. I. General plan and procedures: growth and food utilization. J. Nutrit. 60: 367 Oser, B.L., and Oser, M. (1956b). Nutritional studies on the diets containing high levels of partial ester emulsifiers. II. Reproduction and lactation. J. Nutrit. 60: Oser,B.L., and Oser, M. (1957a). Nutritional studies on the diets containing high levels of partial ester emulsifiers. III. Clinical and metabolic observations. J. Nutrit. 61: 149. Oser, B.L., and Oser, M. (1957b). Nutritional studies on the diets containing high levels of partial ester emulsifiers. IV. Mortality and post-mortem pathology: general conclusions. J. Nutrit. 61:

222 Wick, A.N., Joseph, L. (1953). The metabolism of sorbitan monostearate. Food Res. 18: 79 4

223 Toxicity Profile Terpenes and Terpenoids Terpenes and Terpenoids, Sweet Orange Oil (CAS No ) The chemical category designated terpenes and terpenoids are hydrocarbons that can include C10 isomeric monocyclic terpene hydrocarbons (d-limonene, dl-limonene, and terpinolene) and two C10 acyclic terpene hydrocarbons (beta-myrcene and dihydromyrcene). Terpenes and terpenoids, sweet orange oil (CAS ) is composed of 91-95% d- limonene, 1-3% beta-myrcene, and 1-2% alpha-pinene. Acute Toxicity The oral and dermal LD50 values for limonene, myrcene, terpinolene, dihydromyrcene and sweet orange peel oil indicate a low order of both oral and dermal toxicity. All rabbit and rat dermal, and mouse and rat oral LD50 values are >4,000 mg/kg with the majority of values being >5,000 mg/kg. Irritation No data were located. Sensitization No data were located. Repeated Dose Toxicity/Carcinogenicity Selected repeat dose toxicity studies were included in this toxicity profile. For summaries on all conducted studies, see the U.S. HPVIS. Groups of ten Sprague Dawley rats of each sex were administered 0, 240, 600 or 1,500 mg/kg sweet orange oil in 1% methyl cellulose by gavage daily for 30 days. Observations included survival, clinical observations, body weights, food consumption, clinical pathology, gross pathology, organ weights and histopathology. No treatment related effects were reported for survival, clinical observations, body weights or food consumption. Decreases in glucose levels related to treatment were reported in the middose females and high-dose males and females. Increases in serum albumin and total 1

224 serum protein were observed in all treated females and the high-dose males. Histopathology revealed treatment related lesions in the nonglandular stomach of the high-dose males and females and in the kidney of all treated male groups. Kidney weights were also increased in all of the treated male groups and in the high-dose female group. Liver weight increases related to treatment were reported for the high-dose females and all treated male groups. The authors concluded that the no-observed-effectlevel (NOEL) under conditions of this study was less than 240 mg/kg/day for both male and female rats. The authors noted that the kidney changes observed in the male rat at all dose levels were expected given the known interaction between limonene and alpha-2- microglobulin. Limonene is the principal constituent of orange oil. In a NTP 13-week study, male and female F344/N rats were dosed by oral gavage 0, 150, 300, 600, 1,200 or 2,400 mg/kg d-limonene in corn oil for 5 days/week. At 2,400 mg/kg, 9/10 female rats and 5/10 male rats (5/10) died within the first week of the study. The final mean body weights of male rats receiving the three highest doses (600, 1,200 or 2,400 mg/kg) were reported to be 6%, 12%, or 23% lower than that of the controls, respectively. Rough hair coats, lethargy, and excessive lacrimation were observed for all animals in the 1,200 and 2,400 mg/kg dose groups. Kidney effects were seen in all treated male rats, with a dose-related increase in severity. The kidney effects were characterized by degeneration of epithelium in the convoluted tubules, granular casts with tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets were observed in the epithelium of the proximal convoluted tubules in all groups of male rats including vehicle controls. In a NTP 13-week study, male and female B6C3F1 mice were dosed by oral gavage with 0, 125, 250, 500, 1,000 or 2,000 mg/kg d-limonene for five days/week. At 2,000 mg/kg, 1/10 males and 2/10 females died before the end of the study; 1/10 females in the 500 mg/kg group also died before study termination. Several other animals also died as a result of gavage error. Mean body weights were 10% lower than control for male mice and 2% lower than control for female mice for the 1,000 and 2,000 mg/kg groups. Clinical signs of rough hair coats and decreased activity were reported for the two highest dose levels. Male rats were dosed by oral gavage with 0, 2, 5, 10, 30 or 75 mg/kg d-limonene 5 days/week for 13 weeks. Linear regression analyses indicated increased relative kidney and liver weights at 30 and 75 mg/kg. Histological examination showed changes in the kidney characterized by hyaline droplet formation, granular casts and multiple cortical changes. Exacerbation of hyaline droplet formation was reported at the earliest necropsy eight days after administration at the 10 mg/kg dose level. In a two-year NTP bioassay, male and female F344/N rats were dosed with 0, 75 or 150 mg/kg or 0, 300 or 600 mg/kg d-limonene for five days/week. Mean body weights for the 150 mg/kg male rats were generally 4-7% lower than vehicle controls from week 2 to study termination. Mean body weights of the 600 mg/kg females were generally 4-7% lower than vehicle controls from week 28 to study termination. No treatment-related clinical signs were reported for the duration of the study. Survival of the 150 mg/kg male 2

225 group was significantly greater than that of the vehicle alone after week 81. Survival of the 600 mg/kg female group was significantly lower than that of the vehicle controls after week 39. In the male rat kidneys, a dose-related increase was observed in the incidence of mineralization and epithelial hyperplasia. A dose-related increase in the severity of spontaneous nephropathy was reported in the treated male rats. Increased incidences in tubular cell hyperplasia and neoplasia were also reported in dosed male rats. Tubular cell adenoma incidence in 150 mg/kg male rats and tubular cell adenoma or tubular cell carcinomas (combined) in dosed male rats were significantly greater than vehicle controls. There was no evidence of carcinogenic activity of d-limonene for female rats receiving 300 or 600 mg/kg. The NOAEL for male rats (excluding the kidney effects) is 150 mg/kg/day (see below on the relevance of the male rat kidney effects for human risk assessment). The NOAEL for female rats is 300 mg/kg/day based on low survival in the 600 mg/kg dose group. In a NTP two-year bioassay, male and female B6C3F1 mice were dosed by oral gavage with 0, 250, or 500 mg/kg or 0, 500 or 1,000 mg/kg d-limonene, respectively, five days a week. Mean body weights for the 1,000 mg/kg female mice administered 1,000 mg/kg were generally 5-15% lower than vehicle controls from week 28 to study termination. No treatment related clinical signs were reported for the duration of the study. Survival of the 250 mg/kg male group was significantly lower than that of the vehicle controls by study termination. In the 500 mg/kg males, livers exhibited presence of cells with abnormal numbers of nuclei and cytomegaly. There was no evidence of carcinogenic activity of d-limonene for male or female B6C3F1 mice at the dose levels tested. The NOAEL for male mice is 250 mg/kg/day based on liver effects at 500 mg/kg/day. Although there was reduced survival in the 250 mg/kg male mice, NTP did not considered this to be dose-related. The NOAEL for female mice is considered to be 500 mg/kg/day based on decreased survival and lower body weights (5-15%) in the 1,000 mg/kg dose group. The kidney changes seen in male rats administered limonene have been well characterized, and are known to be specific to the male rat and of no significance in human risk assessment. Genotoxicity Given the structural similarity between the members of this chemical category, the substances in this category exhibit low genotoxic potential in vitro. Mutagenicity/genotoxicity testing has been performed on three members of this chemical category, including a complete battery of in vitro genotoxicity tests using limonene. No evidence of mutagenicity was observed when limonene was incubated with Salmonella typhimurium strains TA98, TA100, TA102, TA1535, TA1537, and TA1538 with and without S-9 metabolic activation at concentrations up to and including 150,000 μg/plate. Limonene did not induce chromosomal aberrations when incubated with Chinese hamster ovary cells at a concentration of μg/ml, nor did it induce sister chromatid 3

226 exchanges in Chinese hamster ovary cells at concentrations of μg/ml. In a mouse lymphoma forward mutation assay, limonene was negative in L5178Y cells with and without S-9 metabolic activation up to a maximum concentration of 100 μg/ml. When incubated with Syrian hamster embryo cells up to 100 μg/ml or 3 mm, limonene did not induce statistically significant cell transformation. The effects of limonene on gap junction intercellular communications were also tested at concentrations up to 1 mm in Syrian hamster embryo cells, and showed no effects. In an in vitro chromosome aberration test with human lymphocytes, myrcene did not induce chromosomal aberrations at concentrations up to 1,000 μg/ml with and without metabolic activation. When incubated with Chinese hamster ovary cells in a V79- HPRT Gene Mutation Assay, myrcene was not mutagenic with or without metabolic activation. In sister chromatid exchange (SCE) tests with human lymphocytes, myrcene did not induce sister chromatid exchanges at concentrations up to 1,000 μg/ml with or without metabolic activation. Additionally, there was no evidence of genotoxicity when myrcene was incubated with V79 and hepatic tumor (HPT) Chinese hamster cells at concentrations up to 500 μg/ml in SCE assays. In fact, myrcene reduced the SCE inducing effect of S-9 mix activated cyclophosphamide in human lymphocytes and Chinese hamster ovary (CHO) cells, and aflatoxin B1 in V79 and HTC Chinese hamster cells in a dose dependent manner. No evidence of mutagenicity was observed when sweet orange peel oil was incubated with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with and without S-9 metabolic activation at concentrations up to 5000 μg/plate. In a mouse lymphoma forward mutation assay, sweet orange peel oil was positive in L5178Y cells with and without S-9 metabolic activation up to a maximum concentration of 75 μg/ml but only at highly toxic concentrations. The authors noted that that positive results in this assay may be associated with changes in physiologic culture conditions (ph and osmolality). Negative results were obtained with sweet orange peel oil in the Rec DNA repair assay using Bacillus subtilis strains H17 and M45. In eighteen separate in vitro tests on the mutagenicity and genotoxicity of limonene, myrcene, and sweet orange peel oil; the majority was negative, with the exception of the mouse lymphoma assay using sweet orange peel oil. This result is questionable given the culture conditions present and the negative results of the mouse lymphoma assay using limonene since limonene is the majority (greater than 90%) constituent of sweet orange peel oil. Two in vivo genotoxicity assays are available for two substances in this chemical category. In an in vivo mammalian spot test, no evidence of mutagenicity was reported when mouse embryos were treated in utero with 215 mg/kg/day limonene on days of gestation. In an in vivo cytogenetic bone marrow assay, beta-myrcene (100, 500 or 1,000 mg/kg) was orally administered via gavage to up to male and female Wistar rats. A dose- related increase in the mitotic index in bone marrow cells was reported for rats administered the 4

227 test substance. The authors commented that this might be an interaction between betamyrcene, which is known to induce CYP-450 enzymes, and colchicine, which arrests cell division at metaphase. beta-myrcene may have increased the bioavailability of colchicine leading to the increase in mitotic index observed in the experiment. No significant increases in chromosomal aberrations were reported in the treated animals at either 24 or 48 hours. The authors concluded that given the results, beta-myrcene was not clastogenic to the rat when orally administered at dose levels up to 1,000 mg/kg. Based on the results of this in vivo genotoxicity assay and the numerous in vitro genotoxicity assays, it is unlikely that any of these materials would exhibit a significant genotoxic potential in vivo. Reproductive Toxicity Selected repeat dose toxicity studies were included in this toxicity profile. For summaries on all conducted studies, see the U.S. HPVIS. beta-myrcene was administered via gavage to female Wistar rats at dose levels of 250, 500, 1,000 and 1,500 mg/kg of beta-myrcene from the 15th day of gestation until weaning of the offspring which was day 21 postnatal. Fertility was impaired in female offspring exposed to 1,000 or 1,500 mg/kg beta-myrcene. In a one-generation reproductive toxicity study, male and female Wistar rats were dosed by oral gavage with 0, 100, 300 or 500 mg/kg beta-myrcene. The exposure period was 91 days prior to and during the mating period for the males and 21 days prior to and during the mating period for females, pregnancy, and lactation until 21 days post parturition. Neither deaths nor signs of toxicity were reported in male rats at any dose level. No statistically significant differences in body weight gain were reported between control and test animals. A slight increase in liver and kidney weights was reported for treated male (highest dose only) and female rats. No morphological alterations of the liver or testis tissue were revealed upon microscopic examination. No effects were reported on the number of spermatids in the testis or on the number of spermatozoa in the cauda epididymis. No adverse effects on body weight gain and no other signs of toxicity were observed in treated female rats during the pre-mating or mating periods. No treatment related effects were reported on fertility as measured by the mating index and pregnancy index upon comparison to controls. At the highest dose level, a slight increase in the resorption rate and a parallel decrease in the ratio of live fetuses per implantation site were reported. Increases in the occurrence of fetal skeleton abnormalities between control and treated groups were reported at the 500 mg/kg level. No adverse effects were reported on duration of pregnancy, labor, pup mortality, and maternal or offspring weight changes. Slight delays in incisor eruption (300 mg/kg) and eye opening (100, 300 mg/kg) were reported but were not dose-related. The authors attributed the increase in skeletal abnormalities at the highest dose level tested to known strain-specific anomalies including increases of dislocated sternums, and lumbar extra ribs. The authors concluded that the NOAEL for toxic effects on fertility and general reproductive performance via the oral route was 300 mg /kg for beta-myrcene. 5

228 Female rats were dosed by oral gavage with 0, 375, 750 or 1,500 mg/kg sweet orange peel for seven days prior to and through cohabitation, gestation, delivery and a four day lactation period. No deaths occurred at any dose level. Statistically significant numbers of rats from all three dose groups experienced excess salvation during the pre-mating and gestation periods, and during the lactation period for high-dose animals. The dosed rats had decreased weight gains compared to the control rats during the seven day premating period. Absolute and relative maternal food consumption was significantly decreased for the 750 and 1,500 mg/kg dose groups during the seven day premating period. No treatment related effect on mating performance or fertility was reported at any dose level. A significant increase in stillbirths and pup deaths was reported for the highest dose group compared to the control group (see next section for developmental effects). Given the results of three reproductive toxicity assays using sweet orange peel oil predominantly composed of d-limonene and beta-myrcene, it may be concluded that the substances within this chemical category exhibit low reproductive toxicity potential. Developmental Toxicity Pregnant female Wistar rats were dosed by oral gavage with 0, 591 or 2,869 mg/kg d- limonene on days 9-15 of gestation. At the highest dose level, increases in maternal mortality and decreases in maternal and fetal body weights were reported. Additionally at the highest dose level, delayed ossification of fetal metacarpal bones and proximal phalanx and decreased weights of the thymus, spleen, and ovaries were reported. The NOAEL for both maternal and offspring toxicity was reported to be 591 mg/kg. Pregnant Japanese white rabbits were administered 0, 250, 500 or 1,000 mg/kg d- limonene on days 6 to 18 of gestation. Increased maternal mortality was reported at the highest dose level. Significant decreases in maternal body weight gain and food consumption were temporarily observed at the 500 and 1,000 mg/kg dose levels. No treatment related effects were reported for the offspring. The NOAEL for maternal toxicity was reported to be 250 mg/kg/day. The NOAEL for offspring toxicity was reported to be greater than 1,000 mg/kg/day. Pregnant ICR mice were administered 0, 591 or 2,363 mg/kg d-limonene on days 7 to 12 of gestation. Significant decreases in body weight gain were reported for pregnant ICR mice administered the highest dose level of d-limonene. In the offspring, increased incidence of fused ribs compared to that of the controls, delayed ossification of some bones and decreased body weight gain were reported at the highest dose level tested. The NOAEL for both maternal and offspring toxicity was reported to be 591 mg/kg/day. Pregnant Wistar rats were administered 0, 250, 500 or 1,200 mg/kg beta-myrcene on gestational days The vehicle was corn oil. Decreased maternal weight gain was reported at the 1,200 mg/kg dose. Increased fetal skeletal malformations were reported at the 1,200 mg/kg dose level. The NOAEL for both maternal and offspring toxicity was 6

229 reported to be 500 mg/kg/day. beta-myrcene was administered via gavage to female Wistar rats at dose levels of 250, 500, 1,000 or 1,500 mg/kg from pregnancy day 15 until weaning of the offspring, which was day 21 postnatal. Mortality, weight gain and post-natal development were evaluated. Reproductive capacity was assessed in the exposed offspring upon reaching maturity (120 days). No adverse effects were noted in the offspring at the lowest dose level tested. Decreased body weight, increased perinatal mortality, and delayed developmental landmarks were noted at the 500, 1000 and 1500 mg/kg dose levels. Fertility was impaired in female offspring exposed to the two highest doses of beta-myrcene. The NOAEL for peri- and post-natal development was set at 250 mg/kg/day. Female rats were dosed by oral gavage with 0, 375, 750 or 1,500 mg/kg sweet orange peel for seven days prior to and through cohabitation, gestation, delivery and a four day lactation period. No deaths occurred at any dose level. Statistically significant numbers of rats from all three dose groups experienced excess salvation during the premating and gestation periods, and during the lactation period for high-dose animals. The dosed rats had decreased weight gains compared to the control rats during the seven-day premating period. Absolute and relative maternal food consumption was significantly decreased for the 750 and 1500 mg/kg/day dose groups during the seven day premating period. No treatment related effects were reported on maternal body weight, changes in body weight, and absolute and relative feed consumption during the lactation period. No treatment related effect on mating performance or fertility was reported at any dose level. A significant increase in stillbirths and pup deaths was reported for the highest dose group compared to the control group. The treatment with sweet orange oil had no effect on the incidence of malformations or gross lesions in the pups. The NOAEL for this study was reported to be less than 375 mg/kg/day for maternal toxicity and 750 mg/kg/day for offspring development. Given the results of six developmental toxicity assays using limonene, sweet orange oil and beta-myrcene, it may be concluded that the substances within this chemical category exhibit low developmental toxicity potential. Key Study/Critical Effect for Screening Criteria The toxicity data on d-limonene will be used as read-across to CAS No The kidney changes seen in male rats administered limonene have been well characterized, and are known to be specific to the male rat and of no significance in human risk assessment. Thus, the male rat kidney effects will not be considered in the determining the critical effect for a drinking water guideline value. In the NTP two-year rat bioassay, the NOAELs for male and female rats were 150 and 300 mg/kg/day, respectively. In the NTP two-year mouse bioassay, the NOAELs for male and female mice were 250 and 500 mg/kg/day, respectively. 7

230 The lowest NOAEL is 150 mg/kg/day for male rats in the NTP 2-year bioassay based on liver effects. Adjusting for 5 days/week to 7 days/week, results in a NOAEL for continuous exposure of 107 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 107/100 = 1.1 mg/kg/day Drinking water guidance value = 3.8 ppm Reference U.S. EPA HPVIS database: 8

231 Toxicity Profile Tetrakis(hydroxymethyl)phosphonium sulfate (CAS No ) Toxicity information is provided on tetrakis(hydroxymethyl)phosphonium sulfate (THPS) and its structural analog tetrakis(hydroxymethyl)phosphonium chloride (THPC) Metabolism A metabolism study on rats has been conducted using 14 C-radiolabelled THPS. THPS was not found in rat urine. However, three metabolites were present, identified as trihydroxymethyl phosphine oxide, bishydroxymethylphosphonic acid and possibly a formaldehyde adduct of the trihydroxy compound (IPCS, 2000). Acute Toxicity No female mice died when given an oral dose of 200 mg/kg THPS (IPCS, 2000). The oral LD50 value of THPC (as a 75% solution) was reported to be 282 mg/kg in male rats; 575 mg/kg in male and female rats combined; 185 mg/kg in male rats; and 161 mg/kg in female rats (IPCS, 2000). No deaths occurred when rats were treated with THPS at 2,000 mg/kg (IPCS, 2000). The 4-hour LC50 in rats was 5.5 mg/l when exposed nose-only to a respirable aerosol (IPCS, 2000). Irritation THPS was not irritating to the skin of rabbits; however, repeated application resulted in severe skin irritation (IPCS, 2000). THPS is a severe eye irritant in rabbits (IPCS, 2000). Sensitization THPS is a skin sensitizer to guinea-pigs (IPCS, 2000). Repeat Dose Toxicity In a 13-week NTP oral gavage study, male and female F344/N rats were given 0, 5, 10, 20, 40 or 60 mg/kg THPS five days/week. Death occurred in the 60 mg/kg male rats (3/10) prior to the end of the study; all other rats survived to the end of the studies. Final mean body weights were 5%, 15%, and 22% lower than those of the vehicle controls for 1

232 males that received 20,40,or 60 mg/kg, and from 7% to 19% lower for all groups of dosed female rats. Diarrhea occurred in all groups of dosed rats during weeks 3 and 4. Vacuolar degeneration of the hepatocytes occurred in all males receiving >10 mg/kg, in all females receiving 40 or 60 mg/kg, and in 5/10 females receiving 20 mg/kg. The severity of this lesion was greatest in the 60 mg/kg group. In other groups, the severity was generally minimal to mild. Lymphoid depletion in the spleen or thymus was observed in the three males in the 60 mg/kg group which died before the end of the studies. Bone marrow hypoplasia was diagnosed in 3/10 male and 4/10 female rats in the 60 mg/kg groups. In a 13-week NTP oral gavage study, male and female B6C3F1 mice were given 0, 5, 10, 20, 40 or 60 mg/kg THPS 5 days/week. Females (1/10) that received 60 mg/kg and 2/10 males and 1/10 females that received 40 mg/kg died before the end of the studies. Final mean body weights of mice that received 20, 40, or 60 mg/kg were 4%, 7%, and 11% lower than those of the vehicle controls for males and 3%, 5%, and 11% lower for females. Periportal vacuolar degeneration occurred in all mice that received 60 mg/kg (minimal to moderate severity), 10/l0 male and 9/10 female mice that received 40 mg/kg, and 8/10 male mice that received 20 mg/kg (minimal to mild severity). In the 2-yr NTP study, male and female F344/N rats were given by oral gavage 0, 5, or 10 mg/kg THPS, 5 days/week for 103 to 104 weeks. Survival of male rats was reduced for the low dose (after week 102) and the high dose (after week 67) groups given THPS compared with that of the vehicle controls; survival at terminal kill was as follows: vehicle control, 28/50; low dose, 13/50; high dose, 16/50. Mean body weights of rats dosed with THPS were comparable to those of the vehicle controls. There was an increase in the incidence of hepatocellular lesions, primarily cytoplasmic vacuolization, which was dose-related. The incidence of these lesions were: in males, 2/50, 4/50, 9/49, at 0, 5, 10 mg/kg; in females, 1/49, 3/50, 8/49 at 0, 5, and 10 mg/kg. The incidences of mononuclear cell leukemia in 5 mg/kg male rats was somewhat greater than those in the vehicle controls (THPS: 30/50; 36/50; 20/50). This marginal increase in the incidences of hematopoietic system tumors was not considered related to chemical exposure, since they were significant only by the life table tests and were not dose related. The incidence of cystic degeneration of the liver was also increased in male rats (7/50, 15/50, and 14/49 at 0, 5, and 10 mg/kg). The LOAEL for this study is 5 mg/kg with no NOAEL. In the 2-yr NTP study, male and female B6C3F1 mice were given by oral gavage 0, 5, or 10 mg/kg THPS, 5 days/week for 103 to 104 weeks. There was no difference in survival or mean body weights between the vehicle controls and mice dosed with THPS. There was an increase in the incidence of hepatocellular lesions, primarily cytoplasmic vacuolization in female mice only, but it was not dose-related. The incidences of this lesion were: in males, 0/48, 1/49, 0/50 at 0, 5, and 10 mg/kg; in females, 6/50, 13/50, and 7/49 at 0, 5, and 10 mg/kg. The LOAEL for this study was 5 mg/kg, with no NOAEL. There was also focal hyperplasia of the adrenal medulla in the 10 mg/kg male mice. The 5 mg/kg male mice had an increased incidence of malignant lymphomas when compared with vehicle controls (2/50; 9/50; 0/50). This marginal increase in the incidences of 2

233 hematopoietic system tumors was not considered related to chemical exposure, since they were significant only by the life table tests and were not dose related. Genotoxicity Salmonella microsomal assays in the presence or absence of metabolic activation have shown uniformly negative results for THPS (IPCS, 2000). THPS induced forward mutations in mouse lymphoma L5178Y cells without metabolic activation; it was not tested in the presence of S9 (NTP, 1987). Another mouse lymphoma assay showed mutations both in the presence and absence of metabolic activation (IPCS, 2000). Increased chromosomal aberrations were detected at metaphase in CHO cells treated with THPS in the presence or absence of S9 from liver of Aroclor-treated rats (IPCS, 2000). Anaphase analysis of THPS-treated CHO cells also showed that chromosomal aberrations were produced along with abnormal spindles (IPCS, 2000). The results of an in vitro assay for unscheduled DNA synthesis in a primary culture of rat hepatocytes were negative (IPCS, 2000). THPS was administered to mice by gavage or dermal application or by mixing treated cloth with the animals feed. The urine of these dosed mice was analyzed for mutagenicity in the Salmonella microsome assay, and frequencies of micronuclei and chromosomal aberrations were evaluated in bone marrow cells. None of these investigations demonstrated mutagenic activity for THPS (NTP, 1987; IPCS, 2000). THPS was negative in rats and mice in dominant lethal assays (IPCS, 2000). Carcinogenicity Two-year NTP studies have been conducted in F344/N rats and B6C3F1 mice by administering 0, 5, or 10 mg/kg THPS by oral gavage, 5 days per week for 103 or 104 weeks (NTP, 1987). The incidences of mononuclear cell leukemia in the low dose male rats administered THPS was somewhat greater than those in the vehicle controls. Low dose male mice administered THPS had an increased incidence of malignant lymphomas when compared with vehicle controls (2/50; 9/50; 0/50). These marginal increases in the incidences of hematopoietic system tumors were not considered related to chemical exposure, since they were significant only by the life table tests and were not doserelated. Reproductive/Developmental Toxicity Pregnant CD rats were given by oral gavage 0, 15, 30 or 60 mg/kg THPS (as a 75% solution) during gestational days 6 to 15. Maternal body weight gain was significantly lower from gestational day 12 until the end of the study in the 60 mg/kg group. There 3

234 were minor signs of maternal toxicity in the 30 mg/kg group. The incidence of fetuses showing extra thoraco-lumbar ribs was significantly higher in the 60 mg/kg group compared to controls. The maternal and developmental NOAELs for this study were 30 mg/kg-day (IPCS, 2000). Pregnant New Zealand rabbits were given by oral gavage 0, 6, 18 or 60 mg/kg THPS (as a 75% solution) during gestational days 7 to 19. Maternal body weight gain was significantly lower in the 60 mg/kg group compared to the controls. There was increased incidence (42/120) of fetuses with eye malformation and some with additional hydrocephaly or limb/phalangeal reduction defects in the 60 mg/kg group. An increased incidence of specific skeleton variation was also observed. No adverse effects were noticed in the 6 or 18 mg/kg groups. The maternal and developmental NOAELs for this study is 18 mg/kg-day (IPCS, 2000). Key Study/Critical Effect for Screening Criteria The critical effect for a drinking water guideline value is liver effects in male and female rats and female mice at 5 mg/kg/day in the two-year study. To adjust for continuous exposure, the LOAEL is adjusted by converting 5 days/week to 7 days/week. LOAELadjusted = 5 * 5/7 = 3.6 mg/kg/day Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (LOAEL to NOAEL) Oral RfD = 4/1,000 = mg/kg/day Drinking water guideline: 0.01 ppm References IPCS (2000). Flame Retardants: Tris(2-butoxyethyl)phosphate, Tris (2-ethylhexyl) phosphate and tetrakis (hydroxymethyl)phosphonium Salts, Environmental Health Criteria 218, International Programme on Chemical Safety, United Nations Environment Programme, International Labour Organisation, World Health Organization. NTP (1987) NTP Technical Report on the Toxicology and Carcinogenesis Studies of Tetrakis(hydroxymethyl)phosphonium sulfate (THPS) (CAS No ) and Tetrakis(hydroxymethyl)phosphonium chloride (THPSC) (CAS No ) in F344/N Rats and B6C3F1 mice (Gavage Studies), NTP TR 296, NIH Publication No , National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. 4

235 F.3 HALLIBURTON CLEANSTIMAU APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES

236 Toxicity Profile Aluminum sulfate (CAS No ) This environmental assessment on aluminum sulfate has been extracted from the 2010 Priority Substances List Assessment Report. Follow-up to the State of the Science Report, 2000 for Aluminum Chloride, Aluminum Nitrate, Aluminum Sulphate, prepared by Environment Canada and Health Canada (Canada, 2010). The report can be downloaded from: Aluminum sulfate, also know as alum, has the chemical formula is Al2(SO4)3. It is often represented as Al2(SO4)3 14H2O. It may be found in different hydrated forms. The commercial product, called cake alum or patent alum, is an octadecahydrate aluminum sulfate, Al2(SO4)3 18H2O. Aluminum sulfate is used primarily as a coagulant and flocculant in water and wastewater treatment. Toxicokinetics Even at moderately elevated levels in the environment, exposure of aluminum leads to only small increases of aluminum in human tissues due to its low bioavailability through all routes of exposure. Acute Toxicity An oral LD50 in mice was reported to be 6,200 mg Al/kg for Al2(SO4)3,although it unclear whether the value refers to Al or to the salt (Sorenson et al., 1974). An oral LD50 was estimated to be >730 mg Al/kg in rats and mice in a 14-day study of oral and intraperitoneal administration (Llobet et al., 1987). Genotoxicity Comprehensive reviews of the genotoxicity data of various aluminum compounds are available (Krewski et al., 2007; ATSDR, 2008). Generally, aluminum compounds have not been mutagenic in a variety of in vitro genotoxicity studies. Aluminum chloride has been shown to induce micronuclei formation and sister chromatid exchange in human blood lymphocytes (Krewski et al., 2007). In a more recent study, aluminum chloride was cytotoxic, reduced the mitotic index and induced DNA damage in a comet assay and a chromosomal aberration assay conducted with cultured human 1

237 lymphoctyes (Lima et al., 2007). In an in vivo rat chromosomal aberration study, male rats dosed with 17 to 171 mg Al/kg in drinking water for 21 days showed increased chromosomal aberrations, characterized mostly by chromatid breaks, with translocations at the higher doses (Roy et al., 1991). EFSA (2008) has proposed that the genotoxic effects of aluminum occurs by an indirect mechanisms involving cross-linking of DNA with chromosomal proteins, interaction with microtubule assembly and mitotic spindle functioning, induction of oxidative damage, and damage of lysosomal membranes with liberation of DNAase to explain the induction of structural chromosomal aberrations, sister chromatid exchanges, chromosome loss and formation of oxidized bases in experimental systems. It was concluded that these effects occur at relatively high levels of exposure and would not likely be of relevance for humans exposed to aluminum via the diet. Carcinogenicity An increase in gross tumors was reported in male rats and female mice in a one-dose study; few study details were reported (Schroeder and Mitchener, 1975a,b; as reported in ATSDR 2008). Two other studies reported no increased incidence of tumors in rats and mice exposed orally to aluminum compounds (Hackenberg, 1972; Oneda et al., 1994). Subchronic/Chronic Toxicity Studies (Oral Exposure) Canada (2010) reviewed the results of 49 subchronic and chronic toxicity studies in adult mice, rats, rabbits, monkeys and dogs. Aluminum was administered orally in drinking water, in the diet or by gavage. The aluminum salts include lactate, chloride, sulfate, nitrate, hydroxide, citrate, maltolate, fluoride and KASAL (basic sodium aluminum phosphate). Many of the subchronic and chronic toxicity studies did not quantify the concentration of aluminum in the base diet. In those studies where the administered dose was substantially less than the possible baseline dose in the diet, the uncertainty associated with the actual combined dose was increased. Neurobehavioral effects were reported in adult mice and rats following oral exposure for 90 days or more. Decreased spontaneous motor activity was reported, with the lowest LOEL being 1 mg Al/kg-day in male Sprague-Dawley rats who received aluminum maltolate at this dose in drinking water over a period of one year (Huh et al., 2005). In contrast, Domingo et al. (1996) and Colomina et al. (2002) found no differences in field activity of Sprague-Dawley rats given 100 mg Al/kg-day of aluminum nitrate (with citrate) in drinking water for periods of four to six months. Other studies have reported decreased motor coordination as measured by performance in the rotarod test, decreased grip strength, and effects on temperature sensitivity and negative geotaxis. 2

238 Learning and memory deficits (maze performance, passive avoidance tests) have also been reported. The LOEL for these effects was 6 mg Al/kg-day in Long Evans rats exposed for 90 days to aluminum chloride (plus citrate) via gavage, although there was some ambiguity in the reporting of doses in this study (Bilkei-Gorzo, 1993). However, other investigators did not find effects on similar learning or memory tests. For instance, von Linstow Rolloff et al. (2002), reported no effects in male Lister hooded rats given 140 mg Al/kg as aluminum sulfate in drinking water. Histopathological effects reported in rats and mice included increased damaged or abnormal neurons in specific brain regions (e.g., cerebral cortex and hippocampus, neurofibrillary degeneration and vacuolization of nuclei, and vacuolated astrocytes and vacuolization of neuronal cytoplasm. The LOEL in which these effects were observed was <1 mg Al/kg-day in the Varner et al. (1998) and Varner et al. (1993) studies in which aluminum nitrate and sodium fluoride (to form aluminum fluoride) was administered in drinking water to male Long Evans rats for periods of 45 to 52 weeks. Petterson et al. (1990) observed mild to moderate histopathological effects in testes, liver and kidney, including hepatocyte vacuolization, seminiferous tubule germinal epithelial cell degeneration and tubular-glomerular nephritis in beagle dogs receiving a dose of 75 mg Al/kg-day of sodium aluminum phosphate. In this same study, no significant differences between exposure groups and controls were observed at the lower doses of 4 to 27 mg Al/kg-day. A number of biochemical effects have also been reported in subchronic and chronic studies. The LOEL associated with significant effects on biochemical endpoints was 1 mgal/kg-day as administered as aluminum maltolate in drinking water for one year (Huh et al., 2005). Reproductive/Developmental Toxicity Canada (2010) reviewed the results of 49 studies investigating gestational, lactational and/or post-weaning exposure of rats, mice and guinea pigs to aluminum salts through diet, through drinking water or by gavage. The aluminum salts administered in these studies included chloride, nitrate, sulfate, lactate and hydroxide. In a few studies, citrate or ascorbic acid was added to enhance absorption of aluminum. The lack of information on base diet for some studies is a major source of uncertainty with respect to the potential combined dose, particularly when the administered dose was low in comparison to the possible base diet dose. There is also uncertainty associated with reported LOELs that are of the same magnitude as the reported LD50 for the administered salt. The most commonly observed neurobehavioral effects in the developmental studies were decreased grip strength, reduced temperature sensitivity, reduced or delayed auditory startle responsiveness, and impaired negative geotaxis response. Decreased activity levels, locomotor coordination, as well as impaired righting reflex were also observed, although not consistently. The LOEL for these effects was 100 mg Al/kg-day, observed 3

239 in Wistar rats administered aluminum lactate in the maternal diet during gestation (Bernuzzi et al., 1989), as well as in Swiss Webster mice administered aluminum lactate in the maternal diet during gestation, lactation and then in the diet of offspring throughout the lifespan (Golub et al., 2000). The observations on the effects on learning and memory of developmental exposure to aluminum salts also varied considerably. Improved performance in the maze tasks was observed in some studies, while in others impaired performance or no change was found. Developmental exposure of mice and rats to aluminum salts also produced some evidence of disturbances in brain biochemistry. The LOEL for these biochemical effects was approximately 20 mg Al/kg-day (Kim 2003). Neurodevelopmental histopathological effects have been reported at doses of mg Al/kg-day. In the few reproductive toxicity studies, there was reduced maternal weight gain was reported, but not in others. Some of the studies reported decreased pup body weight, while other studies reported no effects. The LOEL for reproductive parameters, including fetal growth, was 13 mg Al/kg-day (Paternain et al., 1988; Domingo et al., 1987), in which Sprague-Dawley rat dams received this dose via gavage as aluminum nitrate. Other observed reproductive and developmental effects included decreased number of corpora lutea and number of implantation sites as well as skeletal malformations. Colomina et al. (2005) reported a delay in sexual maturation in both males and females, although this effect was produced at different dose levels in the two sexes (at 50 mg Al/kg-day in females and at 100 mg Al/kg-day in males). Misawa and Shigeta (1993) observed delayed pinna detachment and eye opening in female pups. No significant maternal or developmental toxicity, as measured by fetal weight gain, reproductive parameters or fetal malformations, was observed by McCormack et al. (1979) at a combined dietary dose of aluminum chloride of 50 mg Al/kg-day, nor by Gomez et al. (1990) where 265 mg Al/kg-day of aluminum hydroxide was administered to dams via gavage during gestation. Human Studies Neurotoxic effects have been reported in humans from aluminum exposure, as well as bone and blood toxicity, during medical treatment in which the gastrointestinal barrier is bypassed (e.g., aluminum-induced encephalopathy through dialysis treatment in patients with renal failure). Some epidemiological evidence exists for long-term cognitive impairment in pre-term infants receiving aluminum-containing nutritional solution intravenously and also associated with occupational exposures. These exposures are not applicable to the general population, although it does support the identification of neurotoxicity and developmental neurotoxicity as endpoints of concern in the human health risk assessment for aluminum. With respect to the conditions of exposure in the general population, the most relevant available information is provided by the epidemiological investigations into the 4

240 association between exposure to aluminum through drinking water and Alzheimer s disease (AD) and other forms of dementia. The hypothesis of aluminum in drinking water as a risk factor for AD or impaired cognitive function in the elderly is controversial in the scientific community. Recent reviews of the epidemiological literature have reiterated the limitations of the epidemiological data base, in its entirety, in regard to the causality of the occurrence of aluminum in the environment and AD, while also maintaining that the hypothesis cannot be rejected at this time (InVS-Afssa-Afssaps 2003; ATSDR 2008; JECFA 2006; Krewski et al., 2007). As a result of these limitations, JECFA (2006) and ATSDR (2006) chose not to base their regulatory values for aluminum intake on epidemiological studies. Critical Effect(s)/Basis for Screening Level Criteria The main focus on aluminum toxicity has been on neurotoxicity and reproductive and developmental toxicity, principally because of the evidence from human case studies and epidemiological studies indicating that these effects may be of concern. Canada (2010) evaluated a total of 138 toxicological studies, published from 1979 to 2007, reporting on neurotoxicity and reproductive/developmental effects of oral aluminum exposure in rodents, monkeys and dogs. No single or limited number of studies provided an adequate basis for characterizing the dose-response relationship. Thus, the entire database was considered in determining the lower range of doses for the adverse effects on the critical endpoints of concern. Exposure-response analysis Two subsets of the studies, based primarily on exposure period, were evaluated: (a) neurotoxic effects in adults following subchronic or chronic exposure (>90 days); and (b) neurodevelopmental and reproductive effects in prenatal/lactation exposure studies. These subsets include studies with highly diverse experimental conditions, notably with respect to the animal species and strain, type of aluminum salt administered, exposure vehicle as well as other aspects of the experimental methodology. There is also variability in the reporting of doses. Some researchers adjust the concentration in drinking water for a constant dose in mg Al/kg bw/d and report this value, while others estimate doses in terms of mg Al/kg bw/d based on measures of animal body weight and food and water intake, but keep the same concentration in the diet throughout the experiment. In other cases, the dose is reported only as a concentration administered via diet, drinking water or gavage, and the intake in mg Al/kg bw/d has been estimated using Health Canada (1994) reference values for animal body weight and intake. In the case of the developmental studies, the LOELs are reported as the maternal dose at the beginning of gestation. In the studies where the concentration in drinking water or the diet remained constant, this dose would generally be lower than the received dose, due to increased food and water intake during gestation and lactation. For the purpose of human health risk assessment, however, the maternal dose at the beginning of pregnancy was considered, as this provided a common point of comparison between studies. 5

241 One condition that was applied to both subsets of studies was that the experimental administered dose constitutes the principal contribution to total aluminum. As previously discussed, the concentration of aluminum in standard laboratory rodent chow may be significant. In the majority of studies, this base diet concentration is not measured. Base diet concentration would considerably impact the exposure-response analysis if: (a) the bioavailability of the aluminum contained in the chow was of a similar magnitude to the bioavailability of the administered aluminum; and (b) the lab chow were to contribute a large percentage of the total aluminum exposure. While it could be hypothesised that the aluminum in the lab chow, associated with ligands in the food matrix, would be less soluble and therefore less bioavailable than added aluminum, no experimental data were identified to assess the relative bioavailabilities of aluminum in lab chow and added aluminum salts. Therefore, with regard to those studies where base diet was not quantified, studies were included in the two subsets only if the administered dose (Da) likely exceeded the base diet dose (i.e., Da > 10 mg Al/kg-day for rats and Da > 30 mg Al/kg-day for mice). This approach limits the influence of the unknown base diet aluminum concentration on the exposure-response analysis, but does introduce a bias against inclusion of low dose studies in the exposure-response analysis. The following observations concerning the exposure-response relationship for aluminum may be made based on the studies selected for the exposure response analysis: (1) there is a wide variation in reported LOELs (from 1 to 663 mg Al/kg-day); (2) there is a predominance of single dose studies or studies where the LOEL was observed at the lowest dose; (3) for the 16 subchronic and chronic exposure studies for neurotoxicity in adults (neurobehavioral and biochemical endpoints), the LOELs range between 1 and 500 mg Al/kg-day (administered and combined doses Da and Dc considered together). (4) For the 22 studies of exposure during gestation and lactation (reproductive, developmental, neurodevelopmental, histopathological and biochemical endpoints), the LOELs (Da and Dc) vary between 2 and 663 mg Al/kg-day. The following discussion focuses particularly on studies documenting LOELs at the lowest doses, and evaluates the findings in relation to three issues: (a) use of a low administered dose; (b) toxicological significance of different endpoints; and (c) methodological strengths and limitations and consistency of study findings. (a) Use of a low administered dose The lowest LOEL was observed by Huh et al. (2005), which reported apoptosis and the activation of the catalytic activity of monoamine oxidases A and B in the brains of Sprague-Dawley rats at a reported combined dose of 1 mg Al/kg-day. The aluminumexposed group received aluminum maltolate in drinking water over a period of 12 months. This study reported an aluminum concentration of 11.5 ppm in the base diet. Although this is a relatively low value for laboratory chow, it does constitute an aluminum dose (0.6 mg Al/kg-day) of nearly twice that of the administered dose (0.38 mg Al/kg-day). The use of an administered dose less than the base diet dose raises the question of exposure misclassification of individual animals, as the normal variability in 6

242 intake between animals may create overlap between the two groups with respect to the dose received. This is considered to be a major limitation of this study. In spite of the extremely low administered dose, the animals receiving aluminum maltolate were found, after one year, to have approximately four times the amount of aluminum in the brain (462 ng/g) as compared to the controls (110 ng/g). This finding suggested a comparable increase in both the fraction of aluminum absorbed into the bloodstream and/or the amount of aluminum distributed to the brain when the aluminum is administered as the maltolate salt. Recently, Zhou et al. (2008) found differences in aluminum oral bioavailability, which were not statistically significant, between the citrate, maltolate and fluoride salts in drinking water. The measured bioavailabilities of all the salts were low (estimated means of 0.5%, 0.61% and 0.35% for maltolate, citrate and fluoride, respectively) and approximately twice the estimated bioavailability of aluminum in food (0.1% to 0.3%) as measured with the same experimental protocol. These findings suggest that while aluminum maltolate may be more bioavailable, the increase would not be sufficient to explain the results of Huh et al. (2006). In light of the uncertainty associated with the reported increased brain concentrations in the Huh et al. (2005) study, in addition to the methodological limitation of testing an administered dose that is less than the base diet dose, the study by Huh et al. (2005) was not retained for the purpose of estimating the lower range of aluminum doses at which neurological effects may be expected to occur. Other investigations with relatively low doses over periods of 12 weeks or longer have also reported neurotoxic effects. These studies were not considered in the exposureresponse analysis as the aluminum content in the laboratory chow was not reported, and thus, unlike the study by Huh et al. (2005), the relative contribution of the aluminum in the base diet could not be evaluated. The LOELs for these studies ranged from 0.07 to 22 mg Al/kg-day (administered dose) and were associated with a significant increase in brain aluminum levels as well as significant increases in neurobehavioral or histopathological effects. (b) Toxicological significance of different endpoints Considering the 16 subchronic and chronic adult exposure studies, the LOELs range between 19 and 500 mg Al/kg-day (administered and combined doses Da and Dc considered together, and excluding the Huh et al. (2005) study). For neurobehavioral endpoints (Morris water maze performance, impaired learning in the shuttle box and motor activity), the LOELs of the seven relevant studies vary between 40 to 500 mg Al/kg-day (Da and Dc), with four studies having LOELs at Das of 40 to 70 mg Al/kg-day (Commissaris et al. 1982; Lal et al. 1993; Gong et al. 2005; Mameli et al. 2006). The neurobehavioral endpoints examined constitute standard elements of neurobehavioral testing and impaired performance is considered to be toxicologically significant in the experimental animal. The biochemical effects observed in the remaining studies included alterations in 7

243 neurotransmission systems, alterations in synaptosomal membrane fluidity and increased lipid peroxidation in the brain, and were associated with LOELs varying from 19 to 420 mg Al/kg-day. These observations provide supportive evidence for neurotoxicity observed via other endpoints as well as information on mechanisms of action, but are more difficult to evaluate with respect to toxicological significance. For this reason, studies with these endpoints were given less weight in the exposure-response evaluation, in comparison to studies that include neurobehavioral endpoints. Considering the 22 studies of exposure during gestation and lactation, the LOELs (Da and Dc) varied between 29 and 663 mg Al/kg-day. For neurobehavioural endpoints (grip strength, auditory startle, negative geotaxis and other reflexes, maze learning and thermal sensitivity, and motor development), the LOELs (administered doses) ranged from 50 to 155 mg Al/kg-day, with the LOELs of two studies falling in the range of 50 to 60 mg Al/kg-day (Colomina et al. 2005; Golub and Germann 2001b). With respect to reproductive parameters, the lowest LOEL was reported by Belles et al. (1999), where aluminum nitrate was administered to pregnant mice via gavage at a dose of 29 mg Al/kg-day and observed an increase in the number of early deliveries and reduced fetal body weight. Reduced birth or fetal weight was also observed by Colomina et al. (1992) and Sharma and Mishra (2006) at LOELs ranging between 50 and 70 mg Al/kg-day. Morphological effects in offspring were also observed in the latter two studies. The motor, reflex and learning endpoints examined in the developmental studies as well as the reproductive parameters of fetal growth and morphological variations are all standard endpoints included in neurodevelopmental testing procedures, and considered to be toxicologically significant. (c) Evaluation of methodology and consistency of results in studies with LOELs of <70 mg Al/kg-day. The methodologies and findings of the abovementioned studies with LOELs of less than 70 mg Al/kg-day for neurobehavioral or reproductive/developmental endpoints were compared in order to characterize the strength of evidence for the effects observed at these dose levels. Adult Neurobehavioral Effects Of the four neurobehavioral studies in adults, all were carried out in rats using aluminum chloride, in drinking water (Gong et al. 2005; Mameli et al. 2006; Lal et al. 1993), or in the diet (Commissaris et al. 1982), for periods varying between 90 days and 11 months. Several weaknesses were identified in the investigations of Commissaris et al. (1982) and Gong et al. (2005). First, exposure information in these two reports was expressed as concentrations in the food or drinking water, and no information was included on intake rates or body weight of the animals. Thus the administered doses (50 and 60 mg Al/kg- 8

244 day, respectively) were calculated on the basis of default intake and body weight values (refer to Health Canada 1994), and are therefore associated with greater uncertainty than had the doses been reported by the researchers on the basis of experimental observations. Moreover, the concentration of aluminum in the base diet was not reported in the two studies, and so the combined dose could not be calculated. The investigations of Commissaris et al. (1982) and Gong et al. (2005) were also limited by the use of a single aluminum dose and the absence of a group receiving sodium chloride. Thus, a dose-response relationship could not be examined, and the observed effects could not be definitively attributed to the aluminum ion. It should be added that these two investigations were carried out with the primary objective of examining the influence of other test substances on aluminum toxicity parathyroid hormone and Ginkgo biloba leaf extract, respectively and not for the purpose of evaluating aluminum toxicity at different dose levels for different endpoints. In the study of Lal et al. (1993), adult male Druckrey albino rats were exposed to an administered dose of 52 mg Al/kg-day for 180 days in drinking water. Although this dose was not reported directly in this form, information on daily water consumption and average body weight was provided, allowing for calculation of the dose based on experimental data. The investigation included a range of behavioral, biochemical and histopathological endpoints. The researchers observed reduced spontaneous motor activity and impaired learning in the shuttle box and maze tests, in addition to increased lipid peroxidation and decreased Mg2+ and Na+K+-ATPase activities in the brain. The aluminum concentration in different brain regions was significantly increased in the aluminum-exposed animals, but no pathological alterations were observed. In the context of evaluating the exposure-response relationship, the study by Lal et al. (1993) is more informative than the Commissaris et al. (1982) and Gong et al. (2005) studies, in that the dose is more accurately reported, brain aluminum content was measured and a range of endpoints were examined, with generally consistent findings reported for the different endpoints. Its limitations include the use of a single dose, the absence of a group exposed to sodium chloride and the lack of information on the aluminum concentration in the base diet. Assuming a concentration of 250 ppm of aluminum in the laboratory chow (ATSDR 2008), the corresponding approximate aluminum dose would be 13 mg Al/kg-day, leading to an estimated combined dose for the Lal et al. (1993) study of 65 mg Al/kg-day. It should be noted that NOELs for impaired learning in the maze and shuttle box tests in aluminum-exposed adults have been observed at doses of 100 and 140 mg Al/kg-day, respectively by Domingo et al. (1996) and VonLinstow Roloff et al. (2002). In the study by Domingo et al. (1996) the aluminum was administered to rats as Al nitrate, with added citrate, in drinking water for a period of 6.5 months. Von Linstow Roloff (2002) administered Al sulfate in drinking water to rats for a period of seven months. Of these four studies, only Mameli et al. (2006) included more than one dose group, and were thereby able to establish a LOEL of 43 mg Al/kg-day and a NOEL of 22 mg Al/kg- 9

245 day. At this administered dose the researchers found impairment of the vestibulo-ocular reflex in male rats of different ages (3, 10 and 24 months old) exposed to aluminum chloride in drinking water. Significant increases of aluminum were observed in brain regions (brainstem-cerebellum and cerebrum). This study, which used 20 animals per dose per age group, also included an exposure group for the salt, in this case sodium chloride, such that the observed effects could be more clearly attributed to the aluminum and not the chloride ion. It should be noted, however, that evidence from other studies supporting the effects of aluminum on the vestibulo-ocular reflex is not available, as this endpoint has not been evaluated by other researchers. In the study by Mameli et al. (2006), the base diet aluminum concentration was measured but not clearly reported, nor was food intake measured. The LOEL of 43 mg Al/kg-day is thus the administered dose. The combined dose may be estimated at approximately 50 mg Al/kg-day, based on default values for rat dietary intake. Considering the observations of LOELs and NOELs associated with neurobehavioral effects in adults as well as the probable combined doses, alterations in learning and reflexes may be observed at approximately 50 to 65 mg Al/kg-day, based on the LOELs of Mameli et al. (2006) and Lal et al. (1993) expressed as estimated combined dose. Reproductive Effects The lowest LOEL presented is from the study by Belles et al. (1999). Mice were exposed to aluminum nitrate via gavage from gestational day 6 to 15 at a dose of 29 mg Al/kgday. In addition to the control group, one group received sodium nitrate at a similar nitrate dose. A high mortality (52%) in the aluminum-exposed pregnant mice was observed in this study, which was not observed in other developmental studies in which aluminum nitrate or other aluminum salts were administered at similar or greater doses. Other observations included reduced body weight gain in the dams during gestation and reduced fetal body weight. The number of early deliveries was also increased in the aluminum-exposed animals as compared to the control group, but there was no significant difference in this regard when compared to the sodium nitrate-exposed group. This study is limited to a single dose, and the aluminum content in the base diet was not measured. The lack of information on base diet is particularly important in studies with mice because of their small body weight. A laboratory chow containing 250 ppm of aluminum would be equivalent to a dose of approximately 33 mg Al/kg-day, which is higher than the administered dose in this investigation. Reduced maternal body weight gain and reduced fetal weight in aluminum-exposed animals were also observed at the LOELs associated with the Sharma and Mishra (2006) and Colomina et al. (1992) studies. A significant reduction in pup weight was also observed at the higher doses tested in the studies of Golub and Germann (2001b) and Colomina et al. (2005), at approximately 100 mg Al/kg-day. In the study by Sharma and Mishra (2006), rats received 70 mg Al/kg-day as aluminum chloride via gavage during gestation and lactation. In addition to the effects on fetal 10

246 weight, the authors observed an increase in skeletal malformations and in oxidative stress in the brains of mothers, fetuses and sucklings. The dose level in this study is based on the measured maternal weights. However, no information on base diet was included. The combined dose, based on a concentration of 250 ppm of aluminum in a typical lab chow and default values of Health Canada (1994), is estimated at approximately 83 mg Al/kgday. Colomina et al. (1992) administered aluminum lactate to mice through gavage. A LOEL of 57.5 mg Al/kg-day (administered dose) was observed for an increased incidence of morphological effects (cleft palate, delayed ossification of parietals), in addition to reduced fetal weight. This study did not report the aluminum content in the base diet. Considering the reported concentration in the laboratory chow used by this research group in other experiments of 42 ppm of aluminum, the estimated base diet dose would be approximately 5.5 mg Al/kg-day, based on Health Canada (1994) default values for body weight and food intake in mice. The combined dose would then be estimated at 63 mg Al/kg-day. In contrast to the findings mentioned above, in the study of McCormack et al. (1979), rats were fed aluminum chloride in the diet at maternal dose levels of 25 and 50 mg Al/kgday during gestation, and no differences in fetal growth or skeletal anomalies were observed. Colomina et al. (1994) found no differences in dam body weight, fetal growth or morphological variations in mice exposed via gavage to 104 mg Al/kg-day of aluminum hydroxide, during gestation. The latter finding may have resulted from the lower solubility and therefore the lower bioavailability of the hydroxide salt. Considering the observations of LOELs and NOELs associated with reproductive effects, and the probable combined doses, reductions in fetal and pup body weight may be observed beginning at approximately 60 mg Al/kg-day (e.g., Colomina et al. (1992)). The study of Belles et al. (1999), in which a LOEL of 29 mg Al/kg-day was observed for reduced fetal weight, is given less weight in this evaluation, in light of the uncertainty associated with the high maternal mortality rate observed in the exposed animals, and the elevated contribution of the base diet to aluminum exposure as compared to the administered dose. Neurodevelopmental Effects The lowest LOELs are from the studies of Colomina et al. (2005) and Golub and Germann (2001b). Both of these studies included exposure through gestation and lactation. The experimental conditions of the two studies, however, differed in many other respects, and these are described briefly below. Colomina et al. (2005) exposed female Sprague-Dawley rats to 0, 50, or 100 mg Al/kgday as aluminum nitrate in drinking water with citric acids, in combination with a base diet dose of approximately 3 mg Al/kg-day. Aluminum exposure was maintained through gestation, lactation and the life of the dams. The maternal effects of aluminum administration included decreased food intake (with reduced body weight) during gestation and lactation and decreased water intake during lactation in the 100 mg Al/kg- 11

247 day dose group. No effects were observed with respect to the length of gestation, the number of litters or the number of fetuses per litter. With respect to the pups, there was a significant increase in the number of days until sexual maturation in males in the 100 mg Al/kg-day dose group and in females at both 50 and 100 mg Al/kg-day. A significant reduction in forelimb grip strength in males was observed in the 100 mg Al/kg-day dose group on PND 11 compared controls. In the water maze task, assessing spatial learning, the performance of aluminum treated rats (50 mg Al/kg-day) was significantly improved in comparison to the control group. The pups in the 100 mg Al/kg-day dose group were not tested in the water maze test, because of altered maternal food and water intakes in this group. No differences in aluminum-exposed animals were observed with respect to surface righting, negative geotaxis or activity in an open field. The authors also measured aluminum concentration in brain regions but did not find increased levels in any regions in the aluminum-exposed animals. Golub and Germann (2001b) investigated the long-term consequences of prenatal exposures to aluminum in Swiss Webster mice, in conjunction with a suboptimal base diet. The base diet was designed to simulate the usual diet of young women in the U.S., with respect to estimated phosphate, calcium, iron, magnesium, and zinc intakes. Following breeding, dams were exposed to aluminum in the diet as aluminum lactate. The doses were equivalent to approximately < 1, 10, 50 and 100 mg Al/kg-day, as estimated at the beginning of gestation. The dams were exposed throughout gestation and lactation. Following weaning at 21 days, the pups were fed the same diet as the dams for two weeks (although the per kg dose levels were higher). No effects were observed in the number of dams completing pregnancy, gestation length, weight gain of the dams (GD0 to GD15), litter size or birth weight. By weaning, both males and females in the two highest dose groups weighed significantly less than the controls, although by PND35 only the highest dose group showed this effect. The female offspring of the highest dose group (maternal exposure of 100 mg Al/kg-day) were found to be slower in maze learning at three months old, as indicated by longer latencies during the first three sessions of the four-session learning series. All aluminum treated groups were similar to controls by the fourth session. Differences in aluminum exposed groups were also observed in the cue relocation trials, in which average trial latency was significantly increased at the two highest dose levels (50 and 100 mg Al/kg-day) as compared to the control group. In the motor testing of male offspring at five months old, males in the highest dose group (maternal exposure of 100 mg Al/kg-day) had significantly lower hindlimb grip strength and greater number of rotations in the rotarod test (animal losing footing). When body weight was taken into account, only the findings for the rotarod test remained significant. The investigations by Colomina et al. (2005) and Golub and Germann (2001b) are methodologically superior in many respects to the majority of the studies. Both include two dose levels in addition to the control group, quantify the aluminum dose associated with the base diet, and examine a range of reproductive and neurodevelopmental endpoints. The Colomina et al. (2005) study includes measurement of aluminum concentration in different brain regions. The Golub and Germann (2001b) study, however, used an experimental protocol designed to test the influence of a suboptimal 12

248 diet, which limits comparisons of the findings with other investigations of aluminum toxicity, particularly as no groups were included with equivalent aluminum dose levels and a standard diet. Interpretation of cognitive and motor test findings in the studies investigating the effects of aluminum exposure is also complicated by a possible biphasic dose-response relationship. For example, in the study by Roig et al. (2006), rats received aluminum nitrate in drinking water during gestation and lactation at administered doses of 50 and 100 mg Al/kg-day. No difference in the motor activity of aluminum-exposed pups and controls was found. However, the animals exposed to 50 mg Al/kg-day showed an improved performance in maze learning. The performance of animals exposed to 100mgAl/kg-day was significantly reduced as compared to the animals exposed to 50 mg Al/kg-day, but not significantly different from controls. Colomina et al. (2005) also observed improved maze performance in aluminum-exposed animals, although the highest exposure group in that study was not tested for this endpoint. Considering the neurodevelopmental studies described above, diminished performance in learning or motor tests may be observed in animals exposed prenatally or through lactation at maternal combined doses beginning at approximately 50 mg Al/kg-day. There is, however, considerable variability in various study results with respect to these endpoints, which also suggest a possible biphasic dose-response relationship in relation to maze learning. Conclusion A dose of 50 mg Al/kg-day, expressed as a combined dose of total aluminum, was considered the level at which neurological and reproductive/developmental effects begin to be repeatedly observed in animal studies. While 50 mg Al/kg-day is an estimation of the lower end of a broad range of LOELs observed under different experimental conditions, it is not considered to be an overly conservative estimate of the effect level of concern. There are two sources of bias against consideration of lower values of LOEL in the above characterization: (a) low-dose studies were not considered if the administered dose was less than the probable base diet dose; and (b) LOELs from single-dose studies may be overestimates of the actual effect levels. Thus, 50 mg Al/kg-day has, however, produced neurotoxic, reproductive and developmental effects in laboratory animals more consistently under a wide range of experimental conditions, as compared to lower doses. This exposure level is therefore retained for the purpose of the characterization of human health risks as the level of concern for neurotoxic, neurodevelopmental and reproductive effects. LOEL [Al] = 50 mg/kg/day Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (LOEL to NOAEL) Oral RfD [Al] = 50/1,000 = 0.05 mg/kg/day 13

249 Drinking water guideline value [Al] = 0.18 ppm Australian drinking water guideline [sulfate] = 500 ppm (health); 250 ppm (aesthetic) References ATSDR (2008). Toxicological Profile for Aluminum, U.S. Dept. of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry. Belles, M., Albina, M.L., Sanchez, D.J., and Domingo, J.L. (1999). Lack of protective effects of dietary silicon on aluminium-induced maternal and developmental toxicity in mice. Pharmacol. Toxicol. 8: 1-6. Bernuzzi, V., Desor, D., and Lehr, P.R. (1989). Developmental alternations in offspring of female rats orally intoxicated by aluminum chloride or lactate during gestation. Teratology 40: Bilkei-Gorzo, A. (1993). Neurotoxic effect of enteral aluminium. Food Chem. Toxicol. 31: Canada (2010) Priority Substances List Assessment Report. Follow-up to the State of the Science Report, 2000 for Aluminum Chloride, Aluminum Nitrate, Aluminum Sulphate, prepared by Environment Canada and Health Canada (Canada, 2010), Colomina, M.T., Gomez, M., Domingo, J.L., Llobet, J.M., and Corbella, J. (1992). Concurrent ingestion of lactate and aluminum can result in developmental toxicity in mice. Res. Commun. Chem. Pathol. Pharmacol. 77: Colomina, M.T., Gomez, M., Domingo, J.L., and Corbella, J. (1994). Lack of maternal and developmental toxicity in mice given high doses of aluminium hydroxide and ascorbic acid during gestation. Pharmacol. Toxicol. 74: Colomina, M.T., Roig, J.L., Sanchez, D.J., and Domingo, J.L. (2002). Influence of age on aluminum- induced neurobehavioral effects and morphological changes in rat brain. Neurotoxicology 23: Colomina, M.T., Roig, J.L., Torrente. M., Vicens, P., and Domingo, J.L. (2005). Concurrent exposure to aluminum and stress during pregnancy in rats: Effects on postnatal development and behavior of the offspring. Neurotoxicol. Teratol. 27: Commissaris, R.L., Cordon, J.J., Sprague, S., Keiser, J., Mayor, G.H., and Rech, R.H. (1982). Behavioral changes in rats after chronic aluminum and parathyroid hormone administration. Neurobehav. Toxicol. Teratol. 4:

250 Domingo, J.L., Llorens, J., Sanchez, D.J., Gomez, M., Llobet, J.M., and Corbella J. (1996). Age-related effects of aluminum ingestion on brain aluminum accumulation and behavior in rats. Life Sci. 58: Domingo, J.L., Paternain, J.L., Llobet, J.M., and Corbella J. (1987). Effects of oral aluminum administration on perinatal and postnatal development in rats. Res Commun Chem. Pathol. Pharmacol. 57: [EFSA] European Food Safety Authority. (2008). Safety of aluminium from dietary intake. Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Food Contact Materials (AFC). The EFSA Journal 754: 1-4. Golub, M.S., and Germann, S.L. (1998). Aluminum effects on operant performance and food motivation of mice. Neurotoxicol. Teratol. 20: Golub, M.S., Germann, S.L., Han, B., and Keen, C.L. (2000). Lifelong feeding of a high aluminum diet to mice. Toxicology 150: Gomez, M., Bosque, M.A., Domingo, J.L., Llobet, J.M., and Corbella, J. (1990). Evaluation of the maternal and developmental toxicity of aluminum from high doses of aluminum hydroxide in rats. Vet. Hum. Toxicol. 32: Gong, Q.H., Wu, Q., Huang, X.N., Sun, A.S., Nie, J., and Shi, J.S. (2006). Protective effect of Ginkgo biloba leaf extract on learning and memory deficit induced by aluminum in model rats. Chin. J. Integr. Med. 12: Hackenberg, U. (1972). Chronic ingestion by rats of standard diet treated with aluminum phosphide. Toxicol. Appl. Pharmacol. 23: Health Canada. (1994). Human Health Risk Assessment for Priority Substances. Huh, J.W., Choi, M.M., Lee, J.H., Yang, S.J., Kim, M.J., Choi, J., Lee, K.H., Lee, J.E., and Cho, S.W. (2005). Activation of monoamine oxidase isotypes by prolonged intake of aluminum in rat brain. J. Inorg. Biochem. 99: [InVS-Afssa-Afssaps] Institut de veille sanitaire-agence française de sécurité sanitaire des aliments-agence française de sécurité sanitaire des produits de santé Évaluation des risques sanitaires liés à l'exposition de la population française à l'aluminium: eaux, aliments, produits de santé p. Kim, K. (2003). Perinatal exposure to aluminum alters neuronal nitric oxide synthase expression in the frontal cortex of rat offspring. Brain Res. Bull. 61: Krewski, D., Yokel, R.A., Nieboer, E., Borchelt, D., Cohen, J., Harry, J., Kacew, S., Lindsay, J., Mahfouz, A.M., and Rondeau, V. (2007). Human health risk assessment for 15

251 aluminium, aluminium oxide, and aluminium hydroxide. J. Toxicol. Environ. Health B Crit. Rev.10 (Suppl 1): Lal, B., Gupta, A., Gupta, A., Murthy, R.C., Ali, M.M., and Chandra, S.V. (1993). Aluminum ingestion alters behaviour and some neurochemicals in rats. Indian J. Exp. Biol. 31: Lima, P.D., Leite, D.S., Vasconcellos, M.C., Cavalcanti, B.C., Santos, R.A., Costa- Lotufo, L.V., Pessoa, C., Moraes, M.O., and Burbano, R.R. (2007). Genotoxic effects of aluminum chloride in cultured human lymphocytes treated in different phases of cell cycle. Food Chem. Toxicol. 45: Llobet, J.M., Domingo, J.L., Gómez,M., Tomás, J.M., and Corbella, J. (1987) Acute toxicity studies of aluminum compounds: antidotal efficacy of several chelating agents. Pharmacol. Toxicol. 60: Mameli, O., Caria, M.A., Melsi, P., Zambenedetti, P., Ramila, M., and Zatta, P. (2006). Effect of aluminum consumption on the vestibule-ocular reflex. Metab. Brain Dis. 21: McCormack, K.M., Ottosen, L.D., Sanger, V.L., Sprague, S., Mayor, G.H., and Hook J.B. (1979). Effects of prenatal administration of aluminum and parathyroid hormone on fetal development in the rat. Soc. Exp. Biol. Med. 161: Misawa, T., and Shigeta, S. (1993). Effects of prenatal aluminum treatment on development and behavior in the rat. J. Toxicol. Sci. 18: Oneda, S., Takasaki, T., Kuriwaki, K., Ohi, Y., Umekita, Y., Hatanaka, S., Fujiyoshi, T., Yoshida, A., and Yoshida, H. (1994). Chronic toxicity and tumorigenicity study of aluminum potassium sulfate in B6C3F1 mice. In Vivo 8: Pettersen, J.C., Hackett, D.S., Zwicker, G.M., and Sprague, G.L. (1990). Twenty-six week toxicity study with Kasal (basic sodium aluminum phosphate) in beagle dogs. Environ. Geochem. Health 12: Roig, J.L., Fuentes, S,, Teresa Colomina, M., Vicens, P., and Domingo, J.L. (2006). Aluminum, restraint stress and aging: behavioral effects in rats after 1 and 2 years of aluminum exposure. Toxicology 218: Roy, A.K., Sharma, A., and Talukder, G. (1991). Effects of aluminum salts on bone marrow chromosomes in rats in vivo. Cytobios 66: Sharma, P., and Mishra, K.P. (2006). Aluminum-induced maternal and developmental toxicity and oxidative stress in rat brain: response to combined administration of Tiron and glutathione. Reprod. Toxicol. 21:

252 Sorensen, J.R., Campbell, I.R., Tepper, L.B., and Lingg, R.D. (1974). Aluminum in the environment and human health. Environ. Health Perspect. 8: Varner, J.A., Huie, C.W., Horvath, W., Jensen, K.F., and Isaacson, R.L. (1993). Chronic ALF, Administration: II Selected histological observations. Neurosci. Res. Commun. 13: Varner, J.A., Jensen, K.F., Horvath, W., and Isaacson, R.L. (1998). Chronic administration of aluminum- fluoride or sodium-fluoride to rats in drinking water: alterations in neuronal and cerebrovascular integrity. Brain Res. 784: von Linstow Roloff, E., Platt, B., and Riedel, G. (2002). Long-term study of chronic oral aluminum exposure and spatial working memory in rats. Behav. Neurosci. 116: Zhou, Y., Harris, W.R., and Yokel, R.A. (2008). The influence of citrate, maltolate and fluoride on the gastrointestinal absorption of aluminum at a drinking water-relevant concentration: a 26 Al and 14 C study. J. Inorg. Biochem. 102:

253 Toxicity Profile Cellulase (CAS No ) Enzymes are catalytic proteins or polypeptides and consist of amino acid residues coupled via peptide bonds. They are primarily characterized by their biocatalytic or enzymatic activity. There is a broad range of detergent cellulases used both from fungal as well as from bacterial origin; all are characterized by ß-1,4-endoglucanase activity. Acute Toxicity The oral LD50 of a cellulase enzyme was determined to be 8,000 mg/kg for mice and >10,000 mg/kg for rats (HERA, 2005). The inhalation LC50 of the same enzyme in rats is >3.48 mg/l, with a mass mean diameter of 7.8 μm and a geometric standard deviation of 2.1 μm (HERA, 2005). Irritation Cellulase enzymes are mild to non-irritating to the skin and eyes of rabbits (HERA, 2005). The amount of protease contamination in the enzyme test preparation, however, may affect the irritation potential, particularly with regards to eye irritation. Sensitization A cellulase enzyme was not a skin sensitizer to guinea pigs (HERA, 2005). Repeated Dose Toxicity Male and female rats were dosed by oral gavage with 0, 300, 1,000 or 3,000 mg/kg of a cellulase enzyme for 4 weeks. There were no treatment-related clinical signs, but a slight dose-related increase in body weight gain (approx. 7%) was seen in the males. No treatment-related changes were noted in the hematology or clinical chemistry. There were no gross or histopathologic changes that were considered treatment-related (HERA, 2005). Male and female rats were given in their feed concentrations of 0, 120, 600 or 3,000 mg/kg of this enzyme for 13 weeks. There were no treatment-related clinical signs. A reduction in body weight gain was seen in both sexes at the 3,000 mg/kg level (10% in males and 20% in females). A slight reduction in food consumption was also noted for the 3,000 mg/kg dose group. Serum alkaline phosphatase activity was increased in the 600 and 3,000 mg/kg females in a dose-dependent manner. However, there were no organ 1

254 weight changes and no effects seen in the gross or histopathologic examination that were considered treatment-related. The NOAEL for this study is considered to be 600 mg/kg/day (HERA, 2005). Genotoxicity In an in vitro bacterial mutation assay, a cellulase enzyme was not mutagenic to Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA98 in the presence or absence of metabolic activation. In an in vivo bone marrow micronucleus assay, a cellulase enzyme was not genotoxic when given to rats by oral gavage (HERA, 2005). Carcinogenicity No data were found. Reproductive Toxicity No data were found. Developmental Toxicity No data were found. Key Study/Critical Effect for Screening Criteria In a 13-week dietary study, there was reduced body weight gain in rats given 3,000 mg/kg of this enzyme. Although there was a dose-related increase in serum alkaline phosphatase activity, there were no significant changes to liver weights, nor were there any accompanying gross or histopathologic changes in the liver. Thus, the changes in serum alkaline phosphatase activity were not considered an adverse effect and the NOAEL for this study is 600 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral Reference Dose = 600/1,000 = 0.6 mg/kg/day Drinking water guideline value = 2.1 ppm 2

255 Reference Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: α-amylase, CAS No ; Cellulase (β-(1,4)-glucanase), CAS No ; Lipase, ,

256 Toxicity Profile Lactose (CAS No ) Lactose is a disaccharide sugar composed of galactose and glucose. It is naturally synthesized in the mammary gland of virtually all mammals, occurring in milk at levels between 1 and 7%. Commercially, lactose is extracted from the whey of cow s milk (whey is the residual liquid of milk following cheese and casein production) and is purified extensively to remove fats, proteins, vitamins and minerals (Baldrick and Bamford, 1997; Goodhart, 1994). Pharmacokinetics/Metabolism Lactose per se is not readily absorbed by the oral route. After dietary intake, lactose is readily hydrolyzed to galactose and glucose by the enzyme lactase (Hansen, 1974; Williams, 1986). Lactase is a β-galactosidase found in the brush border of the intestinal epithelium. Once hydrolyzed, the monosaccharides are then readily absorbed. Lactose can also be hydrolyzed by other enzymes, such as acid β-galactosidase. Acute Toxicity The acute oral LD50 in rats is >10,000 mg/kg (Baldrick and Bamford, 1997). Irritation No data were located. Sensitization No data were located. Repeated Dose Toxicity/Carcinogenicity Wistar rats were fed 20% lactose for 12 weeks before mating, and during pregnancy and lactation. At weaning, the animals were fed the same diet and the study was terminated at week 130. There were low body weights; increased blood alkaline phosphatase and urinary calcium; decreased ph of cecum contents; enlargement/increased weight of the cecum; pelvic nephrocalcinosis of the kidneys; increased proliferation of the bile ducts; adrenal medullary hyperplasia and pheochromocytomas; and Leydig cell tumors (Sinkeldam et al., 1992a). 1

257 Similar findings were also observed in another study in which rats were fed 20% lactose in their diet for 130 weeks. Findings included loose feces (week one and two only); decreased ph of the feces; low body weights; increased food and water consumption; increased blood alkaline phosphatase and decreased bicarbonate; increased calcium concentration and excretion; reduced urinary ph; increased cecum weight; mineralization of the renal pelvis; increased incidence of basophilic cell foci in the adrenal medulla; and Leydig cell hyperplasia (De Groot et al., 1995). Genotoxicity Lactose was negative in an in vitro chromosomal aberration study with cultured human lymphocytes in the presence and absence of metabolic activation (Sinkeldam et al., 1992a). Reproductive/Developmental Toxicity In a multi-generation study, rats were fed 20% lactose over three successive generations. There were no adverse effects on fertility or reproductive performance. There was a slight decrease in the number of pups born alive, litter size at birth, viability index and pup weight for F0 and F1 offspring. There were signs of low body weights in the parental animals, and enlargement and increased weight of the cecum (Sinkeldam et al., 1992b). Pregnant mice were given 10,000 mg/kg lactose during gestation days No maternal toxicity was observed and there were no effects on litter size, body weight on days 1 and 3, or neonate survival (Seidenberg et al., 1986). Key Study/Critical Effect for Screening Criteria Many of the findings from the oral studies on lactose can be related to the fact that it is poorly absorbed from the small intestine. This results in a large proportion reaching the cecum and colon where it is readily fermented by the intestinal microflora. Consequently, there is increased production of volatile fatty acids and lactic acid, and a lowering of the ph and increased osmotic pressure. These changes facilitate calcium absorption. The adrenal tumors are thought to be secondary to increased calcium absorption from the intestine. Increased Leydig cell tumors in rats are of questionable relevance to humans. Adverse effects were noted in rats given 20% in their diet for 130 weeks. Since there was only one dose level, a NOAEL was not identified. LOAEL (mg/kg/day) = 20,000 ppm * 0.05 = 1,000 mg/kg/day 2

258 Where 0.05 is the fraction of body weight that is consumed per day as food for the mouse (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (LOAEL to NOAEL) Oral Reference Dose = 1,000/1,000 = 1.0 mg/kg/day Drinking water guideline value = 3.5 ppm References Baldrick, P., and Bamford, D.R. (1997). A toxicological review of lactose to support clinical administration by inhalation. Food Chem. Toxicol. 35: DeGroot, A.P., Lina, B.A.R., Hagenaars, A.J.M., Hollanders, V.M.H., Andringa, M., and Feron, V.J. (1995) Effects of a dietary load of acid or base on changes induced by lactos in rats. Food. Chem. Toxicol. 33: Goodhart, F.W. (1994). Lactose. In: handbook of Pharmaceutical Excipients, 2 nd Edition, pp , The Pharmaceutical Press, London. Hansen, R.G. (1974) Metabolism of lactose and galactose. In: Nutrition Foundation Monograph Series. Sugars in Nutrition, Edited by H.L. Sipple and K.W. McNutt, pp , Academic Press, New York. Seidenberg, J.M., Anderson, D.G., and Becker, R.A. (1986). Validation of an in vivo developmental toxicity screen in the mouse. Teratogen. Carcinogen. Mutagen. 6: Sinkeldam, E.J., Woutersen, R.A., Hollanders, V.M.H., Til, H.P., Van Garderen- Hoetmwer, A., and Bär, A. (1992a). Subchronic and chronic toxicity/carcinogenicity feeding studies with lactitol in rats. J. Amer. Coll. Toxiciol. 11: Sinkeldam, E.J., Hollanders, V.M.H., Woutersen, R.A., Koeter, H.B.W.M., and Bär, A. (1992b). Multi-generation reproduction study of lactitol in rats. J. Amer. Coll. Toxicol. 11: Williams, C.A. (1986) Metabolism of lactose and galactose in man. Prog. Biochem. Pharmacol. 21:

259 Toxicity Profile Maltodextrin (CAS No ) Maltodextrin is a nonsweet nutritive saccharide polymer that consists of D-glucose unites linked primarily by α,1 4 bonds and that has a dextrose equivalent (D.E.) of <20. Typical commercial maltodextrins contain as few as three and as many as nineteen linked dextrose units. It is prepared as a white powder or concentrated solution by partial hydrolysis of corn starch, potato starch, or rice starch with safe and suitable acids and enzymes. Maltodextrin is considered to be a biopolymer that is broken down in the gastrointestinal tract to its monomeric units and absorbed. The corresponding monomers are found naturally in the body. Thus, maltodextrin is expected to be safe to human health. The U.S. FDA lists maltodextrin as GRAS (Generally Recognized as Safe). No toxicity data could be found on maltodextrin and therefore no oral reference dose or drinking water standard was determined. References 1

260 Toxicity Profile Potassium chloride (CAS No ) Potassium chloride (KCl) has been reviewed in the OECD-SIDS program (OECD, 2001). Potassium chloride (KCl) is an inorganic salt that readily dissociates in aqueous media to K + and Cl -. Acute Toxicity The oral LD50 in rats has been reported to be 3,020 mg/kg (Boyd and Shanas, 1961). Irritation Potassium chloride did not produce an irritant response in an in vitro skin irritation OECD 439 test or an in vitro eye irritation test (ECHA). Repeated Dose Toxicity Fourteen female rats were given KCl in their drinking water (approximately 5,250 mg/kg/day) for 105 days. Ten rats were sacrificed after 105 days of exposure for examination of the heart, kidneys and the adrenals; four rats (recovery group) were kept for an additional month. KCl exposure resulted in decreased heart weight, increased kidney weight, and enlargement of part of the adrenals. All changes were reversible within one month of exposure (Bacchus, 1951). F344/Slc male rats were given 0, 110, 450 or 1,820 mg/kg/day KCl in feed for two years. At the end of the study, survival rates were 48%, 64%, 58% and 84% in the controls, 110, 45 and 1,820 mg/kg/day groups. Nephritis was reported to be predominant in all groups, including the controls. The only treatment-related effect observed was gastritis (inflammation of the stomach lining). The incidence of gastritis and ulcers were 6%, 18%, 18% and 30% in the controls, 110, 450 and 1,820 mg/kg/day groups (Imai et al., 1968). Male and female Wistar rats were fed diets containing 0 or 3% KCl over a total period of 30 months: Examination after 13 weeks (10 rats/sex/group), after 18 months (15 rats/sex/group) and after 30 months (50 rats/sex /group). Due to the reduction of feed intake the mean test substance intake and mean body weight decreased in time. After 30 months of treatment, there was hypertrophy of the zona glomerulosa in the adrenals (24/50 treated rats versus 4/50 in controls); and cystitis in the urinary bladder (males: 3/59; females 3/50) and single epithelial hyperplasia of the bladder (males 3/50; females 1

261 2/50) (Lina et al., 1994; Lina and Kuijpers, 2004). Genotoxicity Potassium chloride was not mutagenic to Salmonella typhimurium strains TA100, TA 1535, TA 1537 and TA 98 strains in an in vitro bacterial mutation assay in the presence or absence of metabolic activation (Mortelmans et al., 1986). KCl was also not mutagenic to E. coli in a SOS chromotest (Olivier and Marzin, 1987). In two different studies, KCl was found to be weakly mutagenic to mouse lymphoma L5178Y cells at high concentrations (Myhr and Caspary, 1988; Mitchell et al., 1988). Potassium chloride induced a significant increase in chromosomal aberrations in Chinese Hamster lung fibroblasts (V79) cells only at the highest test dose (12,000 µg/ml) in the absence of a metabolic activation system. Measurements of the osmotic pressure of the medium revealed a two-fold increase at this test compound concentration when compared to the normal medium (530 mosmol/ kg versus 253 mosmol/kg (Hasegawa et al., 1984). There are two other reports on the effect of KCl on formation of chromosome aberrations in Chinese hamster ovary cells (CHO). In these studies KCl concentrations of 75 and 80 mm (approximately 5500 and 6000 µg/ml) resulted in 19% and 28% aberrant cells respectively. Increased number of chromosome aberrations was observed with KCl concentrations that reduced cell survival 40% and more. It is reasonable to conclude that the increases in mutagenicity and chromosome aberrations observed in these studies are related to cytotoxicity resulting from the high KCl concentrations used. This argument is supported by the studies on the effect of increased osmolarity on genotoxicity in cultured mammalian cells. The reported mutagenic effect of KCl most probably results from a disruption of osmotic balance of cells with a subsequent interference with chromosomal stability. This may result in the clastogenic effects (DNA breakage and chromosome structural instability) due to K + effects on sequestering of Mg ++ ions required for normal maintenance of chromatin integrity. Other chemicals may also exert such effect (e.g., NaCl, sucrose). Carcinogenicity F344/Slc male rats were given 0, 110, 450 or 1,820 mg/kg/day KCl in feed for two years. At the end of the study, survival rates were 48%, 64%, 58% and 84% in the controls, 110, 45 and 1,820 mg/kg/day groups. There was no increased incidence of tumors that were considered to be treatment-related (Imai et al., 1968). Reproductive/Developmental Toxicity Pregnant female Wistar rats were given by oral gavage 3.1 to 310 mg/kg KCl during gestation days 5 through 15, and sacrificed on gestation day 20. There were no effects on 2

262 survival, total number of corpora lutea, implant sites, resorptions, soft tissue observations (urogenital tract), or live offspring. No treatment-related effects were seen in the offspring with regards to survival, sex ratio, average offspring weight, external congenital abnormalities, soft tissue or skeletal defects. The maternal and developmental NOAELs were 310 mg/kg/day (FDRL, 1975). Female CD-1 mice were given by oral gavage 2.35 to 235 mg/kg KCL during gestation days 5 through 15, and sacrificed on gestation day 17. There were no effects on survival, total number of corpora lutea, implant sites, resorptions, soft tissue observations (urogenital tract), or live offspring. No treatment-related effects were seen in the offspring with regards to survival, sex ratio, average offspring weight, external congenital abnormalities, soft tissue or skeletal defects. The maternal and developmental NOAELs were 235 mg/kg/day (FDRL, 1975). Key Study/Critical Effect for Screening Criteria In a two-year rat feeding study, there was an increased incidence of gastritis and ulcers at dose levels of >110 mg/kg/day (Imai et al., 1968). There was no NOAEL. Thus, the LOAEL for this study is 110 mg/kgday. Since the gastritis and ulcers are the result of a localized irritation effect of the test substance (site of contact) in the gastrointestinal tract, an uncertainty factor for interspecies variability is deemed unnecessary. For systemic effects, the NOAEL for the two-year rat feeding study is considered to be 1,820 mg/kg/day, the highest dose tested. Uncertainty factors: 10 (intraspecies variability); 10 (interspecies variability); 1 (intraspecies variability) Oral Reference Dose = 1,820/100 = 18.2 mg/kg/day Drinking water guideline: 64 ppm References Bacchus, H. (1951). Decrease of cardiac mass following excess dietary potassium chloride in the rat. Amer. J. Phys. 166 : Boyd, E.M., and Shanas, M.N. (1961). The acute oral toxicity of potassium chloride. Arch. Int. Pharmacodyn. Ther. 133: Brusick, D. (1988). Genotoxic effects in cultured mammalian cells produced by low ph treatment conditions and increased ion concentrations. Environ. Mutag. 8:

263 FDRL (1975). Teratological evaluation of FDA (KCl) in mice and rats. Final report prepared under DHEW. Contract No. FDA by Food and Drug Research Laboatories Inc. Waverly, New York. Hasegawa, M.M., Nishi, Y., Ohkawa, Y., and Inui, N. (1984). Effects of sorbic acid and its salts on chromosome aberrations, sister chromatid exchanges and gene mutations in cultured Chinese Hamster cell. Food Chem. Toxicol. 22: Imai, S., Morimoto, J., Sekiya, N., Shima, M., Kiyozuka, Y., Nakamori, K., and Tsubura, Y. (1986). Chronic toxicity test of KCl and NaCl in F344/Scl rats. J. Nara Med. Ass. 37: Lina, B.A.R., Hollanders, V.M.H., and Kuijpers, M.H.M. (1994). The role of akalizing and neutral potassium salts in urinary bladder carcinogenesis in rats. Carcinogenesis 15: Lina, B.A.R., and Kuijpers, V.M.H. (2004). Toxicity and carcinogenicity of acidogenic or alkalogenic diets in rats; effects of feeding NH(4)Cl, KHCO(3) or KCl. Food Chem. Toxicol. 42: Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., Zeiger, E. (1986). Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Env. Mutagenesis 8: Myhr, B.C., and Caspary, W.J. (1988). Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: Intralaboratory results for sixty-three coded chemicals tested at Litton Bionetics, Inc. Env. Molecular Mutagenesis. 12: Mitchell, A.D., Rudd, C.J., and Caspary, W.J. (1988). Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: Intralaboratory results for sixty-three coded chemicals tested at SRI international. Environ. Mol. Mutag. 12: OECD (2001a). IUCLID Data Set for Potassium chloride (CAS No ), UNEP Publications. OECD (2001b). Screening Information Assessment Report (SIAR) for Potassium chloride (CAS No ), UNEP Publications. Olivier, P., and Marzin, D. (1987). Study of the genotoxic potential of 48 inorganic derivates with the SOS chromotest. Mutat. Res. 189: Seeberg, A.H., Mosesso, P., and Forster, R. (1988). High-dose-level effects in mutagenicity assays utilizing mammalian cells in culture. Mutagenesis 3:

264 Toxicity Profile Shellac, Ammonium salt (CAS No ) Lac is the hardened resinous secretion of the insesct Kerria lacca (Douglas, 1971; CIR, 1986). The secreted lac or sticklac contains lac resin, erythrolaccin, wax, inset debris, wood materials, sand, dust and water-soluble impurities. The semirefined seedlac of commerce is the yellowish granular material obtained from the processing of sticklac (Douglas, 1971; Misra and Sengupta, 1970; CIR, 1986). Unrefined shellac can contain shellac wax and erythrolaccin (the yellow material), which both comprise about 6% of Shellac (CIR, 1986). The removal of these constituents yields lac resin that has a soft (30%) and hard (70%) components (CIR, 1986). Mild alkaline hydrolysis of this resin results in a complex mixture of hydroxyaliphatic and alicyclic acids and their polyesters. The composition of the mixture varies with the lac source and the time of collection (CIR, 1986). The major component of the aliphatic fraction is aleuritic acid. The alicyclic fraction consists of sesquiterpenic acids, primarily jalaric acid and its derivatives. The native form of the pure lac resin molecule has a molecular weight of 2,095, which contains four terpene acids (mostly jalaric acid) and four aleuritic acid units in repeating ester linkage (Singh et al., 1974). The proposed structure for lac resin respresents an average situation since the molecule may not always be homogenous with respect to sequence or ratio of jalaric acid to other sesquiterpenic acid substitutes (Singh et al., 1974). The sesquiterpenic acids in lac resin are jalaric acid, shellolic acid, epishellolic acid, laksholic acid, and epilaksholic acid. Aqueous shellac solutions can be prepared by neutralization with ethanolamines, alkalies, or borax (CIR, 1986). Acute Toxicity The acute oral LD50 in rats is >5,000 mg/kg (CIR, 1986). Irritation No data were located. Sensitization No data were located. 1

265 Repeated Dose Toxicity Male and female Sprague-Dawley rats were fed 0, 1,000, 3,000 or 10,000 ppm Regular Bleached Shellac in commercial feed for 28 days. The rats were then mated, and a single litter (F1) was delivered. Twenty-five weaned F1 animals from each group were then fed with the same F0 diets for an additional 90 days. There was no evidence of treatmentrelated effects in either generation, including fertility, reproductive performance, and pup development. The NOAEL for this study is 10,000 ppm (CIR, 1986). Genotoxicity Shellac and shellac wax were not mutgenic to Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 with and without metabolic activation (CIR, 1986). Shellac wax was not mutagenic to S. typhimurium strains TA1535, TA1537 and TA1538, or to Saccharomyces cerevisiae strain D4 with or without metabolic activation (CIR, 1986). Carcinogenicity No studies could be found. Reproductive/developmental Toxicity See Repeated Dose Toxicity section. Key Study/Critical Effect for Screening Criteria No treatment-related effects were noted in an one-generation rat reproductive toxicity study in which rat were fed shellac in the diet at concentrations up to 1% (10,000 ppm). NOAEL (mg/kg/day) = 10,000 ppm * 0.05 = 500 mg/kg/day Where 0.05 is the fraction of body weight that is consumed per day as food for the rat (U.S. EPA). Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral Reference Dose = 500/1,000 = 0.5 mg/kg/day Drinking water guideline value = 1.75 ppm (shellac, ammonium salt may be insoluble environmental ph) 2

266 References CIR (1986) Final report on the safety assessment of shellac. J. Amer. College Toxicol. 5: Douglas, J.S. (1971). Gums, resins and balsams. Soap Perfume Cosmetics 44: Misra, G.S., and Sengupta, S.C. (1970) Shellac. Encyclopedia of Polym. Sci. Technol. 12: Singh, A.N., Upadhye, A.B., Mhaskar, V.V., Dev., S., Pol, A.V., and Naik, V.G. (1974). Chemistry of lac resin VII. Pure lac resin 3: Structure. Tetrahedron 30:

267 Toxicity Profile Sodium Carboxymethyl Cellulose (CAS No ) Sodium carboxycellulose is the sodium salt of carboxymethylcellulose. Carboxymethyl cellulose is a cellulose derivative with carboxymethyl groups (-CH2COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. Sodium carboxycellulase is a listed as GRAS (Generally Regarded as Safe) by the U.S. Food and Drug Administration (FDA GRAS database). It is an approved food additive in the EU (EC, 1995) and may be added to all foodstuffs following quantum satis principle, except in products for the dietary management of metabolic disorders, where the limit of use is 10 g/l or kg (EC, 1999). The Joint FAO/WHO Expert Committee on Food Additives has determined an Acceptable Daily Intake (ADI) for sodium carboxymethyl cellulose of Not Specified (no upper limit) (JECFA, 1989). References EC (1995). European Parliament and Council Directive No 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners, OJ L 61, , p EC (1999). Food additives permitted in dietary foods for infants and young children for special medical purposes as defined in Directive 1999/21/EC (Commission Directive 1999/21/EC of 25 March 1999 on dietary foods for special medical purposes, (OJ L 91, , p. 29). FDA GRAS Database: JECFA (1989). 1

268 Toxicity Profile Sodium Lauryl Sulfate (CAS No ) Sodium lauryl sulfate has been reviewed in the OECD-SIDS program (OECD, 2007a,b). Acute Toxicity The acute oral LD50 for sodium lauryl sulfate in rats was reported to be 1,200 mg/kg (Henkel KGaA, 1983). The acute dermal LD50 of a 33% solution of sodium lauryl sulfate was reported to be 600 mg/kg; this corresponds to 200 mg active substance/kg (Carson and Oser, 1964). Irritation A 25% solution of sodium lauryl sulfate was severely irritating to the skin of rabbits when applied for 4 hours under occlusive conditions (Henkel KGaA, 1987a). A 5% solution of sodium lauryl sulfate was moderately to strongly irritating to the skin of rabbits when applied for 4 hours under semi-occlusive conditions (Unilever Research, 1982). In a human repeated 4-hour patch test, a 20% solution of sodium lauryl sulfate was considered moderately irritating to the skin (Proctor and Gamble, 1998). Sodium lauryl sulfate, as a 25% solution, was severely irritating to the eyes of rabbits (Henkel KGaA, 1987b). Sensitization Sodium lauryl sulfate has tested positive in two out of three mouse local lymph node assays or LLNAs (Ikarashi et al., 1993; Basketter et al., 1994; Montelius et al., 1994). The positive response in the LLNA is thought to be due to a mechanism associated with the irritation potential of sodium lauryl sulfate and not due to an allergic response (Montelius et al., 1994; Basketter et al., 1994, 2002). Ammonium lauryl sulfate did not show any allergic response in a human repeat insult patch test (OECD, 2007a,b), and other structurally related alkyl sulfates also tested negative in human repeat insult patch tests (OECD, 2007a,b). There have been a few reported contact sensitization cases seen under conditions where also other, possibly sensitizing agents were used or on compromised skin (OECD, 2007a,b). Repeated Dose Toxicity Male and female rats were given 0, 40, 200, 1,000 or 5,000 ppm (corresponding to 3, 17, 86 or 430 mg/kg-day) sodium lauryl sulfate in their feed for 90 days. There were 1

269 increased liver weights in the high-dose females animals; no other adverse effects were observed. The NOAEL for this study is 1,000 ppm (86 mg/kg/day) (Walker et al., 1967). Male and female rats were given by oral gavage 0, 30, 100 or 300/600 mg/kg of a 90% aqueous solution of sodium lauryl sulfate for 28 days. The high-dose of 300 mg/kg was changed to 600 mg/kg after 10 days of treatment. At the high-dose, feed intake and body weight were reduced, and water intake was increased. There was bleeding and ulceration of the stomach, and transient alterations of the tongue and myocard. Hematological findings included increased leucocytes and alanine aminotransferase (ALT) activity; and decreased hematocrit and erythrocyte volume (MCV). Relative weights of adrenals, kidneys, brain, gonads and liver were increased; the relative thymus weight was decreased. There were no effects at the 100 mg/kg dose level. The NOAEL for this study is 90 mg/kg/day (90% of the 100 mg/kg dose level) (Henkel KGaA, 1987c). A sodium C12-15 alkyl sulfate was investigated in 13-week rat dietary study. The dietary concentrations were 0, 0, 0.07, 0.14, 0.28, 0.56, 1.13 or 2.25% (0, 58, 1134, 228, 470, 961, or 1,944 mg/kg/day for males; 0, 66, 131, 261, 506, 1,070 or 2,218 mg/kg/day for females). The LOAEL for this study was 0.28% (245 mg/kg/day) based on hypertrophy of the liver, and the NOAEL was 0.14% (122 mg/kg/day) (Unilever Research, 1976d). In two separate studies, male and female Wistar rats were given 0, 0.015, 0.15 or 1.5% sodium C12-15 alkyl sulfate in their diet (approximately 0, 11, 113 or 1,125 mg/kg/day) for two years. The findings in both studies were very similar. The survival rate in the high-dose animals for both studies was about 70%. Animals dosed with 1.5% showed decreased growth rates, with also decreases in food and water intake. In the high-dose animals, there was increased absolute and relative liver weights, changes in serum liver enzymes, hypertrophy of the liver parenchyma, and a decrease in the incidence and severity of chronic nephropathy and nephrocalcinosis. The NOAEL for these two studies were considered to be 113 mg/kg/day (Unilever Research, 1995a,b). Genotoxicity Sodium lauryl sulfate was not genotoxic in a variety of in vitro and in vivo genotoxicity tests (OECD, 2007a,b). Carcinogenicity In two separate studies, male and female Wistar rats were given 0, 0.015, 0.15 or 1.5% sodium C12-15 alkyl sulfate in their diet (approximately 0, 11, 113 or 1,125 mg/kg/day) for two years. The findings in both studies were very similar. The survival rate in the high-dose animals for both studies was about 70%. There were no increased incidence of tumors that were considered treatment-related (Unilever Research, 1995a,b). 2

270 Reproductive Toxicity In a dominant lethal study, male Swiss albino mice were fed either 1 % sodium lauryl sulfate for 2 weeks or with 0.1 % sodium lauryl sulfate for 6 weeks. Body weights were significantly decreased at the 1% dose level, while the treatment caused no adverse effects on fertility (i.e., impairment of epididymal spermatozoa) (Hemsworth et al., 1981). In the 13-week oral toxicity study with sodium C12-15 alkyl sulfates (CAS No ), there were no indications of adverse effects on the reproductive organs. (Unilever Research, 1976). Developmental Toxicity Pregnant female rats were dosed by oral gavage with 0, 0.2, 2, 300 or 600 mg/kg sodium lauryl sulfate during gestational days Mortality (3/20 dams) occurred at 600 mg/kg. There was slight to moderate maternal toxicity at >300 mg/kg. No developmental toxicity was noted at any dose level. The maternal and developmental NOAELs were 2 and 600 mg/kg/day, respectively (Palmer et al., 1975). Pregnant female mice were dosed by oral gavage with 0, 0.2, 2, 300 or 600 mg/kg sodium lauryl sulfate during gestational days There were 4/20 maternal deaths at 600 mg/kg, and 1/20 maternal deaths at 300 mg/kg. At 600 mg/kg, there was total resorption and/or increased incidence of litter loss. At <300 mg/kg, there were no adverse effects on fetal morphogenesis. The maternal and developmental NOAELS were 2 and 300 mg/kg/day, respectively (Palmer et al., 1975). Pregnant female rabbits were dosed by oral gavage with 0, 0.2, 2, 300 or 600 mg/kg sodium lauryl sulfate during gestational days There were 11/13 maternal deaths at 600 mg/kg, and 1/13 maternal deaths at 300 mg/kg. Maternal toxicity at 300 mg/kg included diarrhea, anorexia, weight loss, cachexia, and fetal loss. Developmental effects were seen at 600 mg/kg and included abortion, total resorption and/or increased incidence of litter loss. At 300 mg/kg, there were no adverse effects on fetal morphogenesis. The maternal and developmental NOAELs are 2 and 300 mg/kg/day, respectively Palmer et al., 1975). Key Study/Critical Effect for Screening Criteria Two chronic rat dietary studies were conducted on sodium lauryl sulfate. The LOAEL for these two studies was 1,125 mg/kg/day based on liver effects. The NOAELs were considered to be 113 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 113/100 = 1.1 mg/kg/day 3

271 Drinking water guideline value = 4 ppm References Basketter, D.A., Scholes, E.W., and Kimber, I. (1994). The performance of the local lymph node assay with chemicals identified as contact allergens in the human maximization test. Food Chem. Toxicol. 32: Basketter, D.A., York, M., McFadden, J.P. and Robinson, M.K. (2004). Determination of skin irritation potential in the human 4-h patch test. Contact Derm. 51: 1-4. Carson, S. and Oser, B.L. (1964). Dermal toxicity of sodium lauryl sulfate. J. Soc. Cosmet. Chem 15: Hemsworth, B.N. (1981). Male mouse fertility after ingestion of spermicidal detergents. Soc. Occup. Med. 9: Henkel KGaA (1983). Texapon L 100. Pruefung auf akute orale Toxizitaet an Ratten (Unpublished Report No. TBD ). Henkel KGaA, Duesseldorf, 2 pp. Henkel KGaA (1987a). Aniontenside, Kationtenside, Amphotenside, Niotenside - Pruefung auf primaere Hautirritation (Unpublished Report No. TBD ). Henkel KGaA, Duesseldorf, 21 pp. Henkel KGaA (1987b). Texapon L 100, Lanette E, Maranil A Paste 75, Texapon T 42, Texapon N 25, Texapon SB 3, Texapon ASV, Nifancol LBE, Forlanit E und Akypro RLM 100. Pruefung auf primaere Schleimhautirritation am Kaninchenauge (Unpublished Report No. TBD ). Henkel KGaA, Duesseldorf, 20 pp. Henkel KGaA (1987c). Texapon K Tage-Test mit wiederholter oraler Verabreichung an Ratten (Unpublished Report No ). Henkel KGaA, Duesseldorf, 67 pp. Ikarashi, Y., Tsuchiya, T., and Nakamura, A. (1993). A sensitive mouse lymph node assay with two application phases for detection of contact allergens. Arch. Toxicol. 67: Montelius, J., Wahlkvist, H., Boman, A., Fernstroem, P., Grabergs, L., and Wahlberg, J.E. (1994). Experience with the murine local lymph node assay: inability to discriminate between allergens and irritants. Acta Derm. Venereol. 74: OECD (2007a). IUCLID Data Set for Category of Alkyl sulfates, Alkane sulfonates and α-olefin sulfonates. 4

272 OECD (2005b). Screening Information Dataset (SIDS) Initial Assessment Report for Category of Alkyl sulfates, Alkane sulfonates and α-olefin sulfonates. Palmer, A.K., Readshaw, M.A., and Neuff, A.M. (1975). Assessment of the teratogenic potential of surfactants. Part II - AOS. Toxicology 3: Procter and Gamble (1998) Assessment of Acute Skin Irritation by Human Patch Test. (Unpublished Report No. BT98C017-DB51; P&G Accession #50559). Unilever Research (1976). Feeding studies on Dobanol-25-sulphate, high conversion, bleached. 2. Thirteen-week test in rats (Unpublished Report No. PCW ). Unilever Research, Colworth/Welwyn, 70 pp. Unilever Research (1982). Rabbit covered patch skin irritation test (Unpublished Report No. CPS 82.21). Unilever Research, Colworth/Welwyn, 27 pp. Unilever Research (1995a). Two year rat feeding study with Dobanol-25-sulphate, low conversion, unbleached (LCU) (Unpublished Report No. RG 953). Unilever Research, Colworth/Welwyn, 109 pp. Unilever Research (1995b). Two year rat feeding study with Dobanol-25-sulphate, high conversion, bleached (HCB) (Unpublished Report No. RG 954). Unilever Research, Colworth/Welwyn, 131 pp. Walker, A.I.T., Brown, V.K.H., Ferrigan, L.W., Pickering, R.G., and Williams, D.A. (1967). Toxicity of sodium lauryl sulphate, sodium lauryl ethoxysulphate and corresponding surfactants derived from synthetic alcohols. Food Cosmet. Toxicol. 5:

273 Toxicity Profile Sorbitan, monododecanoate poly(oxy-1,2-diethanediyl) (CAS No ) Sorbitan, monododecanoate poly(oxy-1,2-diethanediyl) or PEG sorbitan monolaurate is a hydrophilic, nonionic surfactant that is also called Polysorbate 20. It has been approved by the U.S. FDA as a direct and indirect food additive for human consumption with certain restrictions. Metabolism The metabolism of radiolabeled polysorbates has been studied in rats. After oral administration, the ester linkage of the polysorbate molecule is hydrolyzed by pancreatic lipase and the released fatty acid is absorbed and metabolized. Essentially all of the fatty acid in polysorbate 80 fed as 10% of the diet is hydrolyzed and absorbed (Oser and Oser, 1957). In another study, the labeled lauric acid moiety of polysorbate 20 was rapidly absorbed and oxidized by rats (Treon et al., 1967). After 24 hours, about 75% of the lauric acid was oxidized and expired as CO2; 4% was not absorbed from the gastrointestinal tract. The approximate distribution of radioactivity 24 hours after oral administration of polysorbate 20 (with radiolabeled lauric acid) was: 80% expired as CO2; 12% with the carcass; 4% unabsorbed from the gastrointestinal tract; 2.5% in the urine; and 1.2% in the liver (Nelson et al., 1966). The sorbitol-polyoxyethylene moiety is poorly absorbed and is mostly excreted in the feces. In a study with the radiolabel in the polyoxyethylene portion of polysorbate 20, 90% was excreted in the feces and 8% in the urine. No radioactivity was found in the liver, carcass or expired CO2 (Nelson et al., 1966). Following intravenous administration of polysorbates to rats, the ester linkage of the polysorbates is hydrolyzed by blood lipases. Intravenous studies using polysorbate 20 (with radiolabeled lauric acid moiety) in rats showed that the fatty acid portion was metabolized and excreted mostly as expired CO2. The sorbitol-polyoxyethylene moiety was not catabolized since no radioactivity was recovered as CO2 when this portion of the molecule was labeled. Most of the radiolabeled polyoxyethylene moiety was excreted in the urine, although a small amount was found in the feces, indicating some biliary excretion; trace amounts were detected in the carcass and liver (Treon et al., 1967). Metabolism of polysorbates in humans appears to be similar to that in rats. Analysis of urine and feces from human volunteers who consumed 4.5 g of polysorbate 80 daily showed that 95% of the polyoxyethylene portion was excreted in the feces and 5% in urine; apparently, none was retained in the body (Culver et al., 1951). 1

274 Acute Toxicity The acute oral LD50 in rats range as high as >38,900 mg/kg (CIR, 1986) and the acute dermal LD50 is >3,000 mg/kg (CIR, 1986). Irritation Undiluted Polysorbate 20 produced essentially no irritation to the skin of rabbits or humans (CIR, 1986). When tested on human eyes, Polysorbate 20 up to concentrations of 40% produced no adverse effects (Marsh and Maurice, 1971). In rabbits, undiluted Polysorbate 20 produced minimal, transient eye irritation (CIR, 1986). Sensitization Polysorbate 20 was a moderate to strong skin sensitizer in the Magnusson-Kligman guinea pig maximization test (CIR, 1986). However, other Polysorbates (65 and 80) were not skin sensitizers when tested in the guinea pig skin sensitization assay (CIR, 1986). The following is from the CIR report on polysorbates (CIR, 1986): Extensive clinical skin testing in the Schwartz prophetic patch test showed little potential for human skin irritation and no evidence of skin sensitization in a total of 580 subjects. A total of 1206 patients with eczema were tested in a chamber method 24-hour occlusive patch test for allergic contact dermatitis to a mixture of 5 percent Polysorbate 60 and 5 percent Polysorbate 80 in petrolatum; allergic reactions were shown by only 2 of the patients ( < 0.2 percent). Several product formulations containing the Polysorbates have been tested for human skin sensitization on a total of 3481 subjects using a variety of testing methods; there were no reactions indicative of sensitization to any of the Polysorbates in these assays. Investigations with patients known to have skin disease revealed isolated instances of skin sensitization to Polysorbate 40 or 80. Intravenous injection of Polysorbate 80 produced hemodynamic changes in 5 patients. Repeated Dose Toxicity Numerous long-term feeding studies have been conducted on polysorbates using a variety of animal species; only the key studies which have been the basis for the ADIs on polysorbates are summarized below. When mixed feed including polysorbate 60 (2%, 5%, 10% or 25%) was administered to both male and female rats for two years, marked diarrhea was observed in the 10% and 25% groups, slight to moderate distention of the cecum in the 25% group, more slight 2

275 distention of the cecum in the 10% group and slightly suspicious changes in fat in the liver in the 25% group. The NOAEL is a dietary level of 5% (Fitzhugh et al., 1959). Rats were given in their feed 0, 5, 10 or 20% Polysorbate 60, 65 or 80 for two years and over four successive generations. At the 20% dose level, the most notable effect was diarrhea; there were also some effects on postnatal survival, lactation efficiency, breeding activity, growth rate, and longevity. The 10% dose level produced only diarrhea. The problems with diarrhea and reproduction at high dose levels were alleviated by the addition of fat to the diet. There were no adverse effects at the 5% level. No other adverse effects were noted. The NOAEL is a dietary level of 5% (Oser and Oser, 1957). Groups of F344/N rats and B6C3F1 mice of each sex were given diets containing 0, 3,100, 6,200, 12,500, 25,000 or 50,000 ppm polysorbate 80. All animals survived to the end of the studies. The final mean body weights of dosed rats and mice were similar to those of the controls. No clinical findings, changes in absolute or relative organ weights, or gross or microscopic lesions in rats or mice were related to polysorbate 80 administration. The NOAEL is 50,000 ppm (NTP, 1992a). It is likely that the diarrhea noted in many feeding studies with the polysorbates resulted from having high concentrations of the unabsorbed polyoxyethylene sorbitan moiety within the intestinal lumen. Genotoxicity No specific studies were located for Polysorbate 20. One report mentioned a review in which stated that Polysorbate 20 was not mutagenic, but no information was provided (JFSC, 2007). Based on Evaluation Report of Food Additives for Polysorbates (20, 60, 65 and 80), Polysorbate 60 was not mutagenic in the Ames test. Ames tests using two strains of S. typhimurium (TA98 and TA100) were performed in three independent assays and the results were negative both with and without metabolic activation (Kawachi et al., 1981; Morita et al., 1981; Inoue et al., 1980). In addition, it did not induce in vitro transformation of hamster embryo cells (Inoue et al., 1980). A B. subtilis rec-assay was performed as a DNA repair study for Polysorbate 60, and while the assay was found to be positive for reverse mutation (JFSC, 2007), others obtained negative results (Kawachi et al., 1981; Morita et al., 1981). Several other studies, including clastogenicity studies, were conducted, but the results were negative (Kawachi et al., 1981). Polysorbate 80 was negative in rec-assays for DNA damage repair using B. subtilis and E. coli (Kawachi et al., 1981). Polysorbate 80 was not mutagenic to S. typhimurium strains TA98, TA100, TA1535 and TA1537 with or without metabolic activation (Kawachi et al., 1981; Morita et al., 1981). In a chromosomal aberration study using 3

276 mammalian cells, the results were negative with or without metabolic activation (Kawachi et al., 1981; Ichidate et al., 1977). Polysorbate 80 was not genotoxic in two rodent micronucleus assays (Jenssen and Ramel, 1980; Scott and Topham, 1982); it was also negative in a dominant lethal study (Anderson et al., 1977). Carcinogenicity There are no carcinogenicity studies on Polysorbate 20 that meets current standards. Male and female Wistar rats were given in their feed 0, 5, 10 or 20% Polysorbate 60 for two years. There was no increased incidence of tumors in the Polysorbate 60-treated animals compared to the controls (Oser and Oser, 1957). In another two-year dietary study, there were no increased incidence of tumors in male and female Osborne-Mendel rats given 2, 5, 10 or 25% Polysorbate 60 (Fitzhugh et al., 1959). Male and female F344/N rats were given in their diet 0, 25,000, or 50,000 ppm Polysorbate 80 for up to 103 weeks. There were no significant changes in absolute or relative organ weights. The mean body weights in the treated male and female rats were similar to those of the controls throughout the studies. No clinical findings were associated with administration of Polysorbate 80. The survival of the treated males was lower than that of the controls. The incidence of adrenal medulla pheochromocytoma was marginally increased in high-dose males. The slight increase in incidence of pheochromocytoma in the high-dose males was judged to be of questionable significance based on historical control data. The incidence of adrenal medulla hyperplasia was increased in the 25,000 ppm males, but not in the 50,000 ppm males. The marginal increased incidence of pheochromocytoma in combination with increased incidence of hyperplasia were considered to be an equivocal finding (NTP, 1992a). Male and female B6C3F1 mice were given 0, 25,000, or 50,000 ppm Polysorbate 80 for up to 103 weeks. There were no significant changes in absolute or relative organ weights. Incidences of hyperplasia and inflammation of the forestomach were increased in females that received 50,000 ppm. The mean body weights in the treated males were similar to those of the controls throughout the studies. The final mean body weight of 50,000 ppm females was 11% lower than that of the controls. No clinical findings were associated with administration of Polysorbate 80. Survival were similar between the treated and control mice. No chemical-related increases in the incidences of neoplasms occurred in male or female mice. The incidences of squamous hyperplasia and inflammation of the forestomach were significantly increased in high-dose male and female mice; forestomach ulcers were significantly increased in high-dose females (NTP, 1992a). Reproductive/Developmental Toxicity Pregnant Sprague-Dawley rats were given by oral gavage 0, 500 or 5,000 mg/kg Polysorbate 20 during gestation days (GD) There was no maternal toxicity, and 4

277 there was no significant differences in body weight between treated and control animals. Maternal weight gain during GD 6-15 was decreased by 14% in the high-dose group relative to control animals. There was no treatment-related effects on organs weights, number of corpora lutea per dam, the number of implantation sites per dam or the percent preimplantation loss per litter. There was also no adverse effects on the growth, viability or morphological development of the conceptuses. The maternal and developmental NOAELS for this study were considered to be 500 and 5,000 mg/kg/day, respectively (NTP, 1992b). Key Study/Critical Effect for Screening Criteria In 1973, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated the effects of Polysorbates 20, 40, 60, 65, and 80 when used as food additives. JEFCA established an ADI of 0-25 mg/kg/day for the group of Polysorbates (JECFA, 1973). The study on which the ADI was based was the two-year feeding study by Fitzhugh et al., 1959, in which the NOAEL was determined to be 5% (equivalent to 2,500 mg/kg/day). In 1978, the EU s Scientific Committee on Food (SCF) evaluated the safety of polysorbates, mainly based on the two-year feeding study on Polysorbate 60 (SCF, 1978). SCF assigned an interim ADI of 0-25 mg/kg/day for the polysorbate group, but requested data from a 90-day oral toxicity study and a metabolism study in one animal species, which SCF considered necessary for a final evaluation. In 1983, the SCF conducted a re-evaluation based on a 13-week oral study in which rats were fed 0, 1, 2 or 5% Polysorbate 60 (equivalent to 500, 1,000 or 2,500 mg/kg/day, respectively) (BIBRA, 1981). SCF accepted a review of the existing metabolism data in lieu of the requested data in 1978 (SCF, 1978). A permanent group ADI for the polysorbates was established at 0-10 mg/kg/day, after application of a 100-fold uncertainty factor to the lowest NOAEL of 2% in the diet from the 13-week feeding study. In 1992, SCF further evaluated the ADI for the polysorbates, in light of the recently conducted NTP bioassay on Polysorbate 80 (NTP, 1992), and concluded that the ADI need not be changed (SCF, 1993). The U.S. FDA has established a group ADI for the polysorbates at 1,500 mg/person/day (0-25 mg/kg/day). (FDA, 1999). The ADI of 25 mg/kg/day established by JECFA and the U.S. FDA will be the basis of the oral RfD and drinking water guideline for sorbitan, monododecanoate poly(oxy-1,2- diethanediyl) [Polysorbate 20] since the findings of the 13-week rat feeding study (BIBRA, 1981), which was the basis for the ADI established by SCF, is unavailable for review. Oral RfD = 2,500/100 = 25 mg/kg/day Drinking water guideline value = 87.5 ppm 5

278 References Anderson, D., McGregor, D.B., Purchase, I.F.H., Hodge, M.C.E., and Cuthbert, J.A. (1977). Dominant-lethal test results with known mutagens in two laboratories. Mutat. Res. 43: BIBRA (1981). A Short-term (13 week) Study in Rats with Polyoxyethylene (20) Sorbitan Monostearate (unpublished). CIR [Cosmetics Ingredient Review] (1984). Final report on the safety assessment of Polysorbates 20, 21, 40, 60, 61, 65, 80, 81, and 85. J. Am. Coll. Toxicol. 3: Culver, P.J., Wilcox, C.S., Jones, C.M., and Rose, R.S., Jr. (1951). Intermediary metabolism of certain polyoxyethylene (20) and sorbitan monooleate and of polyoxyethylene (40) monostearate. J. Pharmacol. Exp. Ther. 103: Fitzhugh, O.G., Bourke, A.R., Nelson, A.A., and Frawley, J.P. (1959). Chronic oral toxicities of four stearic acid emulsifiers. Toxicol. Appl. Pharmacol. 1: Inoue, K., Sunikawa, T., and Takayama, S. (1980). Studies of in vitro cell transformation and mutagenicity by surfactants and other compounds. Food Cosmet. Toxicol. 18: Ishidate, M., and Odashima, S. (1977). Chromosome studies with 134 compounds on Chinese hamster cells in vitro a screening for chemical carcinogens. Mut. Res. 48: JEFCA (1973). 17 th Meeting of the Joint FAO/WHO Expert Committee on Food Additives. JFSC (2007). Evaluation Report of Food Additives: Polysorbates (Polysorbates 20, 60, 65 and 80), Japanese Food Safety Commision. Jenssen, G., and Ramel, C. (1980). The micronucleus test as part of a short-term mutagenicity test program for the prediction of carcinogenicity evaluated by 143 agents tested. Mutat. Res. 75: Kawachi, T., Yahagi, T., Kada, T., Tazima, Y,m Ishidate, M., Sasaki, M., and Sugiyama, T. (1981). Cooperative program on short-term assays for carcinogenicity in Japan. IARC Sci. Pub. 27: Marsh, R.J., and Maurice, D.M. (1971). The influence of nonionic detergent and other surfactants on human corneal permeability. Exp. Eye Res. 11:

279 Morita, K., Ishigaki, I., and Abe, T. (1981). Mutagenicity of cosmetic-related substances. J. Soc. Cosmet. Chem. Japan 15: Nelson, M.F., Poulas, T.A., Gongwer, L.E., and Kirschman, J.C. (1966). Preparations of carbon-14 labeled polyoxyethylene (20) sorbitan monolaurate and their metabolic fate in rats. J. Food Sci. 31: NTP (1992a) Toxicology and Carcinogenesis Studies of Polysorbate 80 (CAS No ) in F344/N rats and B6C3F1 Mice (Feed Studies), NTP TR 415, NIH Publication No , National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. NTP (1992b). Abstract for TER91010-Polyoxyethylene Sorbitan Monolaurate (CASRN ). Developmental Toxicology of Polyoxyethylene Sorbitan Monolaurate (CAS # ) in Sprague-Dawley CD Rats, NTIS# PB , National Institute of Environmental Health Sciences. Oser, B.L., and Oser, M. (1957). Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. IV. Mortality and post-mortem pathology. J. Nutr. 61: Oser, B.L., and Oser, M. (1971). Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. III. Clinical and metabolic observations. J. Nutr. 61: SCF (1978). Evaluation of Polysorbates 20, 40, 60, 65, 80, Paragraph 12, Reports of the Scientific Committee for Food (15 th Series). SCF (1993). Opinion on Polyoxyethylene (20) Sorbitan Mono-Oleate (Polysorbate 80), Reports of the Scientific Committee for Food (34 th Series), September 17, Scott, K., and Topham, J.C. (1982). Assay of 4CMB, 4HMB and BC by the micronucleus studiesubcutaneous administration. Mutat. Res. 100: Treon, J.F., Gongwer, L.E., Nelson, M.F., and Kirschman, J.C. (1967). In: Chemistry, Physics, and Application of Surface Active Substances, Vol. III, p. 381, Gordon and Breach, New York. 7

280 Toxicity Profile Sulfuric acid (CAS No ) Sulfuric acid (H2SO4) will dissociate completely in aqueous media at physiological ph to H + and SO4 2-. The pka of sulfuric acid is Acute Toxicity The oral LD50 in rats was reported to be 2,140 mg/kg (Smyth et al., 1969). The acute LC50 values are: to mg/l for rats; 0.6 to 0.85 mg/l for mice; and 1.47 to 1.61 mg/l for rabbits (OECD, 2001). Irritation Undiluted sulfuric acid is corrosive to the skin and eyes (OECD, 2001). A 10% solution of sulfuric acid in water has been shown to be non-irritating to the skin of rabbits, guinea pigs and humans (Nixon et al., 1975; 1990). Instillation of 0.1 ml of a 10% solution of sulfuric acid was shown to be non-irritating to the eyes of rabbits when added to the conjunctival sac (Jacobs and Martens, 1989; Jacobs, 1992). Other studies which added a 10% solution of sulfuric acid directly to the eyes of rabbits showed severe irritation (Griffith et al., 1980; Murphy et al., 1982). Sensitization No animal sensitization studies have been conducted on sulfuric acid. There have been no reported cases of dermal sensitization in humans (OECD, 2001). Repeated Dose Toxicity There have been a number of repeat dose toxicity studies conducted by the inhalation route on sulfuric acid, but no studies were located by the oral or dermal routes. Genotoxicity Sulfuric acid was not mutagenic in the Ames test using various strains of S. typhimurium and in E. coli with and without metabolic activation (OECD, 2001). No in vivo genotoxicity studies have been conducted on sulfuric acid. 1

281 Carcinogenicity Animal carcinogenicity studies have been conducted with sulfuric acid solutions or mists. These studies cannot be used for risk assessment purposes due to significant methodological deficiencies. Reproductive/Developmental Toxicity No studies were found. Key Study/Critical Effect for Screening Criteria No toxicity studies on sulfuric acid were identified. The Australian drinking water guideline values are available for ph (H + ) and sulfate which could applicable to sulfuric acid. References Griffith, G.F., Nixon, G.A., Bruce, R.D., Reer, P.J., and Bannon, E.A. (1980). Doseresponse studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye. Toxicol. Appl. Pharmacol. 55: Jacobs, G.A., and Martens M.A. (1989). An objective method for the evaluation of eye irritation in vivo. Food Chem. Toxicol. 27: Jacobs, G.A. (1992). OECD eye irritation tests on two strong acids. J. Am. Coll. Toxicol. 11: 734. Murphy, J.C., Osterberg, R.E., Seabaugh, V.M., and Bierbower, G.W. (1982). Ocular irritancy responses to various phs of acids and bases Toxicology 23: Nixon, G.A., Bannan, E.A., Gaynor, T.W., Johnston, D.H., and Griffith, J.F. (1990). Evaluation of modified methods for determining skin irritation. Regul. Toxicol. Pharmacol. 12: Nixon, G.A., Tyson, C.A., and Wertz, W.C. (1975). Interspecies comparisons of skin irritancy. Toxicol. Appl. Pharmacol. 31: OECD (2001). Screening Information Dataset (SIDS) Initial Assessment Report for Sulfuric acid (CAS No ), UNEP Publications. 2

282 Smyth, H.F., Jr., Carpenter, C.P., Weil, C.S., Pozzani, U.C., Striegel, J.A., and Nycum, J.S. (1969). Range-finding toxicity data : list VII. Am. Ind. Hyg. Assoc. J. 30:

283 Toxicity Profile Talc (CAS No ) Talc refers to both mineral talc and industrial mineral products that are marketed under the name talc and contain proportions of mineral talc that range from about 35% to almost 100%. Industrial talc generally refers to products that contain abundant minerals other than talc; cosmetic talc now normally contains >98% talc but the content may have been lower in the past. Pharmaceutical talc contains >99% talc. Talcum powder is cosmetic-grade talc. Talc is a mineral composed of hydrated magnesium silicate with the chemical formula Mg3Si4O10(OH)2 or H2Mg3(SiO3)4. Talc is not soluble in water. The U.S. Food and Drug Administration (FDA) regulates talc and states that it is generally recognized as safe for use in color additives in foods, drugs and cosmetics, and in paper, paper products, cotton and cotton fabrics that come into contact with food. The Food Chemical Codex (2003) provides specifications for food-grade talc, including the statement that talc derived from deposits that are known to contain associated asbestos is not food grade. Under the voluntary guidelines initiated in 1976, the Cosmetic, Toiletry, and Fragrances Association stated that all cosmetic talc should contain at least 90% platy talc (hydrated magnesium silicate) that is free from detectable amounts (<0.5%) of fibrous, asbestos minerals. Toxicokinetics (Oral) The absorption and disposition of 3 H-labeled talc in rats, mice, and guinea pigs administered a single oral dose was studied by Phillips et al. (1978). The oral doses were 50 mg/kg for rats, 40 mg/kg for mice, and 25 mg/kg for guinea pigs. In rats, mice, and guinea pigs, more than 95% of the dose was excreted in the feces 3 to 4 days after dosing. Less than 2% of the radioactivity was recovered in the urine. This radioactivity probably reflected contamination of urine samples with feces. No radioactivity was found in the liver or kidneys of these animals. This information suggests that talc is not absorbed from the gastrointestinal tract following oral exposure. Acute Toxicity No data were located. 1

284 Repeated Dose Toxicity/Carcinogenicity Male and female Wistar rats were given in their diet 0 or 50 mg/kg of commercial talc [characteristics unspecified] for the life of the animals (average survival was 702 and 649 days, respectively). There was no significant difference in the talc-fed animals compared with control animals (Gibel et al., 1976). Male and female Wistar-derived rats were given in their diet 100 mg Italian talc (grade 00000; ready milled; mean particle size, 25 μm; containing 92% talc, 3% chlorite, 1% carbonate minerals and 0.5 1% quartz) per rat per day for 5 months (talc-containing diet was actually given for 101 days) and were then maintained on basal diet for life (average survival, 614 days). No differences in tumor incidence were noted between treated animals and control animals (eight/sex for an average survival of 641 days) (Wagner et al., 1977). In humans and experimental animals, the effects of talc are dependent on the route of exposure, and the dose and properties of the talc. Talc pneumoconiosis was somewhat more prevalent and severe among miners exposed to talc containing asbestiform minerals and/or asbestos than among those exposed to talc without such contaminants. However, the role of quartz and asbestos in the observed pneumoconiosis could not be ruled out. Among drug users, intravenous injection of talc present as a filler in the drugs resulted in microembolization in a variety of organs and alterations in pulmonary function. In animal studies, talc has been shown to cause granulomas and mild inflammation when inhaled. Observations of the effects that occurred in the lungs of rats exposed by inhalation to talc suggested that the operative mechanisms may be similar to those identified for carbon black, and talc is known to cause the release of cytokines, chemokines and growth factors from pleural mesothelial cells. IARC: There is inadequate evidence in humans for the carcinogenicity of inhaled talc not containing asbestos or asbestiform fibres. There is limited evidence in experimental animals for the carcinogenicity of talc not containing asbestos or asbestiform fibres. Inhaled talc not containing asbestos or asbestiform fibres is not classifiable as to its carcinogenicity (Group 3). Genotoxicity The IARC (1987) review of talc included unpublished results from a 1974 study conducted by Litton Bionetics that showed no mutagenic activity for talc in vitro or in vivo. Talc did not induce mutations in Salmonella typhimurium strains TA1530 or HisG46, or in the yeast, Saccharomyces cerevisiae. No chromosomal aberrations were observed in human fibroblasts treated with talc in vitro. In vivo tests conducted in rats gave negative results for induction of chromosomal aberrations in bone marrow cells and dominant lethal mutations in germinal cells. 2

285 Three samples of respirable talc failed to elicit significant unscheduled DNA synthesis (10, 20 and 50 μg/cm 2, 24 hours), sister chromatid exchange or aneuploidy (2, 5, 10 and 15 μg/cm 2, 48 hours) in rat pleural mesothelial cells, in contrast to various positive controls. The three samples, i.e. Spanish talc (No. 5725), Italian talc (No. 5726) and French talc (No. 7841), contained 90 95% talc; the remaining contents were chlorite and dolomite. Electron microscopy analysis revealed that talc particles were taken up by the rat pleural mesothelial cells, but no aneuploidy was observed in metaphases (Endo- Capron et al., 1993). Reproductive Toxicity No data are available. Developmental Toxicity No teratologic effects were observed in hamsters, rats, mice, or rabbits after oral administration of talc. The doses used were 1,600 mg/kg for rats and mice on days 6 through 15 of gestation; 1,200 mg/kg for hamsters on day 6 through 10 of gestation; and 900 mg/kg for rabbits on days 6 through 18 of gestation. Key Study/Critical Effect for Screening Criteria There are no adequate studies for which to derive a oral reference dose. Talc is poorly absorbed from the gastrointestinal tract, if at all, and the limited data available by the oral route indicate that talc is essentially non-toxic by the oral route of exposure. References Endo-Capron, S., Renier, A., Janson, X. et al. (1993). In vitro response of rat pleural mesothelial cells to talc samples in genotoxicity assays (sister chromatid exchanges and DNA repair). Toxicol. In Vitro 7: Gibel, W., LOhs, K., Horn, K.H., Wildner, G.P., and Hoffman, F. (1976) Experimental study on carcinogenic activity of asbestos filters (Ger.). Arch. Geschwulstforsch. 46: IARC (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 42, International Agency for the Research on Cancer, World Health Organization, Lyon, France; cited in NTP (1993). IARC (2010). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Carbon Black, Titanium Dioxide, and Talc, Volume 93, International Agency for the Research on Cancer, World Health Organization, Lyon, France. 3

286 NTP (1993). Toxicology and Carcinogenesis of Talc (CAS No ) in F344/N Rats and B6C3F1 Mice (Inhalation Studies), NTP TR 421, NIH Publication No , National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. Phillips, J.C., Young, P.J., Hardy, K., and Gangolli, S.D. (1978). Studies on the absorption and disposition of 'H-labelled talc in the rat, mouse, guinea-pig and rabbit. Food Cosmet. Toxicol. 16: Wagner, J.C., Berry, G., Cooke, T.J., Hill, R.J., Pooley, F.D., and Skidmore, J.W. (1977). Animal experiments with talc. In: Inhaled Particles (W.H. Walton and B. McGovern, Eds.), Vol. IV, Part 2, pp , Pergamon Press, Oxford. 4

287 Toxicity Profile Tributyl Tetradecyl Phosphonium Chloride [TTPC] (CAS No ) Limited toxicity information was located for this alkyl phosphonium salt. Acute Toxicity An oral LD50 in rats was reported to be 1,002 mg/kg. The 4-hour LC50 to rats is <0.9 mg/l (aerosol). Irritation TTPC is corrosive to the skin and eyes. Sensitization TTPC is not a skin sensitizer. Repeated Dose Toxicity No data were found. Genotoxicity TTPC tested negative in an Ames test. Carcinogenicity No data were found. Reproductive Toxicity No data were found. 1

288 Developmental Toxicity No data were found. Key Study/Critical Effect for Screening Criteria No data are available for determining the critical effect and the LOAEL/NOAEL for an oral reference dose. Reference Material Safety Data Sheet for Bellacide 350, BWA Water Additives, SDS No , Report Date: 10/03/

289 APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES F.4 SCHLUMBERGER CLEARFRAC XT

290 Toxicity Profile Acetic acid (CAS No ) Acetic acid dissociates in aqueous media to H + and the acetate anion (CH3CO2 - ). It is naturally occurring as the acid in apple cider vinegar and other fruit-derived products. It and several of its salts are commonly used as food additives (e.g., as flavor enhancers) and are listed as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA). ph: 2.5 at 50 g/l and 20 o C pka: 20 o C Acute Toxicity The oral LD50 in rats is 4,950 mg/kg, and the dermal LD50 in rabbits is 1,060 mg/kg. The 4-hour LC50 in rats is 11.4 mg/l. Irritation In the EU, acetic acid is classified as a skin and eye irritant at concentrations <25%. At >25%, it is classified as corrosive to the skin. Sensitization No data are available. Repeated Dose Toxicity In an 8-month study, rats dosed with 60 mg/kg acetic acid (three times per week) developed hyperplasia in the esophagus and forestomach. Genotoxicity Acetic acid was not mutagenic to Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 in an in vitro bacterial mutation assay with or without metabolic activation. Acetic acid (in media adjusted to a ph of 8.5) was also not genotoxic in an in vitro chromosomal aberration test using Chinese Hamster Ovary cells with or without metabolic activation. 1

291 Carcinogenicity No adequate carcinogenicity studies have been conducted on acetic acid. Reproductive Toxicity No studies could be located. Developmental Toxicity There were no effects on implantations or on maternal or fetal survival in mice, rats, or rabbits at doses up to 1,600 mg/kg. Key Study/Critical Effect for Screening Criteria There are no repeat dose toxicity studies that were considered adequate for human health risk assessment. The only study reported on the toxicity of acetic acid by oral gavage showed effects of irritation (probably due to the ph of acetic acid) at the site of contact in the gastrointestinal tract. No systemic effects were reported. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has maintained a group ADI of not limited for acetic acid and its potassium and sodium salts. The Australian drinking water guidance value for ph may apply to acetic acid. References ECHA REACH: JECFA: U.S. EPA HPVIS database: 2

292 Toxicity Profile Reaction mass of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2Hisothiazolin-3-one [3:1] (CAS No ) Acute Toxicity The oral LD50 of Kathon WT (1.5% a.i.) is >5,000 mg/kg (>75 mg a.i./kg) in male rats and >3,310 mg/kg (~49.6 mg a.i./kg) in female rats. The oral LD50 of Acticide 14 (14% a.i.) is mg/kg (~66 to 69 mg a.i./kg) in rats (combined sexes); and 465 mg/kg (69 mg a.i./kg) in male rats and 393 mg/kg (59 mg a.i./kg) in female rats. The dermal LD50 of Kathon CG is >5,000 mg/kg (>75 mg a.i./kg) in female rabbits. The dermal LD50 of Acticide 14 (14% a.i.) is 1,008 mg/kg (141 mg a.i./kg) in rats (combined sexes). The inhalation LC50 of Kathon 886F (13.9% a.i.) in male and female rats is 2.36 mg/l (0.33 mg a.i./l). Irritation Undiluted Kathon MW (1.5% a.i.) and Acticide 14 (14% a.i.) were corrosive to rabbit skin. Undiluted Kathon CG was severely irritating to the eyes of rabbits. Kathon RH 886T at 100 ppm (0.01% a.i.) was non-irritating to the eyes of rabbits. Sensitization CMI/MI has been tested in the guinea pig Buehler and Magnusson-Kligman tests and has been shown to be a strong skin sensitizer. It is also a strong contact allergen in humans. Repeat Dose Toxicity Rats were exposed to CMI/MI in a powdered commercial diet. CMI/MI concentrations were increased over the 13-week period (initial concentration up to week 2, intermediate concentration week 3-4, final concentration week 5 to 13). Concentrations in the control and CMI/MI groups were: 0/0/0, 40/57/80, 132/187/260, 400/570/800 ppm. There were no mortalities and no effects on body weight or food consumption. In each group, some animals showed slight alopecia or reddened raw or scabbed areas on the skin. There were no other differences in general behavior or appearance. There were no treatment-related changes in hematological, biochemical, urinary parameters nor any pathology. No systemic toxicity was observed up to and including the highest dose of either CMI/MI 1

293 (800 ppm, equivalent 29.1 mg a.i./kg/day). The NOAEL in this study is 800 ppm (29.1 mg a.i. /kg/day). Rats were exposed to CMI/MI via their drinking water at concentrations of 25, 75 or 225 ppm a.i. for three months (equivalent to an average intake of 2.38, 6.28 and 16.3 mg/kg/day in males and 4.06, 10.8, and 24.7 mg/kg/day in females). Two additional groups of rats were given tap water or tap water containing the inorganic ions present in the CMI/MI solution, at a concentration equal to that in the high dose group (225 ppm). This solution is referred to as the ion control solution. There were no mortalities in either sex at any dose level. There were no treatment-related effects on body weight up to the mid dose. A significant decrease in body weight was seen in males at the high dose during the first two weeks of the study. Food consumption was significantly decreased in males at all dose levels and in females mid and high dose groups during the first few weeks of dosing. Water consumption was significantly decreased at all concentrations. No overt clinical signs or ophthalmic were seen in any group throughout the 13 week toxicity or the reproductive phase. No adverse effects were seen in the examinations. No hematological treatment-related changes were seen in either sex at any dose level. There was a significant decrease in globulin and an increase in the albumin/globulin (A/G) ratio in males in both the high concentration and in the ion control groups, after 13 weeks of treatment. A significant decrease in total protein was also seen high concentration group, but was not seen in the ion control group. Females in the high concentration group showed a modest (40%) increase in SGOT (AST, aspartate aminotransferase) levels after 13 weeks of dosing. No changes were observed at any dose in mixed-function oxidase activities of the liver. At the end of 13 weeks of treatment, there was a significant increase in relative liver weight in males and in relative kidney weight in females at the high concentration but without any correlative changes in organ pathology. Histology revealed a local irritation of the glandular mucosa of the stomach in 7 of 15 males and 5 of 15 females at the high concentration. These subtle low level changes did not occur at the low or mid concentration nor were they present in either control group. No other compound related changes were seen. Reproductive organs at all doses were comparable to the control. The NOEL in this study was considered to be 75 ppm a.i. (equivalent to 6.28 and 10.8 mg/kg body weight/day in males and females, respectively), based primarily on irritation of the glandular stomach at the high dose. The No Observed Adverse Effect Level (NOAEL) is 225 ppm a.i. (equivalent to 16.3 and 24.7 mg/kg body weight/day in males and females, respectively), the highest dose tested, since no adverse effects were observed on the histopathology of any tissues or organs distant from the site of dosing. Genotoxicity The results of the in vitro mutagenicity tests were equivocal. In bacterial reverse mutation tests, CMI/MI was positive in Salmonella strain TA100 but was predominantly negative in other strains commonly tested strains. CMI/MI was mutagenic in the mouse lymphoma assay with and without metabolic activation. Effects at the Tk locus was seen both with and without S-9, but were enhanced with S9 mix. CMI/MI was not genotoxic 2

294 in the in vitro unscheduled DNA synthesis (UDS) assay with primary rat hepatocytes or in an in vitro chromosomal aberration test with Chinese hamster lung cells. The in vivo studies indicated that CMI/MI does not have relevant mutagenic potential in vivo. The positive mutagenic effect of CMI/MI found in in vitro gene mutation assays was not confirmed in the sex-linked recessive lethal test in Drosophila melanogaster nor in two unscheduled DNA synthesis (UDS) studies in the rat. CMI/MI also did not show any increase in cells with micronuclei in mice nor did it induce chromosomal aberrations in rat bone marrow cells. Carcinogenicity In a two-year rat chronic study, Kathon 886 is given as 14.2% active ingredient with a ph between 2-3. In the document with compiled batch information for Kathon the active ingredient is 13.2, [10.13 % CMI/ 3.85% MI] with 15.4% magnesium nitrate and 9.0% magnesium chloride. The dose levels were 0, 30, 100 and 300 ppm (equivalent to: 0, 2.0, 6.6, 17.2 mg a.i./kg/day in males and 0, 3.1, 9.8, 25.7 mg a.i./kg/day in females. There were no deaths. There were no treatment-related effects on body weight or body weight gain at doses or food consumption up to and including the mid dose group. A treatmentrelated and concentration-dependent decrease in water consumption was seen in both sexes in all treated groups throughout the study. These decreases ranged from 0-22% at low dose 3-30% at mid dose and 15-40% at high dose. These decreases appear to be due to the unpalatability of the CMI/MI and not its inorganic stabilizer salts since the water consumption in the salt control was comparable to the tap water control throughout the study. Based on the average daily water consumption, the high dose was judged to be a maximum tolerated dose. The decreases in body weight and body weight gain were seen in high dose animals throughout the study and may be secondary to decreased water consumption. No treatment-related clinical effects were recorded. No treatment-related ophthalmic, hematological. biochemical or urinary changes were noted. Organ weights were comparable to the control. No effects on type or incidence of neoplasms were seen at up to and including the high dose (males: 17.2 a.i.; females 25.7 mg a.i./kg/day). Slight to moderate forestomach hyperplasia was seen at both mid and high dose groups. Gastric irritation was the primary effect observed. No adverse effects on the histopathology of any other tissues/organs were observed away from the site of dosing. No systemic effects were observed. The NOEL in this study was considered to be 30 ppm a.i. (2.0 to 3.1 mg a.i./kg/day), based primarily on gastric irritation of the stomach at 100 and 300 ppm a.i.. The NOAEL was considered to be 300 ppm a.i. (17.2 to 25.7 mg/a.i./kg/day), since no evidence of systemic toxicity was observed at any dose and there was no adverse effects on the histopathology of any tissues/organs distant from the site of dosing at any dose. Reproductive/Developmental Toxicity A one-generation reproductive toxicity study was combined with the 13-week drinking 3

295 water study reported above in the repeated dose toxicity section. Reproductive capability was similar in all groups. Litter size and survival at birth was also similar in all groups. One dam at the high concentration lost the entire litter by day 4 due to a lactation problem. This is not uncommon and was not considered treatment related. Pups of the other high concentration group dams, except one, survived and thrived to day 21. Thus, no adverse effects were observed on reproductive capability of male and female rats and no effects were observed on fetal health or pup survival (to day 21) up to and including the high dose (equivalent to 16.3 and 24.7 mg/kg/day in males and females, respectively]. In a two-generation reproductive toxicity study, rats were dosed with CMI/MI (Kathon 886F: 11.1% CMI, 3.7% MI a.i.) at concentrations of 0 (control), 0 (magnesium salt control), 30, 100 or 300 ppm a.i. For the P1 generation, this was equivalent to 0, ; , and mg a.i./kg/day; and in the P2 generation 0, , , and mg a.i./kg/day. There were no treatment related effects on survival, food consumption or overt signs of toxicity. A decrease in bodyweight gain was noted initially in the P1 generation. This was thought to be linked to reduced water consumption since significant dose-related reduction in water consumption was seen at all concentrations in both the P1 and P2 generations, during the premating, gestation and lactation stages. Treatment-related histopathological changes were seen in the stomach in the P1 and P2 generation. These included erosions of the glandular mucosa, edema and inflammation in the submucosa of the glandular and nonglandular stomach, with hyperplasia and hyperkeratosis of the nonglandular stomach at the 100 and 300 ppm a.i. Other histopathological changes were seen but were not dose dependent.the estrus cycle in P1 or P2 females at any treatment level was comparable with the controls, as was the sperm motility, morphology, testicular sperm count or caudal epididymal reserves of P1 or P2 males. All other endpoints (gestation index, gestation length, number of pups per litter or treatment-related gross findings in F1 or F2 pups) were similar to those in the controls in either generation. The study authors considered that rats exposed to CMI/MI in the drinking water through two generations had a NOAEL of 30 ppm a.i. ( mg/kg/day in the P1 animals; mg/kg/day in the P2 animals) for parental animal toxicity, based on the gastric irritation of stomach at higher doses. The NOEL for reproductive toxicity is 300 ppm a.i. ( mg/kg/day in the P1 animals; mg/kg/day in the P2 animals), the highest dose tested. There were no effects on fertility or fetal development at any dose level. In a rat oral gavage developmental toxicity study on Kathon 886 (13.9% a.i.), the developmental NOEL for CMI/MI is >15 mg a.i./kg during organogenesis (highest dose tested). In a rat oral gavage developmental toxicity study on Acticide 14 (10.2% CMi/ 4% MI), the NOAEL for maternal toxicity is <3.95 mg a.i./kg; the NOAEL for teratogenicity is >19.6 mg a.i./kg; and the NOAEL for embryotoxicity is >19.6 mg a.i./kg. In a rabbit oral gavage developmental toxicity study on Acticide 14 (10.2% CMI/ 4% MI), the developmental NOAEL is >5.49 mg a.i./kg; the NOAELs for maternal toxicity and fetal toxicity are 1.41 mg a.i./kg. In a rabbit oral gavage developmental toxicity study on Kathon MW (13.9% a.i.), the NOEL for maternal toxicity is 2 mg a.i./kg; the developmental NOEL is 8 mg a.i./kg, the highest dose based on severe 4

296 maternal toxicity at 20 mg a.i./kg. Key Study/Critical Effect for Screening Criteria A two-year dietary study was conducted in rats with CMI/MI (13.2% active ingredient). No systemic effects were observed. There was, however, gastric irritation of the stomach at 100 and 300 ppm. The NOEL for this study is 30 ppm (equivalent to 2.0 and 3.1 mg a.i./kg/day for males and females, respectively). The NOEL of 2.0 mg/kg/day will be used for deriving a drinking water guidance value. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 2/100 = 0.02 mg/kg/day Drinking water guidance value = 0.07 ppm Reference EU SCCS (2009). Opinion on the mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2H-isothiazol-3-one [3:1], Colipa N o P56, Scientific Committee on Consumer Safety, SCCS/1238/09. 5

297 Toxicity Profile Choline chloride Cholinium chloride (CAS No ) Choline chloride, also known as cholinium chloride, is a quaternary amine salt. It dissociates in water into the corresponding positively charged quaternary hydroxyl alkylammonium ion and the negatively charged chloride ion. Choline chloride has been reviewed in the OECD-SIDS program (OECD, 2004). Acute Toxicity The oral LD50 in rats was reported to be between 3,150 and 5,000 mg/kg (BASF AG, 1963a, 1969). Irritation Application of a 70% aqueous solution to the skin of rabbits for 20 hours under occlusive conditions resulted in only minor skin irritation (BASF AG, 1963b). Slight eye irritation was seen in the eyes of rabbits after instillation of a 70% aqueous solution of choline chloride; no effects were seen one day after exposure (BASF AG, 1963c). Sensitization No data are available in animals. In a Human Repeated Insult Patch Test, there was no evidence of dermal sensitization in two hundred subjects given 0.5% (w/v) aqueous solution of choline chloride during the induction phase and 0.2% (w/v) aqueous solution during the challenge phase (Colgate-Palmolive, 2003). Repeated Dose Toxicity A 72-week feeding study was conducted to investigate the impact of choline chloride on the liver tumor promoting activity of phenobarbital and DDT in diethylnitroamineinitiated Fischer 344 rats (Shivapurkar et al., 1986). Animals received approximately 500 mg/kg-day choline chloride. Following the end of the exposure period, the animals were keep on the same untreated diet as the control group until study termination at week 103. Histopathology was limited to the liver and organs that developed gross abnormalities. There were no significant differences between treated and control animals on survival rates, body weights, and relative liver weights. Neither was there any 1

298 increased number of neoplastic liver nodules, hepatocellular carcinomas, lung tumors, leukemia nor other tumors between treated and control animals. The NOAEL for choline chloride in this study is 500 mg/kg/day. In humans, oral administration of 10,000 mg/day choline chloride in a pilot study treating a small number of patients with Alzheimer s disease, resulted in a slight hypotensive effect (Boyd et al., 1977). This dose was regarded as a LOAEL by the Standing Committee on the Scientific Evaluation of Dietary Reference Intake (2000). Genotoxicity Choline chloride was not mutagenic to bacteria in reverse mutation assays (Haworth et al., 1984; JETOC, 1997; Litton Bionetics, 1977). A small, but statistically significant, and dose-related increase in sister chromatid exchanges (SCEs) in Chinese Hamster Ovary (CHO) cells was reported at 50 and 500 μg/ml choline chloride in the absence of S9 only (Bloom et al., 1982). No higher concentrations were examined. These results could not be confirmed in another study using CHO cells at concentrations of choline chloride up to 5,000 μg/ml. (Galloway et al., 1985). In a gene conversion assay with Saccharomyces cerevisiae strain D4, choline chloride was negative in the presence and absence of metabolic activation (Litton Bionetics, 1977). No in vivo genotoxicity studies were available. Carcinogenicity No studies were located. Reproductive Toxicity No reliable studies have been conducted that address female fertility or reproductive toxicity by a relevant route of exposure. Developmental Toxicity Pregnant female mice were given in their feed 1,250 to 20,000 mg/kg choline chloride during gestational days 1 to 18 (BASF AG, 1966). Maternal body weight gain was reduced in all treated groups except for the 1,250 mg/kg group. Determination of maternal weight gain of dams with embryonic/fetal absorptions showed that there was no 2

299 almost net weight gain at >4,160 mg/kg and net weight loss in the 20,000 mg/kg group. All fetuses were resorbed in the 20,000 mg/kg group. Embryonic/fetal lethality of 35% and 69% were seen in the 4,160 and 10,800 mg/kg groups, respectively. No resorptions occurred in the 1,250 mg/kg group. Developmental toxicity was seen in all but the 1,250 mg/kg group. No statistically significant increases in malformations were observed in any dose group. The NOAELs for maternal and developmental toxicity is 1,250 mg/kg/day. Key Study/Critical Effect for Screening Criteria The Standing Committee on the Scientific Evaluation of Dietary Reference Intakes selected hypotension as the critical effect from the study by Boyd et al. (1977) when deriving a Tolerable Upper Intake Level. Boyd et al. (1977) reported a LOAEL of 10,000 mg/day choline chloride (7,500 mg/day choline). An uncertainty factor of 2 was chosen because of the limited data regarding hypotension and the inter-individual variation in response to cholinergic effects. Thus, the value for the Tolerable Upper Intake Value for repeated exposure of adults to choline is 3,500 mg/day choline. The oral RfD for choline chloride is derived by using the LOAEL of 10,000 mg/day from the Boyd et al. (1977) study, which is divided by an uncertainty factor of 2, to obtain a value of 5,000 mg/day or 71 mg/kg/day for a 70 kg person. Oral RfD = 71 mg/kg/day Drinking water guideline value = 248 ppm References BASF AG (1963a). Acute oral toxicity of choline chloride 70 % in water. Department of Toxicology. Unpublished results. Study No. XIII Jan BASF AG (1963b). Toxicity of choline chloride 70 % in water; skin irritation after exposure to choline chloride. Department of Toxicology. Unpublished results. Study No. XIII Mar BASF AG (1963c). Toxicity of choline chloride 70% in water; eye irritation. Department of Toxicology. Unpublished results. Study No. XIII Mar BASF AG (1966). Study on teratogenic effects of choline chloride in the mouse after oral application. Department of Toxicology. Unpublished results. Study No. XIV/ Oct BASF AG (1969). Acute oral toxicity of choline chloride 50% powder. Department of Toxicology. Unpublished results. Study No. XIX/ Aug

300 Bloom A., Galloway, S., Nakamura, F.T., Teteviri, A., Armstrong, M., Lavappa, K.L., Duk, S., and Ahmed, M.A. (1982). Comparison of results for SCE and chromosome aberrations for eleven compounds tested in two laboratories by standardized methods. Environ. Mutagen. 4: 397. Boyd, W.D., Graham-White, J., Blackwood, G., Glen, I., and McQueen, J. (1977). Clinical effects of choline in Alzheimer senile dementia. Lancet 2: 711. Colgate-Palmolive (2003). Study No. DCR TKL. TKL Research Inc. Paramus, NJ, USA. In: SCCNFP. Scientific Committee on Cosmetic Products and Non-Food Products. Choline Chloride. SCCNFP/0672/03. 9 Dec Galloway, S.M., Bloom, A..D, Resnick, M., Margolin, B.H., Nakamura, F., Archer, P., and Zeiger, E, (1985). Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ. Mutagen. 7: Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. (1983). Salmonella mutagenicity test results for 250 chemicals. Environ. Mutagenesis Suppl. 1: Litton Bionetics (1977). Mutagenic evaluation of compound FDA choline chloride. FCC. Report No. PB Mar OECD (2004). SIDS Initial Assessment Report for Choline chloride (CAS No ), UNEP Publications. Shivapurkar, N., Hoover, K.L., and Poirier, L.A. (1986). Effect of methionine and choline on liver tumor promotion by phenobarbital and DDT in diethylnitrosamineinitiated rats. Carcinogenesis 7: Standing Committee on the Scientific Evaluation of Dietary Reference Intake. Institute of Medicine (2000). Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. National Academy Press, Washington D.C. 4

301 Toxicity Profile Crystalline Silica, Cristobalite (CAS No ) Crystalline Silica, Quartz (CAS No ) Silica is an off-white granule that occurs naturally in various crystalline and amorphous or other non-crystalline forms. Crystalline silica is characterized by silicon dioxide (SiO2) molecules oriented in fixed, periodic patterns to form stable crystals. The primary crystalline form of silica is quartz. Other crystalline forms of silica include cristobalite, tripoli and tridymite. Particle size is a key determinate of silica toxicity, since toxicity is restricted to particles that are small enough to be deposited into the target regions of the respiratory tract. Oral Exposure No oral studies were located; however, crystalline silica is not expected to exhibit toxicity by the oral route. Although absorption studies were not found for crystalline silica, kinetic studies on amorphous silica show no absorption from the gastrointestinal tract. Dermal Exposure No dermal studies were located; however, crystalline silica is not expected to exhibit toxicity by the dermal route. Inhalation Exposure See attached OECD-SIDS Initial Targeted Assessment Profile on Quartz and Cristobalite, SIAM 32, April 2011.

302 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

303 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

304 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

305 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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306 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

307 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

308 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

309 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

310 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

311 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

312 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

313 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

314 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

315 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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316 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

317 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

318 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

319 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

320 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

321 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

322 Toxicity Profile Erucic amidopropyl dimethyl betaine (CAS No ) Erucic acid is the fatty acid of erucic amidopropyl dimethylbetaine. It is a monounsaturated omega-9 fatty acid (22:1 ω9). No toxicity data could be located on erucic amidopropyl dimethyl betaines. Thus, this toxicity profile is based on the data on cocoamidopropyl betaines. Cocamidopropyl betaines are mainly a mixture of C12-C18 fatty acids from hydrolysis of coconut oil. Coconut oil has a mixed fatty acid composition, which varies slightly, as it is a natural product. Lauric acid - resulting in lauramidopropyl betaine - is the major ingredient of coconut oil. Acute Toxicity The acute toxicity of cocoamidopropyl betaine ( % aqueous solution) in rats is low. The oral LD50 is >4,900 mg/kg, and the dermal LD50 is >2,000 mg/kg. Irritation A 30% aqueous solution of cocoamidopropyl betaine was only very slightly irritating to the skin of rabbits. In human studies, up to 3% solutions were weakly irritating. A 5-10% solution of cocamidopropyl betaine produced mild to moderate irritation to the eyes of rabbits; solutions containing 15% were irritating to highly irritating; and a 30% aqueous solution was irritating with irreversible damage. Sensitization The sensitizing potential of cocoamidopropyl betaine in humans is low. Commercial cocoamidopropyl betaine may, however, contain impurities identified as sensitizers (amidoamine and/or 3-dimethylaminopropylamine) which may explain positive results in human patch tests. There is no evidence for a photosensitizing potential. Repeated Dose Toxicity Rats were given a 30% aqueous solution of cocoamidopropyl betaine by oral gavage at doses of 0, 250, 500 or 1,000 mg/kg/day (corresponding to about 0, 75, 150, and 300 mg active substance/kg/day, respectively) 5 days/week for 28 days (Henkel KgA, 1991). The only treatment-related findings were forestomach lesions at the highest dose level, 1

323 probably as a result of the irritant effect of the test substance. The NOAEL for this study is 500 mg/kg/day (150 mg cocoamidopropyl betaine/kg/day). Rats were given a 30% aqueous solution of cocoamidopropyl betaine by oral gavage at doses of 0, 250, 500 or 1,000 mg/kg/day (corresponding to about 0, 75, 150, and 300 mg active substance/kg/day, respectively) five days/week for 90 days (Th. Goldschmidt AG, 1991). The only treatment-related findings were forestomach lesions at the 500 and 1,000 mg/kg dose levels, probably as a result of the irritant effect of the test substance. The NOAEL for this study is 250 mg/kg day (75 mg cocoamidopropyl betaine/kg/day). Genotoxicity There is no evidence for a genotoxic potential of cocoamidopropyl betaine. A 30% aqueous solution of cocoamidopropyl betaine was not genotoxic in vitro in either a bacteria mutation test or in a mouse lymphoma test. A limited intraperitoneal mouse micronucleus test with 27 % active cocamidopropyl betaine was negative. Carcinogenicity No valid carcinogenicity studies have been conducted. Reproductive Toxicity No reproductive toxicity studies have been conducted on cocoamidopropyl betaine. However, there was no evidence of an adverse effect on the reproductive organs in rats given oral doses of up to 1,000 mg/kg/day of a 30% aqueous solution of cocoamidopropyl betaine (300 mg active substance/kg/day) for 90 days (Th. Goldschmidt AG, 1991). Developmental Toxicity A 28.9% aqueous solution of cocoamidopropyl betaine was tested in a rat developmental toxicity at doses of 330, 990, and 3,300 mg/kg/day (corresponding to 95, 286, and 950 mg/kg/day, respectively) (CESIO, 2004). There were dose-related maternal toxic effects (reduced body weights and stomach ulcers) at 990 mg/kg/day and above. Embryotoxic effects (increased numbers of resorptions, decreased number of viable fetuses, decreased fetal body weight) were found only at the maternal toxic dose level of 3,300 mg/kg/day. The NOAEL for maternal toxicity was 330 mg/kg/day (corresponding to 95 mg active substance/kg-day) and the NOAEL for developmental toxicity was 990 mg/kg/day (corresponding to 286 mg active substance/kg/day). 2

324 Key Study/Critical Effect for Screening Criteria The key study is a 90-day rat oral gavage study in which forestomach lesions were seen at dose levels of 500 and 1,000 mg/kg of a 30% aqueous solution of cocoamidopropyl betaine. The NOAEL was 250 mg/kg (75 mg active substance/kg). NOAELadjusted = 75 * 5/7 = 53.6 mg/kg/day Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 53.6/1000 = 0.05 mg/kg/day Drinking water guideline = 0.19 ppm References CESIO (2004) Prenatal development toxicity study in rats with cocamidopropyl betaine by oral administration - according to OECD guideline DRAFT. Essen, LPT Study No /03, Henkel KGaA (1991) Dehyton K; 28-Tage-Test mit wiederholter oraler Verabreichung an Ratten. TED ; Juli 1991, Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Cocoamidopropyl betaine (CAPB) (CAS No.: , , ), 2005; OECD (2006). SIDS Initial Assessment Profile for Alkylamidopropyl betaines. Th. Goldschmidt AG (1991) Tego-Betain. 90 day oral (gavage) subchronic toxicity study in the rat. Essen, Th. Goldschmidt AG, , ,

325 Toxicity Profile Hydrochloric acid (CAS No ) Hydrochloric acid has been reviewed in the OECD-SIDS program (OECD, 2002a,b). Hydrochloric acid (HCl) or hydrogen chloride is readily dissociated in water into hydrated protons and chloride ion. Acute Toxicity The oral LD50 value of hydrogen chloride is reported to be 238 to 277 mg/kg for female rats (Hoechst AG, 1966), and 900 mg/kg for rabbits (Loewy and Munzer, 1923). The lethal dose by dermal exposure is >5,010 mg/kg for rabbits (Monsanto, 1976). The LC50 values for HCl are reported to be mg/l/5min, mg/l/30min and mg/l/60min for rats; and 20.9 mg/l/5min, 3.9 mg/l/30min and 1.7 mg/l/60min for mice (Darmer et al., 1974; Hartzell et al., 1990; MacEwean et al., 1974). Irritation Concentrations >3.3% cause skin irritation, and concentrations >17% cause corrosion in animal studies (OECD, 2002a,b). An aqueous solution (4%) of hydrogen chloride was slightly irritating (Agner and Serup, 1988), and a 10% solution was determined to be Irritating to skin for the EU Dangerous Preparations Directive, in human volunteer experiments (York et al., 1996). 0.1 ml of 10% aqueous solution of HCl was highly irritating to the eyes of rabbits (Jacobs, 1992); 0.03 ml or more of 5% HCl was corrosive to the eyes of rabbits (Griffith et al., 1980); 0.1 ml of a 3.3% aqueous solution of HCL was slightly irritating the eyes of rabbits (Hoechst AG, 1966); and 0.1 ml of a 0.33% aqueous solution of HCl was not irritating to the eyes of rabbits. (Hoechst AG, 1966). Sensitization Hydrogen chloride is not a skin sensitizer to guinea pigs and humans (Gad et al., 1986). Repeated Dose Toxicity Rats were fed diets containing 280 to 1,250 mmol/kg hydrochloric acid (10.2 to 45.6 mg/kg) for 7-12 weeks. There was increased water intake in all treated groups. All 1

326 animals fed diet containing 937 mmol/kg and above for 9 weeks, and half of the animals fed diet containing 900 mmol/kg for 12 weeks died. Also at doses >937 mmol/kg, there was decreased body weight, food consumption, blood ph, femur length, rate of ash in bone (Upotn and L Estrange, 1977). In another study with rats, hydrochloric acid was administered via drinking water at ph 2-3 (study duration not provided). Decreased protein levels in urine and decreased urine volumes were observed in the treatment groups (Clausing and Gottschalk, 1989). Genotoxicity While consistent negative results have been obtained in the bacterial systems, positive results have been obtained in the non-bacterial systems (OECD, 2002a,b). The positive results were observed at high concentrations, but they were considered to be artifacts due to the low ph. Positive results were obtained in a sex-linked recessive lethal study with D. melanogaster; only one dose level was tested (Stumm-Tegethoff, 1966) There are no mammalian studies on in vivo mutagenicity with hydrogen chloride Carcinogenicity (Oral Studies only) A ten-month study has been conducted using mice (Dyer et al., 1946). Because the methods such as strain used, duration and dose of administration, presence of coadministration substance, were not appropriate, it cannot be used for the assessment of carcinogenicity of hydrochloric acid. Reproductive Toxicity No reliable studies were identified by the oral route. Developmental Toxicity No reliable studies were identified by the oral route. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for ph may apply to hydrochloric acid. References Agner, T., and Serup, J. (1988). Contact thermography for assessment of skin damage due to experimental irritants., Acta. Dermatol. Venerol., 68:

327 Clausing, P., and Gottschalk, M.Z. (1989). Effects of drinking water acidification, restriction of water supply and individual caging on parameter of toxicological studies in rats. Versuchstierkd., 32: Darmer, K.I. et al. (1974). Acute toxicity in rats and mice exposed to hydrogen chloride gas and aerosol. Am. Ind. Hyg. Assoc. J.,35: Dyer, H.M. et al. (1946). Effect of administration of hot water, acids, alkali, mecholyl chloride, or atropine sulfate upon the gastric mucosa of mice. J Natl. Cancer Inst. 7:67. Gad, S.C., Dunn, B.J., Dobbs, D.W., Reilly, C., and Walsh, R.D. (1986). Development and validation of an alternative dermal sensitisation test: the mouse ear swelling test (MEST)., Toxicol. Appl. Pharmacol. 84: Griffith, J.F. et al. (1980). Dose-response studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye. Toxicol. Appl. Pharmacol. 55,: Hartzell, G.E. et al. (1990). Toxicity of smoke containing hydrogen chloride. ACS Symp. Ser., 425: Hoechst AG (1966). Farbwerke Hoechst AG Report 150/66. Jacobs, G.A. (1992). OECD eye irritation tests on two acids. J. Am. Coll. Toxicol. 11: 734. Loewy, A., and Munzer, E. (1923). Beiträge zur von der experimentellen Säurevergiftung., Bioch. Z. 134, MacEwean, J.D. et al. (1974). Toxic Hazard Research Unit, Annual Technical Report, NTIS AD- A Monsanto (1976). Unpublished report YO ; cited in European Commission- European Chemical Bureau IUCLID (2000) OECD (2002a). IUCLID Data Set for Hydrogen chloride (CAS No ). OECD (2002b). Screening Information Dataset (SIDS) Initial Assessment Report for Hydrogen chloride (CAS No ), UNEP Publications. Stumm-Tegethoff, B.F.A. (1969). Formaldehyde-induced mutations in Drosophila melanogaster ndependence of the presence of acids. Theor. Appl. Genet. 39: Upotn, P.K., and L Estrange, J.L. (1977). Effects of chronic hydrochloric and lactic acid balance and bone composition of the rat. Quart. J. of Exp. Physiol. 62:

328 York, M.H., Griffiths, A.E., Whittle, E., and Basketter, D.A. (1996). Evaluation of a human patch test for the identification and classification of skin irritation potential. Contact Dermatitis 34:

329 Toxicity Profile 2-Propanol or Isopopranol (CAS No ) Acute Toxicity The acute oral LD50 has been reported as 4,700 mg/kg, 5,300 mg/kg, 5,500 mg/kg, and 5,8400 mg/kg in rats; 4,500 mg/kg in mice; and 5,030 mg/kg, 7,800 mg/kg, and 7,900 mg/kg in rabbits (31, 272, 274, 275, 144, 35 Kimura et al., 1971; WHO, 1990; Smyth and Carpenter, 1948; Lehman and Chase, 1994; Munch and Schwartze, 1925; Munch, 1972). The acute dermal LD50 in rabbits has been reported to be 12,900 mg/kg (Smyth and Carpenter, 1948). The acute inhalation 8-h LC50 in rats was 19,000 ppm in females and 22,500 ppm in males (Laham et al., 1980). Exposure of rats to 16,000 ppm for 8 h resulted in four deaths out of six animals (Smyth and Carpenter, 1948). Irritation Isopropanol applied to the intact or abraded skin of rabbits and guinea pigs produced negligible irritation (Nixon et al., 1975). Liquid isopropanol is moderately irritating to the eyes of rabbits (Griffith et al., 1980; WHO, 1990). Isopropanol produced little irritation when tested on the skin of six human subjects (Bevan, 2012). Sensitization There have been reports of isolated cases of dermal irritation and/or skin sensitization (Bevan, 2012). Except for three case reports, the positive reactions were observed on patch testing patients with contact dermatitis due to ethanol. These patients also had a positive reaction to ethanol. Repeated Dose Toxicity (Oral Studies only) In a drinking water study, rats ingested 0.5 to 10% of isopropanol for 27 weeks and showed decreased body weight gain but no gross or microscopic tissue abnormalities (Lehman and Chase, 1975). Increased formation of hyaline droplets in the proximal tubules was reported in male rats given 1 4% isopropanol in drinking water for 12 weeks (Pilegaard and Ladefoged, 1993). In another study, daily application of a 50% solution to the heads of rats for 187 days produced no apparent injury to the skin (Boughton, 1944). Isopropanol was administered in drinking water to three dogs for 7 months. The alcohol concentration was 4% from the end of the first month until the conclusion of the experiment. Tolerance to the alcohol developed as manifested by an increased degree of neuromuscular coordination at similar blood levels in habituated versus control animals 1

330 and by increased elimination of the alcohol. The only significant histopathological changes were noted in the kidneys of one dog that died (Lehman et al., 1945). Genotoxicity All genotoxicity assays conducted so far with isopropanol have been negative (OECD, 1997a,b; Bevan, 2012). Isopropanol was not mutagenic in the Salmonella microsomal assay using the spot test, in strains TA98, TA100, TA1535, and TA1537 with and without S9 from the livers of Aroclor-induced rats (Abbondandolo et al., 1980). Isopropanol was not mutagenic in Salmonella strains TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538 with and without metabolic activation, when tested using a plate-incorporation modification of this assay (Zeiger et al., 1992). Isopropanol was inactive in mutagenicity tests in Neurospora crassa (Brockman et al., 1984) and isopropanol did not enhance adenovirus (SA7) transformation using Syrian hamster embryo cells (Heidelberger et al., 1983). Isopropanol was inactive in a sister chromatid exchange assay with and without S9 metabolic activation (von der Hude et al., 1987). Isopropanol was inactive in an in vitro CHO/HGPRT gene mutation assay and in an in vivo bone marrow micronucleus assay in mice (Kapp, Jr. et al., 1993). Carcinogenicity No carcinogenicity studies have been conducted by the oral route. CD-1 mice were exposed by inhalation to 0, 500, 2500, or 5000 ppm of isopropanol vapor for 6 h/day, 5 days/week for 18 months. An additional group of mice (all exposure levels) were assigned to a recovery group which were exposed to isopropanol for 12 months and then retained until study termination at 18 months. There was no increased frequency of neoplastic lesions in any of the isopropanol-exposed animals. Nonneoplastic lesions were limited to the testes (males) and the kidney. In the testes, enlargement of the seminal vesicles occurred in the absence of associated inflammatory or degenerative changes. The kidney effects included tubular proteinosis and/or tubular dilatation. The incidence of testicular and kidney effects was not increased in the isopropanol-exposed recovery animals (Burleigh-Flayer et al., 1997). Fischer 344 rats were exposed to 0, 500, 2500, or 5000 ppm of isopropanol vapor for 6 h/day, 5 days/week for 24 months. The mortality rates for all male rats were 82, 83, 91, and 100% for the 0-, 500-, 2500-, and 5000-ppm groups, respectively. The corresponding values for the female rats were 54, 48, 55, and 69%. The main cause of death for the 5000-ppm rats (both sexes), as well as for much of the mortality of the 2500-ppm male rats, was chronic progressive nephropathy. Isopropanol exposure resulted in impaired kidney function, as indicated by various urine chemistry changes in male (2500- and 5000-ppm) and female (5000-ppm) rats. Animals in these groups also exhibited histopathological effects in the kidneys which appeared to be an exacerbated form of 2

331 chronic progressive nephropathy. The only neoplastic lesion noted was increased interstitial (Leydig) cell adenomas in male rats. The frequency of these tumors, although elevated above the control animals, was within the historical control range of the testing facility and within the range reported for control animals from the National Toxicology Program carcinogenicity studies (Burleigh-Flayer et al., 1997). The carcinogenic potential of isopropanol was evaluated via inhalation using three strains of mice. Male mice were exposed to 7.5 ppm of isopropanol for 3 to 7 h/day, 5 days/week for 5 to 8 months. Animals were killed at either 8 or 12 months. There was no significant increase in the number of lung tumors observed. Similarly, no increases in lung tumors were observed in the same strains of mice that received subcutaneous injections of isopropanol once weekly for 20 to 40 weeks (Weil et al., 1952). Reproductive Toxicity In a two-generation reproductive toxicity study, Sprague Dawley rats were dosed by oral gavage with 0, 100, 500, or 1,000 mg/kg isopropanol. There were seven parental deaths that were considered treatment-related: two high-dose F0 females, two F1 high-dose females, one mid-dose F0 female, and two low-dose F1 males. Lactation body weight gain was increased in the 500- and 1000-mg/kg females in both generations, and liver and kidney weights were increased in the 500- and 1,000-mg/kg groups in both sexes. Centrilobular hepatocyte hypertrophy was noted in some 1,000 mg/kg F1 males. There were some kidney effects in the 500- and 1000-mg/kg F0 males and in all treated F1 male rats. The kidney effects were characterized by an increased number of hyaline droplets in the convoluted proximal tubular cells, epithelial degeneration and hyperplasia, and proteinaceous casts. Increased mortality occurred in the high-dose F1 offspring during the early postnatal period; no other clinical signs of toxicity were observed in the offspring from either generation. Offspring body weight, however, in the 1,000-mg/kg group was reduced during the early postnatal period. There was significant mortality in the F1 weanlings (18/70) before the selection of the F1 adults. A statistically significant reduction was observed in the F1 male mating index of the 1,000-mg/kg group (73 versus 97% in the controls). There were no other treatment-related effects on reproduction, including fertility and gestational indices, or histopathology of the reproductive organs (Bevan et al., 1995). A benchmark dose level of 420 mg/kg/day (lower bound on dose associated with a 5% response rate for the decrease in the male mating index was calculated (Allen et al., 1998). In a one-generation reproductive/embryotoxicity study, male and female Wistar rats were given 0, 0.5%, 1.0% or 2.0% isopropanol in their drinking water. The calculated intakes for males were 383, 686 and 1107 mg/kg-day (pre-mating) and 347, 625 and 1030 mg/kg-day (18 weeks of treatment). The calculated intakes for females were 456, 835 and 1206 mg/kg-day (premating); 668, 1330 and 1902 mg/kg-day (gestation); and 1053, 1948 and 2768 mg/kg-day (postpartum). An immediate, statistically significant dosedependent decrease occurred in water intake in the male rats. Intake was reduced ~5-14% (1% group; premating period) and ~30% (2% group; days 7-11 to end of study). 3

332 Overall mean feed consumption was significantly lower in treated versus control animals. Male body weights (2% only) were reduced throughout the study. Water consumption was initially reduced in the 1% and 2% females, but the 2% group recovered to only ~70% of the control values (premating); it continued to be reduced during the gestation and lactation period. Mean maternal body weights were reduced (all treated groups) at the start of gestation, with partial recovery during the gestation period except for the 2% group. Overall weight gain during gestation in these groups were similar to the controls. Following parturition from PND 4 onward, the 2% dams had significantly lower body weights. There were no infertile males in any group, and no treatment-related effect on female fertility or on length of gestation. The number of pups/litter on GD 1 was reduced in the 2% group; because it was not replicated in the embryotoxicity portion, an increase in pup mortality during partuition or GD 0, followed by cannibalism of the dead pups by the dam was suggested. No macroscopic abnormalities were seen in females; nor was there any treatment-related histopathological changes seen in the reproductive tissue in the 2% parental animals. Absolute kidney weight and relative kidney, liver, and spleen weights were increased in the 2% F0 males; increased absolute liver and kidney weights and relative liver weights in the 2% F0 females. In the embryotoxicity portion, there was a statistically significant increase in the total number of preimplantation losses in the 2% animals. Whole body edema was seen in 40% of the fetuses in 3/8 litters in the 2% group. No macroscopic abnormalities of the viscera of these fetuses were detected, and the incidence of edema was not related to gender. In the one-generation portion, postnatal pup survival and in the average pup weight (by PND 7) were decreased in the 2% group. F1 generation animals of both sexes showed increased relative liver weights at all dose levels, and the 2% males had higher relative kidney weights. A slight but significant decrease in absolute brain weight and increase in relative empty cecum weights in both sexes of the 2% F1 generation group. No treatment-related gross abnormalities were observed in the F1 generation animals at necropsy (BIBRA, 1988). The effects of isopropanol (2.5% in drinking water) on the reproduction and growth of rats was assessed in a multigenerational study. No reproductive toxicity was observed (Lehman et al., 1945). Isopropanol was administerd as a 3% solution in drinking water to Wistar rats. Reduced parental body weight gain, food, and water consumption was observed in the treated animals compared with the controls. Fertility, litter size, and pup weights at postnatal days 4 and 21 were reduced in treated animals compared with the controls. In the second generation, the isopropanol concentration was reduced to 2%, and there were essentially no effects (Gallo et al., 1977). Developmental Toxicity Isopropanol was given at concentrations of 0, 0.5%, 1.25% or 2.5% in the drinking water to female Wistar rats on GD 6 to 16. The calculated intakes of isopropanol during GD 6-16 were 596, 1242 and 1605 mg/kg/day. There was an immediate reduction in water intake in the 2.5% dose group, and this was statistically significant throughout the 4

333 treatment period when compared to controls. A smaller reduction in water intake was also seen in the 1.25% females (statistically significant during GD 6-9), with no change in the 0.5% females. Palatability of the drinking water may have been the problem since water intake significantly increased the first day following the end of the treatment period for all dose groups. Feed consumption patterns paralleled the water consumption during and after treatment in the mid- and high-dose groups. Overall, mean body weights of the 2.5% females were lower than the controls from GD 7 to termination. Effects on weight gain in the 0.5% and 1.25% females were limited to a failure to gain weight during the first (0.5%) and second (1.25%) day of treatment. There were no treatment-related effects in postimplantation loss, mean number of implantation sites or live fetuses. There was a slight dose-dependent decrease in mean litter weight and a significant decrease in mean fetal weight in the 1.25% and 2.5% groups. A statistically significant increase in variations was observed, indicative of a lower degree of ossification in the treated animals There was a dose-dependent decrease in the number of fetuses with the 4 th sacral arch and a dose-dependent increase in the number of fetuses with less than 2 caudal arches. The sternum also showed reduced ossification because there were increased numbers of fetuses with small, absent, or incompletely ossified sternebrae (BIBRA, 1987). In a rat developmental study, female Sprague Dawley rats were dosed by oral gavage with either 0, 400, 800, or 1200 mg/kg of isopropanol during gestational days 6 to 15. Two dams (8%) died at 1200 mg/kg and one dam (4%) died at 800 mg/kg. At 1200 mg/kg, maternal body weights were reduced throughout gestation (GS 0-20; 89.9% of control value), associated with reduced gravid uterine weight. There were no other treatment-related effects on the dams. Fetal body weights per litter were also significantly reduced at the 800 and 1200 mg/kg dose levels, but there were no teratogenic effects. There were no adverse maternal or developmental effects at 400 mg/kg (Tyl et al., 1994). In a rabbit developmental study, female New Zealand white rabbits were dosed by oral gavage with either 0, 120, 240, or 480 mg/kg of isopropanol during gestational days 6 to 18. At 480 mg/kg, isopropanol was unexpectedly toxic to pregnant female rabbits, resulting in the deaths of four does (26%). Maternal body weights were significantly reduced during treatment (gestational days 6 18) and were associated with reduced maternal food consumption during this period. Profound clinical signs were noted at 480 mg/kg and included flushed and/or warm ears, cyanosis, lethargy, and labored respiration. No adverse maternal effects were noted at 120 or 240 mg/kg. There were no developmental or teratogenic effects at any dose tested (Tyl et al., 1994). Isopropanol was given by oral gavage to Sprague Dawley rats from gestational days 6 to 21 in doses of 0, 200, 700, or 1200 mg/kg. The dams were allowed to deliver, litters were culled on postnatal day (PND) 4, pups were weaned on PND 22, and their dams were killed. Weaned pups were assessed for day of testes descent or vaginal opening, motor activity, auditory startle, and active avoidance. The pups were killed on PND 68. Some of the pups were taken from each dose group and were perfused in situ for pathological examination of the central nervous system. There were no biologically significant findings in the behavioral tests, no changes in organ weights, and no pathological 5

334 findings of note. Thus, there was no evidence of developmental neurotoxicity from isopropanol exposure (Bates et al., 1994). Key Study/Critical Effect for Screening Criteria The benchmark dose level of 420 mg/kg/day from the two-generation reproductive toxicity study will be used to derive a drinking water guidance value for 2-propanol. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 420/100 = 4 mg/kg/day References Abbondandolo, A. et al. (1980). Mutat. Res. 79 : Allen B., Gentry R., Shipp A., and Van Landingham, C. (1998). Regul. Toxicol. Pharmacol. 28: Bates, H.K. et al. (1994). Fundam. Appl. Toxicol. 22: Bevan, C., Tyler, T.R., Gardiner, T.H., Kapp, R.W., Jr., Andrews, L., and Beyer, B.K. (1995). J. Appl. Toxicol. 15: Bevan, C. (2012). Monohydric Alcohols C1 to C6. In: Patty s Industrial Hygiene and Toxicology, 6th Edition, Volume 3, Chapter 55 (Bingham and B. Cohrssen, Eds.), pp , John Wiley and Sons, Inc., NY. BIBRA (1987). Unpublished report, The British Industrial Biological Research Association; reviewed in Faber, W.D. et al. (2008). Birth Defects Research (Part B) 83, (2008). BIBRA (1988). Unpublished report, The British Industrial Biological Research Association; reviewed in Faber, W.D. et al. (2008). Birth Defects Research (Part B) 83: Boughton, L.I. (1944). J. Am. Pharm. Assoc. 33: Brockman, H.E. et al. (1984). Mutat. Res. 133 : Burleigh-Flayer, H. et al. (1997). Fundam. Appl. Toxicol. 36: Gallo, M.A. et al. (1977). Toxicol. Appl. Pharmacol. 41:

335 Griffith, J.F. et al. (1980). Toxicol. Appl. Pharmacol. 55 : Heidelberger, C. et al. (1983). Mutat. Res. 114: Kapp Jr., R.W. et al. (1993). Environ. Mol. Mutagen. 22: Kimura, E.T. Ebert, D.M., and Dodge, P.W. (1971). Toxicol. Appl. Pharmacol. 19: Laham, S. et al. (1980). Drug Chem. Toxicol. 3: Lehman, A.J., and Chase, H.F. (1944). J. Lab. Clin. Med. 29: Lehman, A.J., Schwerma, H., and Rickards, E. (1945). J. Pharmacol. Exp. Ther. 85: Munch, J.C., and Schwartze, E.W. (1925). J. Lab. Clin. Med. 10: 985. Munch, J.C. (1972). Ind. Med. Surg. 41: Nixon, G.A., Tyson, C.A., and Wertz, W.C. (1975). Toxicol. Appl. Pharmacol. 31 : OECD (1997a). IUCLID Data Set for 2-Propanol (CAS No ), UNEP Publications. OECD (1997b). Screening Information Dataset (SIDS) Initial Assessment Report for 2- Propanol (CAS No ), UNEP Publications. Pilegaard, K., and Ladefoged, O. (1993). In Vivo 7: Smyth, H.F., and Carpenter, C.P. (1948). J. Ind. Hyg. Toxicol. 30: Tyl, R.W. et al. (1994). Fundam. Appl. Toxicol. 22: von der Hude, W. et al. (1987). Environ. Mutagen. 9: Weil, C.S., Smyth, H.F., and Nale, T.W. (1952). Arch. Ind. Hyg. Occup. Med. 5: WHO (1990). Environmental Health Criteria 103, World Health Organization, Geneva. Zeiger, E. et al. (1992). Environ. Mol. Mutagen. 19(Suppl. 21):

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337 Toxicity Profile Magnesium chloride (CAS No ) Acute Toxicity The oral LD50 of magnesium chloride hexahydrate in rats is >2,000 mg/kg. The dermal LD50 of magnesium chloride hexahydrate in rats is >2,000 mg/kg. Irritation Magnesium chloride hexahydrate was applied topically to the EPISKIN-SMätissue for 15 min followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. The test item showed no irritant effects. Single ocular instillation of magnesium chloride hexahydrate to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 6, 4 and 2 days postinstillation in animal no. 1, 2 and 3, respectively. Based on these findings, magnesium chloride hexahydrate is not an eye irritant. Sensitization Magnesium chloride hexahydrate was not a skin sensitizer when tested in guinea pigs. Repeated Dose Toxicity In an OECD 422 study, Wistar rats at dosages of 250, 500 and 1,000 mg/kg magnesium chloride hexahydrate revealed no major toxicological findings. The cause of death of animals during the conduct of the study could not be determined (a plausible cause may be gavage error or regurgitation). The NOAEL for this study is 1,000 mg/kg/day. Groups of 50 male and 50 female B6C3F1 mice were given magnesium hexahydrate at dose levels of 0, 0.5% and 2% in the diet for 96 weeks, after which all animals received the control diet for 8 weeks and were then necropsied. In the 2% females, a decrease in body weight was observed. However, survival rates did not differ between the treatment and control groups for males or females and clinical signs and urinary, hematological or serum clinical chemistry parameters showed no treatment-related effects. Therefore, this change was considered not to be of biological significance. The NOAEL for female and male mice was 2% in feed, which corresponded to 3,930 and 2,810 mg/kg/day, respectively. 1

338 Genotoxicity Magnesium chloride was not genotoxic in the in vitro mouse lymphoma assay and in the in vitro chromosomal aberration assay using human lymphocytes. Carcinogenicity Male and female B6C3F1 mice were given in their fed 0, 0.5% or 2% magnesium chloride hexahydrate for 96 weeks, after which all animals received the control diet for 8 weesk and were then necropsied. Tumors were mainly found in the skin/subcutis, liver and lymphatic system. With the exception of a significant decrease in the incidence of liver tumors among the 2% males, there were no differences in the tumor incidences between the treated and control animals. Reproductive/Developmental Toxicity In an OECD 422 study, Wistar rats at dosages of 0, 250, 500 or 1,000 mg/kg showed no treatment-related reproductive or developmental effects. The NOAEL for this study is 1,000 mg/kg/day. Key Study/Critical Effect for Screening Criteria The Australian drinking water guideline value for chloride may apply to magnesium chloride. Reference ECHA REACH database: 2

339 Toxicity Profile Magnesium nitrate (CAS No ) Acute Toxicity The acute oral LD50 in rats is >5,000 mg/kg. Irritation No studies are available. Sensitization Magnesium nitrate is not a skin sensitizer based on results of a Local Lymph Node Assay (LLNA). Repeat Dose Toxicity Studies No studies are available. Genotoxicity Magnesium nitrate hexahydrate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Carcinogenicity No data are available. Reproductive Toxicity No data are available. Developmental Toxicity No data are available. 1

340 Key Study/Critical Effect for Screening Criteria The Australian drinking water guidance value for nitrates may apply to magnesium nitrate. Reference ECHA REACH database: 2

341 Benzenesulfonic acid, 4-ethyenyl-, sodium salt, homopolymer (CAS No ) Acetic acid ethenyl ester, polymer with ethanol (CAS No ) Vinylidene chloride/methacrylate copolymer (CAS No ) Polyvinyl acetate, partially hydrolyzed (CAS No ) THIS IS NOT THE CORRECT CASRN All of the these substances are polymers. Mammalian toxicity data was located for only for acetic acid, ethenyl ester, polymer with ethanol (CAS No ) on a supplier s MSDS. However, all of the polymers listed above are expected to exhibit similar toxicity profiles. As polymers, they are not expected to be absorbed by the oral, dermal or inhalation routes of exposure due to their large molecular weights. They are also expected to be stable in biological media and resistant to metabolism. This polymers are expected to be essentially acutely non-toxic by the oral, dermal and inhalation routes of exposure. The oral LD50 in rats for CAS No was reported to be >11,000 mg/kg. Under certain conditions, dusts may be formed, which is to be considered as a nuisance dust. The polymers are expected to be non-irritating and not dermal sensitizers. No irritation or sensitization was observed in guinea pigs from CAS No These polymers are not expected to exhibit any systemic toxicity. Reference DuPont (2006). MSDS for ELVANOL 60-30, revised 27-Oct-2006.

342 Toxicity Profile Sodium chloroacetate (CAS No ) Sodium chloroacetate has been reviewed in the OECD-SIDS program (OECD, 1994). Acute Toxicity The oral LD50 is 76 mg/kg in rats (Woodard et al., 1941); 165 mg/kg (Morrison, 1946) and mg/kg (Woodard et al., 1941) in mice; and 79 mg/kg in guinea pigs (Woodard et al., 1941). The dermal LD50 is >2,000 mg/kg in rats (Hofmann and Jung, 1988). A number of cattle died after accidental exposure to sodium chloroacetate. The doses involved were at least 17 to 70 mg/kg and probably in the order of 170 mg/kg. Extensor paralysis of the limbs, tremors, and convulsions were observed in three cows before death (Quick et al., 1983). In cows exposed to 50 mg/kg, lethargy lasting 24 hours was observed. A doubling of the dose resulted in severe symptoms of intoxication (diarrhea, muscular twitching and loss of muscle tone) with recovery after two weeks. Death occurred within nine hours later (Dalgaard-Mikkelsen and Rasmussen, 1961). Irritation When tested in an OECD 404 guideline study, 500 mg of sodium chloroacetate was not irritating to the skin of rabbits (Kreling and Jung, 1988). When tested in an OECD 405 guideline study, 100 mg of sodium chloroacetate was not irritating to the eyes of rabbits (Kreiling and Jung, 1988). Sensitization No data are available. Repeated Dose Toxicity Sprague-Dawley rats (10/sex/dose) were dosed by oral gavage with 0, 15, 30, 60 or 120 mg/kg sodium chloroacetate for 90 consecutive days. At 120 mg/kg, 30% of the females and 80% of the males died within the first two days of treatment. Hemorrhagic and congested lungs (possibly a postmortem change) were observed in early deaths (1-3 days), whereas liver lesions were observed in later deaths. The levels of creatine, blood calcium and blood urea nitrogen were significantly elevated in males in all dose groups, but creatine levels were elevated in the 30 and 60 mg/kg females. Increased serum levels 1

343 of alanine aminotransferase and separate aminotransferase were seen in both males and females, but a significant trend could not be demonstrated. Chronic heart inflammation was present in both sexes, particularly at the higher dose levels. Relative liver and kidney to bodyweight ratios were increased in both sexes. Histopathological examination showed a significant increase in chronic renal nephropathy and increased splenic pigmentation in the 60 mg/kg males. Chronic progressive nephropathy (CPN) is a common spontaneous kidney disease of laboratory rat strains (Gray, 1977) that is both a degenerative and regenerative entity exhibiting a high rate of tubule cell proliferative activity throughout its progression (Hard and Khan, 2004; Short et al., 1989). It is a progressive disease with known physiological factors that modify disease progression, such as high dietary protein. Hard et al. (2009) compared CPN in rats and human nephropathies and concluded that CPN is a distinctive entity in rats with no counterpart in human disease. A number of chemicals have been shown to exacerbate CPN in rats, particularly in Sprague-Dawley rats (Hard et al., 2012). Because of the uniqueness of CPN to rats, the chemical-induced exacerbation of CPN in male rats should not be considered relevant to human health risk assessments (Hard et al., 2012) Thus, the LOAEL for this study is considered to be 60 mg/kg/day based on the increased incidence of splenic pigments, brown and localized in splenic macrophages, which were statistically significant in the 60 mg/kg/day male rats. The NOAEL is 30 mg/kg/day. Genotoxicity No data are available. Carcinogenicity No data are available. Reproductive Toxicity No data are available. Developmental Toxicity An unpublished study was cited in the OECD-SIDS dossier on chloroacetate (OECD, 2004). Female rats were dosed with 0, 17, 35, 70 or 140 mg/kg sodium chloroacetate during GD Mean adjusted percentage weight gain for GD 0 to 20 was reduced in the 140 mg/kg dams. Reduction in weight gain was also observed in the 35, 70 and 140 mg/kg groups for the first three days of dosing. Mean cardiovascular malformations, 2

344 comprising of levocardia, were significantly elevated over controls for the 140 mg/kg group. The mean frequency per litter of soft tissue malformations ranged from 1.2% (controls) to 6.37% (140 mg/kg); there was no dose-related effect. The NOAELs for maternal and developmental effects were 70 mg/kg/day. Key Study/Critical Effect for Screening Criteria A 90-day rat oral gavage toxicity study was conducted. The LOAEL for this study is considered to be 60 mg/kg/day based on the increased incidence of splenic pigments, brown and localized in splenic macrophages, which were statistically significant in the 60 mg/kg/day male rats. The NOAEL is 30 mg/kg/day. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral Reference Dose = 30/1,000 = 0.03 mg/kg/day Drinking water guideline value = 0.11 ppm References Dalgaard-Mikkelsen, S., and Rasmussen, F. (1961). Nordisk Veterinaermedicin (Scandinavian Veterinary Medicine) 13: 271. Daniel, F.B., Robinson, M., Stober, J.A., Page, N.P., and Olson, G.R. (1991). Toxicology 67: Gray, J.E. (1977). Crit. Rev. Toxicol. 5: Hard, G.C., and Khan, K.N. (2004). Toxicol. Pathol. 32: Hard, G.C., Betz, L.J., and Seely, J.C. (2012). Toxicol. Pathol. 40: Hard, G.C. et al. (2013). Toxicol. Sci. 132: Hofmann and Jung (1988). Hoescht AG Kreiling and Jung (1988). Hoescht AG. Morrison, J.L. (1945). J. Pharmacol. Exp. Ther. 86: OECD (1994). OECD-SIDS dossier on Sodium Chloroacetate (CAS No ), UNEP Publications. 3

345 Quick, M.P. et al. (1983) Veterinary Records 113: Short, B.G., Burnett, V.J., and Swenberg, J.A. (1989). Toxicol. Appl. Pharmacol. 101: Woodard, G. et al. (2004). J. Ind. Hyg. Toxicol. 23:

346 Toxicity Profile Talc (CAS No ) Talc refers to both mineral talc and industrial mineral products that are marketed under the name talc and contain proportions of mineral talc that range from about 35% to almost 100%. Industrial talc generally refers to products that contain abundant minerals other than talc; cosmetic talc now normally contains >98% talc but the content may have been lower in the past. Pharmaceutical talc contains >99% talc. Talcum powder is cosmetic-grade talc. Talc is a mineral composed of hydrated magnesium silicate with the chemical formula Mg3Si4O10(OH)2 or H2Mg3(SiO3)4. Talc is not soluble in water. The U.S. Food and Drug Administration (FDA) regulates talc and states that it is generally recognized as safe for use in color additives in foods, drugs and cosmetics, and in paper, paper products, cotton and cotton fabrics that come into contact with food. The Food Chemical Codex (2003) provides specifications for food-grade talc, including the statement that talc derived from deposits that are known to contain associated asbestos is not food grade. Under the voluntary guidelines initiated in 1976, the Cosmetic, Toiletry, and Fragrances Association stated that all cosmetic talc should contain at least 90% platy talc (hydrated magnesium silicate) that is free from detectable amounts (<0.5%) of fibrous, asbestos minerals. Toxicokinetics (Oral) The absorption and disposition of 3 H-labeled talc in rats, mice, and guinea pigs administered a single oral dose was studied by Phillips et al. (1978). The oral doses were 50 mg/kg for rats, 40 mg/kg for mice, and 25 mg/kg for guinea pigs. In rats, mice, and guinea pigs, more than 95% of the dose was excreted in the feces 3 to 4 days after dosing. Less than 2% of the radioactivity was recovered in the urine. This radioactivity probably reflected contamination of urine samples with feces. No radioactivity was found in the liver or kidneys of these animals. This information suggests that talc is not absorbed from the gastrointestinal tract following oral exposure. Acute Toxicity No data were located. 1

347 Repeated Dose Toxicity/Carcinogenicity Male and female Wistar rats were given in their diet 0 or 50 mg/kg of commercial talc [characteristics unspecified] for the life of the animals (average survival was 702 and 649 days, respectively). There was no significant difference in the talc-fed animals compared with control animals (Gibel et al., 1976). Male and female Wistar-derived rats were given in their diet 100 mg Italian talc (grade 00000; ready milled; mean particle size, 25 μm; containing 92% talc, 3% chlorite, 1% carbonate minerals and 0.5 1% quartz) per rat per day for 5 months (talc-containing diet was actually given for 101 days) and were then maintained on basal diet for life (average survival, 614 days). No differences in tumor incidence were noted between treated animals and control animals (eight/sex for an average survival of 641 days) (Wagner et al., 1977). In humans and experimental animals, the effects of talc are dependent on the route of exposure, and the dose and properties of the talc. Talc pneumoconiosis was somewhat more prevalent and severe among miners exposed to talc containing asbestiform minerals and/or asbestos than among those exposed to talc without such contaminants. However, the role of quartz and asbestos in the observed pneumoconiosis could not be ruled out. Among drug users, intravenous injection of talc present as a filler in the drugs resulted in microembolization in a variety of organs and alterations in pulmonary function. In animal studies, talc has been shown to cause granulomas and mild inflammation when inhaled. Observations of the effects that occurred in the lungs of rats exposed by inhalation to talc suggested that the operative mechanisms may be similar to those identified for carbon black, and talc is known to cause the release of cytokines, chemokines and growth factors from pleural mesothelial cells. IARC: There is inadequate evidence in humans for the carcinogenicity of inhaled talc not containing asbestos or asbestiform fibres. There is limited evidence in experimental animals for the carcinogenicity of talc not containing asbestos or asbestiform fibres. Inhaled talc not containing asbestos or asbestiform fibres is not classifiable as to its carcinogenicity (Group 3). Genotoxicity The IARC (1987) review of talc included unpublished results from a 1974 study conducted by Litton Bionetics that showed no mutagenic activity for talc in vitro or in vivo. Talc did not induce mutations in Salmonella typhimurium strains TA1530 or HisG46, or in the yeast, Saccharomyces cerevisiae. No chromosomal aberrations were observed in human fibroblasts treated with talc in vitro. In vivo tests conducted in rats gave negative results for induction of chromosomal aberrations in bone marrow cells and dominant lethal mutations in germinal cells. 2

348 Three samples of respirable talc failed to elicit significant unscheduled DNA synthesis (10, 20 and 50 μg/cm 2, 24 hours), sister chromatid exchange or aneuploidy (2, 5, 10 and 15 μg/cm 2, 48 hours) in rat pleural mesothelial cells, in contrast to various positive controls. The three samples, i.e. Spanish talc (No. 5725), Italian talc (No. 5726) and French talc (No. 7841), contained 90 95% talc; the remaining contents were chlorite and dolomite. Electron microscopy analysis revealed that talc particles were taken up by the rat pleural mesothelial cells, but no aneuploidy was observed in metaphases (Endo- Capron et al., 1993). Reproductive Toxicity No data are available. Developmental Toxicity No teratologic effects were observed in hamsters, rats, mice, or rabbits after oral administration of talc. The doses used were 1,600 mg/kg for rats and mice on days 6 through 15 of gestation; 1,200 mg/kg for hamsters on day 6 through 10 of gestation; and 900 mg/kg for rabbits on days 6 through 18 of gestation. Key Study/Critical Effect for Screening Criteria There are no adequate studies for which to derive a oral reference dose. Talc is poorly absorbed from the gastrointestinal tract, if at all, and the limited data available by the oral route indicate that talc is essentially non-toxic by the oral route of exposure. References Endo-Capron, S., Renier, A., Janson, X. et al. (1993). In vitro response of rat pleural mesothelial cells to talc samples in genotoxicity assays (sister chromatid exchanges and DNA repair). Toxicol. In Vitro 7: Gibel, W., LOhs, K., Horn, K.H., Wildner, G.P., and Hoffman, F. (1976) Experimental study on carcinogenic activity of asbestos filters (Ger.). Arch. Geschwulstforsch. 46: IARC (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 42, International Agency for the Research on Cancer, World Health Organization, Lyon, France; cited in NTP (1993). IARC (2010). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Carbon Black, Titanium Dioxide, and Talc, Volume 93, International Agency for the Research on Cancer, World Health Organization, Lyon, France. 3

349 NTP (1993). Toxicology and Carcinogenesis of Talc (CAS No ) in F344/N Rats and B6C3F1 Mice (Inhalation Studies), NTP TR 421, NIH Publication No , National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. Phillips, J.C., Young, P.J., Hardy, K., and Gangolli, S.D. (1978). Studies on the absorption and disposition of 'H-labelled talc in the rat, mouse, guinea-pig and rabbit. Food Cosmet. Toxicol. 16: Wagner, J.C., Berry, G., Cooke, T.J., Hill, R.J., Pooley, F.D., and Skidmore, J.W. (1977). Animal experiments with talc. In: Inhaled Particles (W.H. Walton and B. McGovern, Eds.), Vol. IV, Part 2, pp , Pergamon Press, Oxford. 4

350 Toxicity Profile Tetramethylammonium chloride (CAS No ) Surrogates: Didecyldimethylammonium chloride (CAS No ) Dodecyltrimethylammonium chloride (CAS No ) No data could be located on tetramethylammonium chloride. Toxicity data are available for didecyldimethylammonium chloride (DDAC) and dodecyltrimethyl-ammonium chloride (DTAC). Thus, these two substances have been used as surrogates for the alkylated quaternary chloride. Acute Toxicity The oral LD50 of 100% DDAC has been reported to be 84 mg/kg in rats and 268 mg/kg in mice. The oral LD50 of a 5-25% solution of DDAC in rats was reported to be 1,190 mg/kg (595 to 297 mg a.i./kg). The acute dermal LD50 of a 80% solution of DDAC in rats is 3,342 mg/kg (2674 mg a.i./kg). The acute oral LD50 of DTAC (24.7% aq. soln.) in rats is 490 mg/kg. The acute oral LD50 of DTAC (37.35% aq. soln.) in rats is 560 mg/kg. Irritation DDAC is a severe skin and eye irritant to rabbits. Sensitization DDAC is not a dermal sensitizer to guinea pigs or to humans. Repeatd Dose Toxicity Mice were given in their diet 0, 100, 300, 600, 1,000 or 3,000 ppm DDAC for days. For the lowest four dose groups, these doses correspond to mean intake levels of approximately 18, 52, 107, and 182 mg/kg for males, and 23, 68, 134, and 224 mg/kg for females. High mortality in the 3,000 ppm groups prohibited calculation of daily intakes. Treatment of mice with 3,000 ppm DDAC in the diet for several days resulted in virtually 1

351 100% mortality in both sexes, with only one male surviving to termination of the study. Death was attributed to treatment-related severe wasting and dehydration resulting from gastrointestinal effects. Treatment with 1,000 ppm DDAC produced a 5% decrease in body weight in males with associated decreases in body weight gain. Similar depressed body weight in females from this group was assumed to be related to DDAC exposure. No other changes were observed in males or females in the other dose groups. The NOEL for this study is 600 ppm (107 and 134 mg/kg/day for males and females, respectively). Rats were given in their diet 0, 100, 300, 600, 1,000 or 3,000 ppm DDAC for days. For the lowest four dose groups, these doses correspond to mean intake levels of approximately 6, 18, 37, and 61 mg/kg for males, and 8, 22, 44, and 74 mg/kg for females. High mortality in the 3,000 ppm group prohibited calculation of mean daily intakes. Dietary exposure to 3,000 ppm DDAC resulted in 80% mortality in both sexes. The three rats of each sex of this group that survived to the end of the study exhibited markedly reduced body weights, fluid- or gas-filled intestines at necropsy, and inflammation of the beginning of the large intestines (typhlitis). Pathological changes in clinical chemistry included decreased serum glucose and protein concentrations in both sexes, decreased albumin and globulin concentrations in females, and increased erythrocyte count and hemoglobin and hematocrit concentrations in males. Administration of 1,000 ppm of less of DDAC resulted in no treatment-related effects. The NOEL for this study is 61 and 74 mg/kg/day for males and females, respectively. Male and female beagle dogs were given DDAC in their diet at doses of 0, 5, 15 and 50 mg/kg for 90 days. There was marked decrease in body weight gain, food consumption and food efficiency in the 50 mg/kg dose group. Clinical chemistry, hematology, urinalysis, and pathological results did not reveal any treatment-related effects. The NOAEL for this study is 15 mg/kg/day. Beagle dogs were dosed orally with 0, 3, 10, or 30 mg/kg DDAC for 52 weeks. During the first four and a half weeks of the study, several dogs in the 30 mg/kg group showed potentially life threatening decreases in body weight and food consumption. The dose was therefore decreased to 20 mg/kg/day. In some cases, depressions in weight and food consumption were so severe that the dogs in this group were removed from treatment completely during study days 31-36, and then reinstated at the 20 mg/kg/day. Administration of 3 or 20 mg/kg/day was not associated with mortality, changes in organ weights, gross pathological findings, ophthalmologic changes, or microscopic changes in selected organs and tissues. The 20 mg/kg/day dose was associated with decreases in mean erythrocyte counts; hemoglobin and hematocrit values; and mean total cholesterol, total protein, and albumin values. The 10 and 20 mg/kg/day doses were associated with an increased incidence of emesis, salivation, and soft/mucoid/liquid feces compared to controls. The NOEL for systemic toxicity was considered to be 10 mg/kg/day. Sprague-Dawley rats were given in their diet 0, 300, 750, or 1,500 ppm DDAC for at least 104 weeks. These doses corresponded to approximate DDAC intakes of 13, 32, and 64 mg/kg/day for males, and 16, 41, and 83 mg/kg/day for females. Treatment-related 2

352 decreases in body weight and food consumption in both males and females were observed in the 1,500 ppm group. In addition, possible treatment-related microscopic changes including hyperplasia of bile ducts in female rats and changes in mesenteric lymph nodes in male and female rats related to blood in the sinuses were observed in the 1,500 ppm group. No treatment-related effects were seen in the type of incidence of clinical signs, survival, palpable masses, clinical pathology, organ weights, gross anatomic pathology, or ophthalmology. The NOEL for toxicity was considered to be 750 ppm (32 and 41 mg/kg/day for males and females, respectively). CD-1 mice were administered in their diet 0, 100, 500, or 1,000 ppm DDAC for at least 78 weeks. The approximate mean intake levels were 15, 76.3, and mg/kg/day for males and 18.6, 93.1, and mg/kg/day for females. Treatment-related findings included decreased body weights and body weight gains in both male and females from the 1,000 ppm groups. There were no treatment-related clinical signs of toxicity, increases in palpable masses, changes in food consumption, differences in organ weights or observations at necropsy or differences in histopathological findings. The NOEL for this study was considered to be 500 ppm (76.3 and 93.1 mg/kg/day for males and females, respectively). Genotoxicity Several studies evaluating the mutagenicity of DDAC have all given negative results. DDAC was not mutagenic to several strains of S. typhimurium in a bacterial reverse mutation assay with or without metabolic activation. DDAC was tested in the CHO/HGPRT forward mutation assay and was not mutagenic in the presence or absence of metabolic activation. DDAC did not induce chromosomal aberrations in CHO cells with or without metabolic activation. In the in vitro rat primary hepatocyte unscheduled DNA synthesis (UDS) assay, DDAC did not induce significant increases in UDS. No chromosomal damage was observed in the bone marrow of rats in an in vivo cyogenetics assay. DTAC (24.7% aq. soln.) was not mutagenic to bacteria in a reverse mutation assay with or without metabolic activation. DTAC (24.7% aq. soln.) was not mutagenic in a mouse lymphoma assay with or without metabolic activation. DTAC was inactive in a rat primary hepatocyte UDS assay. DTAC was not genotoxic when given by oral gavage in a rat bone marrow micronucleus assay. Carcinogenicity Sprague-Dawley rats received DDAC in their diet at concentrations of 0, 300, 750 or 1,500 ppm for at least 104 weeks. These doses corresponded to approximate mean DDAC intakes of 13, 32, and 64 mg/kg/day for males, and 16, 41, and 83 mg/kg/day for females. DDAC was not considered to be carcinogenic under the conditions of this study. 3

353 CD-1 mice were administered in the diet 0, 100, 500, or 1,000 ppm DDAC for at least 78 weeks. The approximate mean intake levels were 15, 76.3, and mg/kg/day for males, and 18.6, 93.1, and mg/kg/day for females. DDAC was not considered to be carcinogenic under the conditions of this study. Reproductive Toxicity A two-generation reproductive toxicity study has been conducted with DDAC. Sprague- Dawley rats were given in their diet 0, 300, 750 or 1,500 ppm for two generations. Results indicate that continuous exposure to DDAC in the diet for two generations resulted in no adverse reproductive effects. Parental toxicity was observed at 1,500 ppm (~112.6 mg/kg/day), limited to body weight reduction, weight gain depression, and decreased food consumption. Postnatal toxicity at 1,500 ppm was indicated by reduced pup body weights. Based on decreased body weight/weight gain and food consumption, the parental Toxicity NOAEL = 750 ppm (56 mg/kg/day); LOAEL = 1500 ppm (113 mg/kg/day). Based on decreased pup body weight/weight gain, the reproductive toxicity NOAEL = 750 ppm (56 mg/kg/day); LOAEL = 1500 ppm (113 mg/kg/day). Developmental Toxicity Female NZ rabbits were given by oral gavage 0, 1.0, 3.0 or 10 mg/kg/day of DDAC during GD Four of the 16 does in the highest group died prior to GD 13. One doe at 10 mg/kg and 2 does at 1.0 mg/kg delivered early and were removed from the study. Nonlethal indications of maternal toxicity were evident at 3 and 10 mg/kg, as evidenced by reduced weight gain and clinical signs during the treatment period. Developmental toxicity, including increased incidences of fetal mortality and reduced fetal body weight per litter, were observed only at the highest dose. No teratogenicity was observed at any dose level. The NOEL for maternal toxicity is 1 mg/kg/day, and the NOEL for developmental toxicity is 3 mg/kg/day. Female Sprague-Dawley rats were given by oral gavage 0, 1, 10 or 20 mg/kg DDAC during GD No females died, aborted, delivered early, or were removed early from the study. Maternal toxicity was indicated at 10 and 20 mg/kg by characteristic clinical signs of audible respiration. Reductions in body weight and food consumption were also observed at 20 mg/kg during the treatment period. No evidence of developmental toxicity including teratogenicity was observed at any dose level. The NOEL for maternal toxicity was 1 mg/kg/day. The NOEL for developmental toxicity is >20 mg/kg/day. 4

354 Key Study/Critical Effect for Screening Criteria The lowest NOAEL was 10 mg/kg/day for dogs in the one-year chronic toxicity study. The LOAEL of 20 mg/kg/day was based on increased incidence of clinical signs in males and females, and decreased total cholesterol levels in females. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral Reference Dose = 20/100 = 0.2 mg/kg/day Drinking water guidance value = 0.7 ppm References Henderson, N.D. (1992). A Review of the Environmental Impact and Toxic Effects of DDAC. Prepared for: BC Environment, Ministry of Environment, Lands and Parks, Victoria, British Columbia. Found as Appendix B of the Robust Summaries for the Fatty Nitrogen Derived (FND) Cationics Category in the U.S. HPV Chemical Challenge Program. U.S. EPA (2006). Reregistration Eligibility Decision for Aliphatic Alkyl Quaternaries (DDAC), EPA739-R , Office of Prevention, Pesticides and Toxic Substances (OPPTS), U.S. Environmental Protection Agency, August 2006 (docket #EPA-HQ-OPP ). U.S. HPVIS (FND Cationics Category): 5

355 F.5 HALLIBURTON ALTERNATIVE FLUID SYSTEM APPENDIX F HUMAN HEALTH TOXICOLOGICAL PROFILES

356 Toxicity Profile Acetic acid (CAS No ) Acetic acid dissociates in aqueous media to H + and the acetate anion (CH3CO2 - ). It is naturally occurring as the acid in apple cider vinegar and other fruit-derived products. It and several of its salts are commonly used as food additives (e.g., as flavor enhancers) and are listed as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA). ph: 2.5 at 50 g/l and 20 o C pka: 20 o C Acute Toxicity The oral LD50 in rats is 4,950 mg/kg, and the dermal LD50 in rabbits is 1,060 mg/kg. The 4-hour LC50 in rats is 11.4 mg/l. Irritation In the EU, acetic acid is classified as a skin and eye irritant at concentrations <25%. At >25%, it is classified as corrosive to the skin. Sensitization No data are available. Repeated Dose Toxicity In an 8-month study, rats dosed with 60 mg/kg acetic acid (three times per week) developed hyperplasia in the esophagus and forestomach. Genotoxicity Acetic acid was not mutagenic to Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 in an in vitro bacterial mutation assay with or without metabolic activation. Acetic acid (in media adjusted to a ph of 8.5) was also not genotoxic in an in vitro chromosomal aberration test using Chinese Hamster Ovary cells with or without metabolic activation. 1

357 Carcinogenicity No adequate carcinogenicity studies have been conducted on acetic acid. Reproductive Toxicity No studies could be located. Developmental Toxicity There were no effects on implantations or on maternal or fetal survival in mice, rats, or rabbits at doses up to 1,600 mg/kg. Key Study/Critical Effect for Screening Criteria There are no repeat dose toxicity studies that were considered adequate for human health risk assessment. The only study reported on the toxicity of acetic acid by oral gavage showed effects of irritation (probably due to the ph of acetic acid) at the site of contact in the gastrointestinal tract. No systemic effects were reported. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has maintained a group ADI of not limited for acetic acid and its potassium and sodium salts. The Australian drinking water guidance value for ph may apply to acetic acid. References ECHA REACH: JECFA: U.S. EPA HPVIS database: 2

358 Toxicity Profile Alcohols, C6-C12, Ethoxylated Propoxylated (CAS No ) No toxicity information could located on alcohols, C6-C12, ethoxylated propoxylated (CAS No ). Toxicity information is available on alcohol ethoxylates. Alcohols, C6-C12, ethoxylated propoxylated is produced by the use of propylene oxide (PO) in addition to ethylene oxide (EO) during the alkoxylation of the alcohol(s). the PO is either incorporated randomly by alkoxylation within the epoxide mixture, or in a block by sequencing the addition of EO and PO to the reactor. Because of the similarity in structures, the toxicity data on the alcohol ethoxylates were used to read-across to alcohols, C6-C12, ethoxylated propoxylated. Alcohol ethoxylates are a class of non-ionic surfactants. The AEs have the basic structure Cx-yAEn. The subscript (x-y) following the C indicates the range of carbon chain units. AEs with carbon unit range between C8 to C18 are most commonly used in household detergent products. The hydrocarbon chain can be either linear or branched. AEs also contain an ethylene oxide (E) chain attached to the alcohol. The degree of ethylene oxide polymerization is indicated by the subscript (n) which indicates the average number of ethylene oxide units. In household products the average ethylene oxide chain length commonly ranges between 3 and 12 units. The toxicity profile has been developed from a risk assessment report on alcohol ethoxylates ranging from C8 to C18 prepared by the household cleaning product and chemical industry (HERA, 2009). Acute Toxicity The acute oral LD50 of C9-11AE2.5 in rats was calculated to be >4,000 mg/kg and <10,000 mg/kg. The acute oral LD50 of C7-9AE6 in rats was determined to be <2,000 mg/kg. The acute oral LD50 of C9-11AE8 in fasted rats was found to be 1,200 mg/kg. The oral LD50 in rats for C12-13AE6.5 is 2,100 mg/kg. The oral LD50 in rats for C12-15AE7 is 1,700 mg/kg. For C14-15AE11, the acute oral LD50 values were reported to be 720 mg/kg (neat) and 1,800 mg/kg (given as 50% (m/v) solution in corn oil). The oral LD50 in rats for C12-15AE11 is >2,00 mg/kg for males and between 1,000 and 2,000 mg/kg for females. The oral LD50 in rats for C14-15AE13 is 1,100 and 1,000 mg/kg in two separate studies. The dermal LD50 of C7-9AE6 was >2,000 mg/kg. The dermal LD50 values for AEs with an alkyl chain length of 9 11 carbon atoms in three different rat studies were determined to be >2,000 mg/kg and >4,000 mg/kg. An acute dermal LD50 value of >2,000 mg/kg was determined for C12-14AE3 and C12-14AE6 in two separate studies. The acute dermal LD50 of C12-15AE7 was determined to be >2,000 mg/kg. 1

359 The acute rat 4-hour LC50 of C9-11AE5 generated as a mist was determined to be >0.22 mg/l. Talmage (1994) reported that alcohol ethoxylates were not acutely toxic to rats at concentrations less than or equal to their saturated vapor concentrations in air. Acute toxic thresholds were reached only when animals were exposed to the undiluted test chemical in the form of a respirable mist or aerosol. Under these conditions, 1- or 4-hour inhalation LC50 values ranged from 1.5 to 20.7 mg/l. Some studies reported no mortalities (1-hour LC50 -study) occurred at concentrations as high as 52 mg/l. Irritation Alcohol ethoxylates with varying alkyl chain lengths and ethoxylation degree were found to be slightly to severely irritating to skin in rabbits and rats. The degree of irritation was dependent on the type of patches used (open application versus full occlusion), the exposure time as well as the concentration of the test material. In humans, AEs are less irritating to skin than in animals. Neat applications of a range AEs in a 4hour human patch test did not warrant these chemicals to be classified as skin irritants under EU legislation, while the same AEs would have been classified for skin irritation on the basis of animal data. Alcohol ethoxylates range from mildly to severely irritating to rabbit eyes. Rinsing the eyes directly after exposure with water for 20 to 30 seconds greatly reduced the severity of the effects such that these products produced only mildly irritating effects. The degree of irritation is concentration-dependent as dilutions in water cause proportionally lower irritation. Generally, concentration of 0.1% were non-irritating, and concentrations of 1 to 10% ranged from slight to moderately irritating. No relationship could be established between the chemical structures of the tested AEs and their eye irritation responses. Sensitization Alcohol ethoxylates should not be considered as skin sensitizers. A substantial amount of skin sensitization studies in guinea pigs following either the Magnusson-Kligman maximization or the Buehler testing protocol are available to evaluate the skin sensitization potential of AEs. Although a mild skin sensitization reaction was observed in a study following the Magnusson-Kligman protocol, the weight of evidence clearly supports the assessment that AEs should not be considered as skin sensitizers. This is further supported by clinical and market data that demonstrate the absence skin sensitization responses to AEs when tested under the conditions of the HRIPT or when used in AE containing consumer products. Repeated Dose Toxicity C10AE5 was fed to rats at doses of 0, 125, 250 or 500 mg/kg for 90 days. There were no 2

360 treatment-related clinical signs, body weight gain, food consumption or feed efficiency. There was a slight increase in absolute liver weights as well as a trend toward a dosedependent increase in the liver weight/body weight ratio, with a statistically significant increase in the high dose group. However, the histological evaluation did not reveal any indication of hepatotoxicity and therefore the increase in liver weights was not interpreted to be a toxicological effect. It can be considered to be an adaptive response as a result of extensive metabolism of the test compound by the liver. There were no other gross or histopathological changes that were considered treatment-related. The NOAEL for this study can be considered to be 500 mg/kg/day. Rats were given in their diet 0, 125, 250, 500, 1,000 or 3,000 ppm C9-11AE6 for 90 days. There were no signs of toxicity at any dose level. The NOAEL is 3,000 ppm (approximately 150 mg/kg/day). Rats were given in their diet 0, 0.04, 0.2 or 1.0% C9-11AE8 for 90 days. Lower body weight gain and decreased food consumption were noted in the 1% males and females and in the 0.2% females from week 1 through the end of the study. Further statistical analyses revealed a significant decrease in the mean body weight gain noted in the 1% females and the decreases in mean food consumption noted in the 1% males and females. The differences noted in the 0.2% females were not statistically significant. The investigators considered these observations to be the result of poor palatability of the test substance. There were no other treatment-related effects. The NOAEL for this study is 1.0% (about 400 mg/kg/day). Rats were fed C12-15AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% or 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the >0.25% groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the >0.25% groups. There were not treatment-related changes in hematology, urinary and clinical chemistry parameters. Liver cell enlargement was seen in the >0.125% groups, suggesting increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (102 mg/kg/day). Rats were fed C12-14AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% or 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the >0.25% groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the >0.25% groups. There were no treatment-related changes in hematology, urinary and clinical chemistry parameters. Liver cell enlargement was seen in the >0.125%, suggesting increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (110 mg/kg/day). Wistar rats were fed C14-15AE7 in the diet at concentrations of 0, 300, 1,000, 3,000, or 10,000 ppm for 90 days. All animals survived until their scheduled necropsy date. 3

361 Significant treatment-related effects on body weight (i.e., reduced mean body weights in males at 10,000 ppm and in females at 3,000 ppm), food intake (i.e., reduced intake in both sexes at 10,000 ppm and at 3,000 ppm for females), organ weights (i.e., increased relative liver weight in both sexes at 3,000 and 10,000 ppm and in females also at 1,000 ppm; increased spleen weight in males at 10,000 ppm; clinical chemistry (i.e., confined to 10,000 ppm dose groups; significantly higher urea, chloride and potassium levels in males; significantly higher urea, chloride and cholesterol levels in females) and hematology (i.e., in both sexes at 10,000 ppm and in males also at 3,000 ppm increased total leukocytes and lymphocytes; females at 10,000 ppm showed depression in numbers of neutrophils, mean cell volume and mean cell hemoglobin) were identified in one or both sexes fed with dietary concentrations of 3,000 and 10,000 ppm. There were no compound-related histopathological effects at any dose level. Minor, but statistically significant changes in liver weight, kidney weights and plasma urea concentration were recorded in female rats in the 1,000 ppm group were not of toxicological significance. The NOAEL for this study is 1,000 ppm (50 mg/kg/day). Rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1%, 0.5% or 1% for 90 days. There were no treatment-related changes in body weight, food intake, and organ weights including those of the reproductive system, clinical chemistry and hematology at any treatment level. The NOAEL is 1% in the diet, which corresponded to 700 and 785 mg/kg-day for males and females, respectively. Rats were fed C12-13AE6.5 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Rats were fed C14-15AE7 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Charles River rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was a dose-related decrease in body weights in the 0.5 and 1% females and in the 1% males, the likely cause being poor palatability of the diet. At study termination, elevated organ-to-body weight ratios were noted for the liver, kidney, heart and thyroid/parathyroid glands in the 1% dietary group. The only significant histopathological finding prevalent in all dose groups was a dose-related increase in incidence of focal myocarditis at 12 months but not at study termination at 2 years. No other treatment-related histopathology was noted. The NOAEL was established at the 4

362 0.5% dietary level, which corresponds to about 162 and 190 mg/kg-day for males and females, respectively. Genotoxicity In all available in vitro and in vivo genotoxicity assays, there was no indication of genetic toxicity of broad range of structurally different alcohol ethoxylates. Carcinogenicity Alcohol ethoxylates as a class are not carcinogenic by the oral route based of available oral long term toxicity/carcinogenicity studies on AEs. Charles River rats were given in their diet 0, 0.1, 0.5 or 1% C14-15AE7 for two years. There was no treatment-related changes in general behavior and appearance. The survival rate of the test animals was comparable if not better than the controls. Body weights of 0.5% females and 1% males and females had significantly lower weight gains than the control. There were no treatment-related effects on organ weights and tumor incidence. Sprague-Dawley rats were fed C14-15AE7 at 0.1, 0.5 or 1% in the diet for two years. A treatment-related body weight depression was observed in females at the two highest treatment levels and in males at the 1% dose level, probably due to the poor palatability of the diet. There was no evidence for any carcinogenic activity. Sprague-Dawley rats were fed C12-13AE6.5 in the diet at doses up to 1% (500 mg/kgday). Reduced food consumption was noted at the higher dose levels (i.e., 0.5 and 1% for females and 1% for males), resulting in a lower body weight gain compared to the control group. No treatment-related histopathology was found and no increase in tumor incidence was observed. Reproductive Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kg-day). No treatment related effects in the parents or pups on general behavior, appearance or survival were observed. Fertility of treated groups was comparable with the controls. The only observation was related to a reduced weight gain of parental rats and pups relative to the control at the 0.5% dose level. The NOAEL for reproduction was therefore set at the highest dose level which was 0.5% dietary level (250 mg/kg-day). In a two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kgday). Three of the groups received the compound continuously during the study. In the 5

363 other three groups the females received the compound only during the 6th through the 15th day of gestation and the males were untreated. No treatment-related changes in behavior or appearance were observed in the parental rats or pups throughout the study. Female rats from the 0.5% continuous treatment group gained slightly less body weight compared to control females. No other consistent differences in body weight were observed. Food consumption was similar for control and treated rats. No compound related differences were seen between control and treated rats with respect to fertility, gestation or viability indices. The average 21-day body weights for pups at the 0.5% continuous treatment group were significantly lower as compared to the average pup body weights in the controls. No other compound-related changes in body weight were observed. None of the deaths of parental rats during the study was considered to be compound-related. Examination of organ weight values revealed that compound-related effects were limited to increased group mean relative liver weights of male and female F1 from the 0.5% continuous feeding group at the 91-day sacrifice, and increases in group mean relative liver weights of males from the 0.5% continuous feeding group of the F2 generation at the 60-day and caesarean section sacrifices. No compound-related histopathological lesions were observed in any of the tissues examined from rats for the F0 and F1 generations. The NOAEL for reproductive toxicity is at least 0.5% in the diet (250 mg/kg/day). Developmental Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% ( about 0, 25, 50 or 250 mg/kg-day). General behavior, appearance and survival were not affected by treatment. At the 0.5% dose level, adults and pups gained less weight than the control rats. In the 0.5% dose group, there was a statistical increase in embryo lethality and soft tissue anomalies and at the 0.1% there was a statistical decrease in mean fetal liver weight. Neither of these effects was considered to be treatment-related by the authors as they showed no dose response characteristics. The NOAEL for maternal toxicity is 50 mg/kg/day. The NOAEL for developmental and teratogenicity is 0.1% (50 mg/kg/day). Pregnant rabbits were given by oral gavage 0, 50, 100 or 200 mg/kg C12AE6 from GD 2 to16. Nine control rabbits and 31 treated rabbits died during the study. Surviving rabbits at the 200 mg/kg dose level generally showed slight losses of body weight. At 100 and 200 mg/kg, ataxia and a slight decrease in body weight was observed in the pregnant animals. In seven treated and two control rabbits early deliveries were recorded. There were no treatment-related effects on corpora lutea, implantations, number of live fetuses and spontaneous abortions. The NOAEL for maternal toxicity is 50 mg/kg/day; the NOAEL for developmental toxicity is 200 mg/kg/day. In two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kg/day). On the 13th day of the gestation period a representative number of female rats from each treatment group of the FC generation (i.e., pups from the 3rd mating of the F0 and F1 parental generation) were sacrificed. Laparotomies were performed and the uterus was 6

364 examined for uterine abnormalities, normal implantation and resorption sites. Remaining females were sacrificed on the 21st day of gestation. Various maternal and fetal parameters showed occasional values that were significantly different from the corresponding controls. However these were not considered related to the material tested as none occurred at the high feeding level and no dose response for these parameters was apparent. With respect to body weight gains, parental female rats and pups of the high dose group did not gain as much body weight as the control rats. Examination of organ weight values reveal compound related effects were limited to increased group mean liver weighs of male and female P1 generation from the 0.5% continuous feeding group at the 91 day sacrifice and increase in group mean relative liver weights of males of the 0.5% continuous feeding group of the P2 generation at the 60 day section sacrifices. The NOAEL for maternal and developmental toxicity was established at the 0.1% in the diet (50 mg/kg/day). Key Study/Critical Effect for Screening Criteria AEs of different structures with regard to the length of the alkyl chain and the degree of ethoxylation have been evaluated in a number of 90-day and two-year oral toxicity studies. The lowest NOAEL of AEs for systemic toxicity was established at 50 mg/kgday in two chronic (two-year) dietary studies on C12-13AE6.5 and C14-15AE7. Effects observed at the LOAEL were related to significantly elevated organ-to-body weight ratios for liver, kidney and heart, although there were no adverse histopathological changes. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 50/100 = 0.5 mg/kg/day Drinking water guidance value = 2 ppm References Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Alcohol Ethoxylates (2009), Talmage, S.S. (1994). Environmental and Human Safety of Major Surfactants Alcohol Ethoxylates and Alkylphenol Ethoxylates. The Soap and Detergent Association. Lewis Publishers, Boca Raton, Florida. 7

365 Toxicity Profile Alcohols, C10-C16, Ethoxylated Propoxylated (CAS No ) No toxicity information could located on alcohols, C10-C16, ethoxylated propoxylated (CAS No ). Toxicity information is available on alcohol ethoxylates. Alcohols, C10-C16, ethoxylated propoxylated is produced by the use of propylene oxide (PO) in addition to ethylene oxide (EO) during the alkoxylation of the alcohol(s). the PO is either incorporated randomly by alkoxylation within the epoxide mixture, or in a block by sequencing the addition of EO and PO to the reactor. Because of the similarity in structures, the toxicity data on the alcohol ethoxylates were used to read-across to alcohols, C10-16, ethoxylated propoxylated. Alcohol ethoxylates are a class of non-ionic surfactants. The AEs have the basic structure Cx-yAEn. The subscript (x-y) following the C indicates the range of carbon chain units. AEs with carbon unit range between C8 to C18 are most commonly used in household detergent products. The hydrocarbon chain can be either linear or branched. AEs also contain an ethylene oxide (E) chain attached to the alcohol. The degree of ethylene oxide polymerization is indicated by the subscript (n) which indicates the average number of ethylene oxide units. In household products the average ethylene oxide chain length commonly ranges between 3 and 12 units. Acute Toxicity The acute oral LD50 of C9-11AE2.5 in rats was calculated to be >4,000 mg/kg and <10,000 mg/kg. The acute oral LD50 of C7-9AE6 in rats was determined to be <2,000 mg/kg. The acute oral LD50 of C9-11AE8 in fasted rats was found to be 1,200 mg/kg. The oral LD50 in rats for C12-13AE6.5 is 2,100 mg/kg. The oral LD50 in rats for C12-15AE7 is 1,700 mg/kg. For C14-15AE11, the acute oral LD50 values were reported to be 720 mg/kg (neat) and 1,800 mg/kg (given as 50% (m/v) solution in corn oil). The oral LD50 in rats for C12-15AE11 is >2,00 mg/kg for males and between 1,000 and 2,000 mg/kg for females. The oral LD50 in rats for C14-15AE13 is 1,100 and 1,000 mg/kg in two separate studies. The dermal LD50 of C7-9AE6 was >2,000 mg/kg. The dermal LD50 values for AEs with an alkyl chain length of 9 11 carbon atoms in three different rat studies were determined to be >2,000 mg/kg and >4,000 mg/kg. An acute dermal LD50 value of >2,000 mg/kg was determined for C12-14AE3 and C12-14AE6 in two separate studies. The acute dermal LD50 of C12-15AE7 was determined to be >2,000 mg/kg. The acute rat 4-hour LC50 of C9-11AE5 generated as a mist was determined to be >0.22 mg/l. Talmage (1994) reported that alcohol ethoxylates were not acutely toxic to rats at concentrations less than or equal to their saturated vapor concentrations in air. Acute 1

366 toxic thresholds were reached only when animals were exposed to the undiluted test chemical in the form of a respirable mist or aerosol. Under these conditions, 1- or 4-hour inhalation LC50 values ranged from 1.5 to 20.7 mg/l. Some studies reported no mortalities (1-hour LC50 -study) occurred at concentrations as high as 52 mg/l. Irritation Alcohol ethoxylates with varying alkyl chain lengths and ethoxylation degree were found to be slightly to severely irritating to skin in rabbits and rats. The degree of irritation was dependent on the type of patches used (open application versus full occlusion), the exposure time as well as the concentration of the test material. In humans, AEs are less irritating to skin than in animals. Neat applications of a range AEs in a 4hour human patch test did not warrant these chemicals to be classified as skin irritants under EU legislation, while the same AEs would have been classified for skin irritation on the basis of animal data. Alcohol ethoxylates range from mildly to severely irritating to rabbit eyes. Rinsing the eyes directly after exposure with water for 20 to 30 seconds greatly reduced the severity of the effects such that these products produced only mildly irritating effects. The degree of irritation is concentration-dependent as dilutions in water cause proportionally lower irritation. Generally, concentration of 0.1% were non-irritating, and concentrations of 1 to 10% ranged from slight to moderately irritating. No relationship could be established between the chemical structures of the tested AEs and their eye irritation responses. Sensitization Alcohol ethoxylates should not be considered as skin sensitizers. A substantial amount of skin sensitization studies in guinea pigs following either the Magnusson-Kligman maximization or the Buehler testing protocol are available to evaluate the skin sensitization potential of AEs. Although a mild skin sensitization reaction was observed in a study following the Magnusson-Kligman protocol, the weight of evidence clearly supports the assessment that AEs should not be considered as skin sensitizers. This is further supported by clinical and market data that demonstrate the absence skin sensitization responses to AEs when tested under the conditions of the HRIPT or when used in AE containing consumer products. Repeated Dose Toxicity C10AE5 was fed to rats at doses of 0, 125, 250 or 500 mg/kg for 90 days. There were no treatment-related clinical signs, body weight gain, food consumption or feed efficiency. There was a slight increase in absolute liver weights as well as a trend toward a dosedependent increase in the liver weight/body weight ratio, with a statistically significant increase in the high dose group. However, the histological evaluation did not reveal any 2

367 indication of hepatotoxicity and therefore the increase in liver weights was not interpreted to be a toxicological effect. It can be considered to be an adaptive response as a result of extensive metabolism of the test compound by the liver. There were no other gross or histopathological changes that were considered treatment-related. The NOAEL for this study can be considered to be 500 mg/kg/day. Rats were given in their diet 0, 125, 250, 500, 1,000 or 3,000 ppm C9-11AE6 for 90 days. There were no signs of toxicity at any dose level. The NOAEL is 3,000 ppm (approximately 150 mg/kg/day). Rats were given in their diet 0, 0.04, 0.2 or 1.0% C9-11AE8 for 90 days. Lower body weight gain and decreased food consumption were noted in the 1% males and females and in the 0.2% females from week 1 through the end of the study. Further statistical analyses revealed a significant decrease in the mean body weight gain noted in the 1% females and the decreases in mean food consumption noted in the 1% males and females. The differences noted in the 0.2% females were not statistically significant. The investigators considered these observations to be the result of poor palatability of the test substance. There were no other treatment-related effects. The NOAEL for this study is 1.0% (about 400 mg/kg/day). Rats were fed C12-15AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% or 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the >0.25% groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the >0.25% groups. There were not treatment-related changes in hematology, urinary and clinical chemistry parameters. Liver cell enlargement was seen in the >0.125% groups, suggesting increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (102 mg/kg/day). Rats were fed C12-14AE7 in the diet at concentrations of 0%, %, %, 0.125%, 0.25%, 0.5% or 1.0% for 90 days. Body weight gain was significantly reduced in male and female rats in the >0.25% groups, and was associated with marked decreases in food and water consumption. Relative liver weights were significantly increased in the 0.5% and 1% male rats fed and in female rats in the >0.25% groups. There were no treatment-related changes in hematology, urinary and clinical chemistry parameters. Liver cell enlargement was seen in the >0.125%, suggesting increased liver metabolism on the basis of increased alkaline phosphatase activity at the higher dose levels. The NOAEL was established at % (110 mg/kg/day). Wistar rats were fed C14-15AE7 in the diet at concentrations of 0, 300, 1,000, 3,000, or 10,000 ppm for 90 days. All animals survived until their scheduled necropsy date. Significant treatment-related effects on body weight (i.e., reduced mean body weights in males at 10,000 ppm and in females at 3,000 ppm), food intake (i.e., reduced intake in both sexes at 10,000 ppm and at 3,000 ppm for females), organ weights (i.e., increased relative liver weight in both sexes at 3,000 and 10,000 ppm and in females also at 1,000 3

368 ppm; increased spleen weight in males at 10,000 ppm; clinical chemistry (i.e., confined to 10,000 ppm dose groups; significantly higher urea, chloride and potassium levels in males; significantly higher urea, chloride and cholesterol levels in females) and hematology (i.e., in both sexes at 10,000 ppm and in males also at 3,000 ppm increased total leukocytes and lymphocytes; females at 10,000 ppm showed depression in numbers of neutrophils, mean cell volume and mean cell hemoglobin) were identified in one or both sexes fed with dietary concentrations of 3,000 and 10,000 ppm. There were no compound-related histopathological effects at any dose level. Minor, but statistically significant changes in liver weight, kidney weights and plasma urea concentration were recorded in female rats in the 1,000 ppm group were not of toxicological significance. The NOAEL for this study is 1,000 ppm (50 mg/kg/day). Rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1%, 0.5% or 1% for 90 days. There were no treatment-related changes in body weight, food intake, and organ weights including those of the reproductive system, clinical chemistry and hematology at any treatment level. The NOAEL is 1% in the diet, which corresponded to 700 and 785 mg/kg-day for males and females, respectively. Rats were fed C12-13AE6.5 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Rats were fed C14-15AE7 in their diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was reduced food consumption in males (1%) and females (0.5 and 1%) which resulted in lower body weight gain compared to controls. At study termination, organ to body weight ratios were increased in the 0.5 and 1% females (liver, kidney and brain), 1% females (heart), and 1% males (liver). A dose-related focal myocarditis was observed in males. The NOAEL was established at 0.1% dietary level based on the relative organ weight increases. This corresponds to 50 mg/kg/day. Charles River rats were fed C14-15AE7 in the diet at concentrations of 0, 0.1, 0.5 or 1% for two years. There was a dose-related decrease in body weights in the 0.5 and 1% females and in the 1% males, the likely cause being poor palatability of the diet. At study termination, elevated organ-to-body weight ratios were noted for the liver, kidney, heart and thyroid/parathyroid glands in the 1% dietary group. The only significant histopathological finding prevalent in all dose groups was a dose-related increase in incidence of focal myocarditis at 12 months but not at study termination at 2 years. No other treatment-related histopathology was noted. The NOAEL was established at the 0.5% dietary level, which corresponds to about 162 and 190 mg/kg/day for males and females, respectively. 4

369 Genotoxicity In all available in vitro and in vivo genotoxicity assays, there was no indication of genetic toxicity of broad range of structurally different alcohol ethoxylates. Carcinogenicity Alcohol ethoxylates as a class are not carcinogenic by the oral route based of available oral long term toxicity/carcinogenicity studies on AEs. Charles River rats were given in their diet 0, 0.1, 0.5 or 1% C14-15AE7 for two years. There was no treatment-related changes in general behavior and appearance. The survival rate of the test animals was comparable if not better than the controls. Body weights of 0.5% females and 1% males and females had significantly lower weight gains than the control. There were no treatment-related effects on organ weights and tumor incidence. Sprague-Dawley rats were fed C14-15AE7 at 0.1, 0.5 or 1% in the diet for two years. A treatment-related body weight depression was observed in females at the two highest treatment levels and in males at the 1% dose level, probably due to the poor palatability of the diet. There was no evidence for any carcinogenic activity. Sprague-Dawley rats were fed C12-13AE6.5 in the diet at doses up to 1% (500 mg/kg/day). Reduced food consumption was noted at the higher dose levels (i.e., 0.5 and 1% for females and 1% for males), resulting in a lower body weight gain compared to the control group. No treatment-related histopathology was found and no increase in tumor incidence was observed. Reproductive Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kg-day). No treatment related effects in the parents or pups on general behavior, appearance or survival were observed. Fertility of treated groups was comparable with the controls. The only observation was related to a reduced weight gain of parental rats and pups relative to the control at the 0.5% dose level. The NOAEL for reproduction was therefore set at the highest dose level which was 0.5% dietary level (250 mg/kg/day). In a two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kg/day). Three of the groups received the compound continuously during the study. In the other three groups the females received the compound only during the 6th through the 15th day of gestation and the males were untreated. No treatment-related changes in behavior or appearance were observed in the parental rats or pups throughout the study. Female rats from the 0.5% continuous treatment group gained slightly less body weight 5

370 compared to control females. No other consistent differences in body weight were observed. Food consumption was similar for control and treated rats. No compound related differences were seen between control and treated rats with respect to fertility, gestation or viability indices. The average 21-day body weights for pups at the 0.5% continuous treatment group were significantly lower as compared to the average pup body weights in the controls. No other compound-related changes in body weight were observed. None of the deaths of parental rats during the study was considered to be compound-related. Examination of organ weight values revealed that compound-related effects were limited to increased group mean relative liver weights of male and female F1 from the 0.5% continuous feeding group at the 91-day sacrifice, and increases in group mean relative liver weights of males from the 0.5% continuous feeding group of the F2 generation at the 60-day and caesarean section sacrifices. No compound-related histopathological lesions were observed in any of the tissues examined from rats for the F0 and F1 generations. The NOAEL for reproductive toxicity is at least 0.5% in the diet (250 mg/kg/day). Developmental Toxicity In a two-generation reproductive toxicity study, Charles River rats were fed C12AE6 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kg/day). General behavior, appearance and survival were not affected by treatment. At the 0.5% dose level, adults and pups gained less weight than the control rats. In the 0.5% dose group, there was a statistical increase in embryo lethality and soft tissue anomalies and at the 0.1% there was a statistical decrease in mean fetal liver weight. Neither of these effects was considered to be treatment-related by the authors as they showed no dose response characteristics. The NOAEL for maternal toxicity is 50 mg/kg/day. The NOAEL for developmental and teratogenicity is 0.1% (50 mg/kg/day). Pregnant rabbits were given by oral gavage 0, 50, 100 or 200 mg/kg C12AE6 from GD 2 to 16. Nine control rabbits and 31 treated rabbits died during the study. Surviving rabbits at the 200 mg/kg dose level generally showed slight losses of body weight. At 100 and 200 mg/kg, ataxia and a slight decrease in body weight was observed in the pregnant animals. In seven treated and two control rabbits early deliveries were recorded. There were no treatment-related effects on corpora lutea, implantations, number of live fetuses and spontaneous abortions. The NOAEL for maternal toxicity is 50 mg/kg/day; the NOAEL for developmental toxicity is 200 mg/kg/day. In two-generation developmental and teratogenicity study, CD rats were fed C14-15AE7 in the diet at dose levels of 0, 0.05, 0.1 or 0.5% (about 0, 25, 50 or 250 mg/kg/day). On the 13th day of the gestation period a representative number of female rats from each treatment group of the FC generation (i.e., pups from the 3rd mating of the F0 and F1 parental generation) were sacrificed. Laparotomies were performed and the uterus was examined for uterine abnormalities, normal implantation and resorption sites. Remaining females were sacrificed on the 21st day of gestation. Various maternal and fetal parameters showed occasional values that were significantly different from the corresponding controls. However these were not considered related to the material tested 6

371 as none occurred at the high feeding level and no dose response for these parameters was apparent. With respect to body weight gains, parental female rats and pups of the high dose group did not gain as much body weight as the control rats. Examination of organ weight values reveal compound related effects were limited to increased group mean liver weighs of male and female P1 generation from the 0.5% continuous feeding group at the 91 day sacrifice and increase in group mean relative liver weights of males of the 0.5% continuous feeding group of the P2 generation at the 60 day section sacrifices. The NOAEL for maternal and developmental toxicity was established at the 0.1% in the diet (50 mg/kg/day). Key Study/Critical Effect for Screening Criteria AEs of different structures with regard to the length of the alkyl chain and the degree of ethoxylation have been evaluated in a number of 90-day and two-year oral toxicity studies. The lowest NOAEL of AEs for systemic toxicity was established at 50 mg/kg/day in two chronic (two-year) dietary studies on C12-13AE6.5 and C14-15AE7. Effects observed at the LOAEL were related to significantly elevated organ-to-body weight ratios for liver, kidney and heart, although there were no adverse histopathological changes. Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability) Oral RfD = 50/100 = 0.5 mg/kg/day Drinking water guidance value = 2 ppm References Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Alcohol Ethoxylates (2009), Talmage, S.S. (1994). Environmental and Human Safety of Major Surfactants Alcohol Ethoxylates and Alkylphenol Ethoxylates. The Soap and Detergent Association. Lewis Publishers, Boca Raton, Florida. 7

372 Toxicity Profile Choline chloride Cholinium chloride (CAS No ) Choline chloride, also known as cholinium chloride, is a quaternary amine salt. It dissociates in water into the corresponding positively charged quaternary hydroxyl alkylammonium ion and the negatively charged chloride ion. Choline chloride has been reviewed in the OECD-SIDS program (OECD, 2004). Acute Toxicity The oral LD50 in rats was reported to be between 3,150 and 5,000 mg/kg (BASF AG, 1963a, 1969). Irritation Application of a 70% aqueous solution to the skin of rabbits for 20 hours under occlusive conditions resulted in only minor skin irritation (BASF AG, 1963b). Slight eye irritation was seen in the eyes of rabbits after instillation of a 70% aqueous solution of choline chloride; no effects were seen one day after exposure (BASF AG, 1963c). Sensitization No data are available in animals. In a Human Repeated Insult Patch Test, there was no evidence of dermal sensitization in two hundred subjects given 0.5% (w/v) aqueous solution of choline chloride during the induction phase and 0.2% (w/v) aqueous solution during the challenge phase (Colgate-Palmolive, 2003). Repeated Dose Toxicity A 72-week feeding study was conducted to investigate the impact of choline chloride on the liver tumor promoting activity of phenobarbital and DDT in diethylnitroamineinitiated Fischer 344 rats (Shivapurkar et al., 1986). Animals received approximately 500 mg/kg-day choline chloride. Following the end of the exposure period, the animals were keep on the same untreated diet as the control group until study termination at week 103. Histopathology was limited to the liver and organs that developed gross abnormalities. There were no significant differences between treated and control animals on survival rates, body weights, and relative liver weights. Neither was there any 1

373 increased number of neoplastic liver nodules, hepatocellular carcinomas, lung tumors, leukemia nor other tumors between treated and control animals. The NOAEL for choline chloride in this study is 500 mg/kg/day. In humans, oral administration of 10,000 mg/day choline chloride in a pilot study treating a small number of patients with Alzheimer s disease, resulted in a slight hypotensive effect (Boyd et al., 1977). This dose was regarded as a LOAEL by the Standing Committee on the Scientific Evaluation of Dietary Reference Intake (2000). Genotoxicity Choline chloride was not mutagenic to bacteria in reverse mutation assays (Haworth et al., 1984; JETOC, 1997; Litton Bionetics, 1977). A small, but statistically significant, and dose-related increase in sister chromatid exchanges (SCEs) in Chinese Hamster Ovary (CHO) cells was reported at 50 and 500 μg/ml choline chloride in the absence of S9 only (Bloom et al., 1982). No higher concentrations were examined. These results could not be confirmed in another study using CHO cells at concentrations of choline chloride up to 5,000 μg/ml. (Galloway et al., 1985). In a gene conversion assay with Saccharomyces cerevisiae strain D4, choline chloride was negative in the presence and absence of metabolic activation (Litton Bionetics, 1977). No in vivo genotoxicity studies were available. Carcinogenicity No studies were located. Reproductive Toxicity No reliable studies have been conducted that address female fertility or reproductive toxicity by a relevant route of exposure. Developmental Toxicity Pregnant female mice were given in their feed 1,250 to 20,000 mg/kg choline chloride during gestational days 1 to 18 (BASF AG, 1966). Maternal body weight gain was reduced in all treated groups except for the 1,250 mg/kg group. Determination of maternal weight gain of dams with embryonic/fetal absorptions showed that there was no 2

374 almost net weight gain at >4,160 mg/kg and net weight loss in the 20,000 mg/kg group. All fetuses were resorbed in the 20,000 mg/kg group. Embryonic/fetal lethality of 35% and 69% were seen in the 4,160 and 10,800 mg/kg groups, respectively. No resorptions occurred in the 1,250 mg/kg group. Developmental toxicity was seen in all but the 1,250 mg/kg group. No statistically significant increases in malformations were observed in any dose group. The NOAELs for maternal and developmental toxicity is 1,250 mg/kg/day. Key Study/Critical Effect for Screening Criteria The Standing Committee on the Scientific Evaluation of Dietary Reference Intakes selected hypotension as the critical effect from the study by Boyd et al. (1977) when deriving a Tolerable Upper Intake Level. Boyd et al. (1977) reported a LOAEL of 10,000 mg/day choline chloride (7,500 mg/day choline). An uncertainty factor of 2 was chosen because of the limited data regarding hypotension and the inter-individual variation in response to cholinergic effects. Thus, the value for the Tolerable Upper Intake Value for repeated exposure of adults to choline is 3,500 mg/day choline. The oral RfD for choline chloride is derived by using the LOAEL of 10,000 mg/day from the Boyd et al. (1977) study, which is divided by an uncertainty factor of 2, to obtain a value of 5,000 mg/day or 71 mg/kg/day for a 70 kg person. Oral RfD = 71 mg/kg/day Drinking water guideline value = 248 ppm References BASF AG (1963a). Acute oral toxicity of choline chloride 70 % in water. Department of Toxicology. Unpublished results. Study No. XIII Jan BASF AG (1963b). Toxicity of choline chloride 70 % in water; skin irritation after exposure to choline chloride. Department of Toxicology. Unpublished results. Study No. XIII Mar BASF AG (1963c). Toxicity of choline chloride 70% in water; eye irritation. Department of Toxicology. Unpublished results. Study No. XIII Mar BASF AG (1966). Study on teratogenic effects of choline chloride in the mouse after oral application. Department of Toxicology. Unpublished results. Study No. XIV/ Oct BASF AG (1969). Acute oral toxicity of choline chloride 50% powder. Department of Toxicology. Unpublished results. Study No. XIX/ Aug

375 Bloom A., Galloway, S., Nakamura, F.T., Teteviri, A., Armstrong, M., Lavappa, K.L., Duk, S., and Ahmed, M.A. (1982). Comparison of results for SCE and chromosome aberrations for eleven compounds tested in two laboratories by standardized methods. Environ. Mutagen. 4: 397. Boyd, W.D., Graham-White, J., Blackwood, G., Glen, I., and McQueen, J. (1977). Clinical effects of choline in Alzheimer senile dementia. Lancet 2: 711. Colgate-Palmolive (2003). Study No. DCR TKL. TKL Research Inc. Paramus, NJ, USA. In: SCCNFP. Scientific Committee on Cosmetic Products and Non-Food Products. Choline Chloride. SCCNFP/0672/03. 9 Dec Galloway, S.M., Bloom, A..D, Resnick, M., Margolin, B.H., Nakamura, F., Archer, P., and Zeiger, E, (1985). Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ. Mutagen. 7: Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., and Zeiger, E. (1983). Salmonella mutagenicity test results for 250 chemicals. Environ. Mutagenesis Suppl. 1: Litton Bionetics (1977). Mutagenic evaluation of compound FDA choline chloride. FCC. Report No. PB Mar OECD (2004). SIDS Initial Assessment Report for Choline chloride (CAS No ), UNEP Publications. Shivapurkar, N., Hoover, K.L., and Poirier, L.A. (1986). Effect of methionine and choline on liver tumor promotion by phenobarbital and DDT in diethylnitrosamineinitiated rats. Carcinogenesis 7: Standing Committee on the Scientific Evaluation of Dietary Reference Intake. Institute of Medicine (2000). Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. National Academy Press, Washington D.C. 4

376 Toxicity Profile Cocoamidopropyl betaine (CAS No ) Cocoamidopropyl betaine has been reviewed in the OECD-SIDS program (OECD, 2006). Acute Toxicity The acute toxicity of cocoamidopropyl betaine ( % aqueous solution) in rats is low. The oral LD50 is >4,900 mg/kg, and the dermal LD50 is >2,000 mg/kg. Irritation A 30% aqueous solution of cocoamidopropyl betaine was only very slightly irritating to the skin of rabbits. In human studies, up to 3% solutions were weakly irritating. A 5-10% solution of cocamidopropyl betaine produced mild to moderate irritation to the eyes of rabbits; solutions containing 15% were irritating to highly irritating; and a 30% aqueous solution was irritating with irreversible damage. Sensitization The sensitizing potential of cocoamidopropyl betaine in humans is low. Commercial cocoamidopropyl betaine may, however, contain impurities identified as sensitizers (amidoamine and/or 3-dimethylaminopropylamine) which may explain positive results in human patch tests. There is no evidence for a photosensitizing potential. Repeated Dose Toxicity Rats were given a 30% aqueous solution of cocoamidopropyl betaine by oral gavage at doses of 0, 250, 500 or 1,000 mg/kg/day (corresponding to about 0, 75, 150, and 300 mg active substance/kg/day, respectively) 5 days/week for 28 days (Henkel KgA, 1991). The only treatment-related findings were forestomach lesions at the highest dose level, probably as a result of the irritant effect of the test substance. The NOAEL for this study is 500 mg/kg/day (150 mg cocoamidopropyl betaine/kg/day). Rats were given a 30% aqueous solution of cocoamidopropyl betaine by oral gavage at doses of 0, 250, 500 or 1,000 mg/kg/day (corresponding to about 0, 75, 150, and 300 mg active substance/kg/day, respectively) five days/week for 90 days (Th. Goldschmidt AG, 1991). The only treatment-related findings were forestomach lesions at the 500 and 1,000 mg/kg dose levels, probably as a result of the irritant effect of the test substance. The NOAEL for this study is 250 mg/kg day (75 mg cocoamidopropyl betaine/kg/day). 1

377 Genotoxicity There is no evidence for a genotoxic potential of cocoamidopropyl betaine. A 30% aqueous solution of cocoamidopropyl betaine was not genotoxic in vitro in either a bacteria mutation test or in a mouse lymphoma test. A limited intraperitoneal mouse micronucleus test with 27 % active cocamidopropyl betaine was negative. Carcinogenicity No valid carcinogenicity studies have been conducted. Reproductive Toxicity No reproductive toxicity studies have been conducted on cocoamidopropyl betaine. However, there was no evidence of an adverse effect on the reproductive organs in rats given oral doses of up to 1,000 mg/kg/day of a 30% aqueous solution of cocoamidopropyl betaine (300 mg active substance/kg/day) for 90 days (Th. Goldschmidt AG, 1991). Developmental Toxicity A 28.9% aqueous solution of cocoamidopropyl betaine was tested in a rat developmental toxicity at doses of 330, 990, and 3,300 mg/kg/day (corresponding to 95, 286, and 950 mg/kg/day, respectively) (CESIO, 2004). There were dose-related maternal toxic effects (reduced body weights and stomach ulcers) at 990 mg/kg/day and above. Embryotoxic effects (increased numbers of resorptions, decreased number of viable fetuses, decreased fetal body weight) were found only at the maternal toxic dose level of 3,300 mg/kg/day. The NOAEL for maternal toxicity was 330 mg/kg/day (corresponding to 95 mg active substance/kg-day) and the NOAEL for developmental toxicity was 990 mg/kg/day (corresponding to 286 mg active substance/kg/day). Key Study/Critical Effect for Screening Criteria The key study is a 90-day rat oral gavage study in which forestomach lesions were seen at dose levels of 500 and 1,000 mg/kg of a 30% aqueous solution of cocoamidopropyl betaine. The NOAEL for this study is 250 mg/kg/day (75 mg active substance/kg/ay). NOAELadjusted = 75 * 5/7 = 53.6 mg/kg/day 2

378 Uncertainty factors: 10 (interspecies variability); 10 (intraspecies variability); 10 (subchronic to chronic) Oral RfD = 0.05 mg/kg/day Drinking water guideline: 0.19 ppm References CESIO (2004). Prenatal development toxicity study in rats with cocamidopropyl betaine by oral administration - according to OECD guideline DRAFT. Essen, LPT Study No /03, Henkel KGaA (1991). Dehyton K; 28-Tage-Test mit wiederholter oraler Verabreichung an Ratten. TED ; Juli 1991, Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: Cocoamidopropyl betaine (CAPB) (CAS No.: , , ), 2005; OECD (2006). SIDS Initial Assessment Profile for Alkylamidopropyl betaines. Th. Goldschmidt AG (1991). Tego-Betain. 90 day oral (gavage) subchronic toxicity study in the rat. Essen, Th. Goldschmidt AG, , ,

379 Toxicity Profile Coffee Beans (Ground and Roasted) The composition of a medium-roasted coffee (Clarke, 1987; IARC, 1991) is shown in the table below: Components Typical avg content (dry basis, %) % extractable Arabica Robusta with water, 100 o C a Alkaloids (caffeine) Trigonelline (including roasted byproducts) Minerals (as oxide ash) Acids Residual chlorogenic Aquinic Aliphatic Sugars Sucrose Reducing Polysaccharides (unchanged from green) Lignin Pectins Proteinaceous compounds Protein Free amino acids Lipids (coffee oil) Caramelized or condensation products (e.g., melanoidins) by difference Volatile substances other than acids a Maier (1981), for normal household brewing The focus of this toxicity profile is on caffeine (CAS No ), which is watersoluble alkaloid. Caffeine has well known central nervous system (CNS) stimulatory properties, and the health benefits as well as the potential health concerns of caffeine have been well studied. The chemical names for caffeine are 1,3,7-trimethyl-2,6- dioxopurine; 1,3,7-trimethylxanthine; 2,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione; and 7-methyltheophylline. Caffeine has been widely consumed by most segments of the population worldwide. The U.S. FDA estimates that the average amount of caffeine consumed by the U.S. population between 2003 and 2008 was approximately 300 mg/person/day (FDA, 2012). Caffeine is probably one of the most studied natural dietary ingredient. In 2004, the Institute of Medicine (IOM) (2004) prepared an extensive review on caffeine, discussing several issues pertaining to safety. Health Canada also released a review of the many studies dealing with caffeine and its health effects (Nawrot et al., 2003). 1

380 Pharmacokinetics Caffeine is rapidly and completely adsorbed in humans, with approximately 99% absorbed within 45 minutes of consumption (Carillo and Benitez, 2000). Plasma caffeine levels may be influenced by the diet or route of exposure, but peak plasma levels occur approximately 15 to 120 minutes after consumption (Mandel, 2002; Beach et al., 1984; Bonati et al., 1984; Collomp et al., 1991). Caffeine is water soluble and is rapidly distributed throughout the body, detected in all bodily fluids, including saliva, breast milk, urine and semen (Beach et al., 1984). Caffeine elimination follows first-order kinetics (Newton et al., 1981), with the plasma half-life of caffeine at approximately 3 to 6 hours in healthy adults and does not accumulate in body fat or other tissues (Kaplan et al., 1997; Nawrot et al., 2003). Caffeine is rapidly metabolized and excreted (1-3 mg/kg/minute) in the urine (Kaplan et al., 1997), and varies between species, with a slightly different metabolic route noted in rats (Nawrot et al., 2003). Acute toxicity The LD50 of caffeine in rodents ranges from 185 to 400 mg/kg, depending on the species and strain (OECD, 2002a,b). In humans, the fatal acute oral dose is estimated at 10 and 14 g (approximately 160 to 230 mg/kg for a 60 kg person) (Hodgman, 1998). The serum caffeine concentration is the most reliable indicator of potential caffeine toxicity, with a serum caffeine concentration >100 μg/ml considered lethal in humans (Mrvos et al., 1989). Human caffeine consumption at up to 10 g has caused convulsions and vomiting, with recovery in six hours (Dreisbach, 1974). An acute dose of one gram caffeine can cause adverse effects, progressing from restlessness, nervousness, and irritability to delirium, emesis, neuromuscular tremors and convulsions (IOM, 2004). However, caffeine consumption through the day at up to 900 mg has been reported without adverse effects (Nawrot et al., 2003). Repeated Dose Toxicity Administration of high levels of caffeine in chronic rodent studies showed decreased weight gain, but in most studies caffeine administration in the diet or in the drinking water did not affect mortality rates of mice and rats, with the exception of two studies that reported a significant reduction in the average life span of rats consuming caffeine (IARC, 1991). Administration of caffeine in chronic toxicity and carcinogenicity studies showed no carcinogenic effects (IARC, 1991). The potential for caffeine to induce genotoxicity has been evaluated in both in vitro an in vivo studies, with in vitro studies indicating both genotoxic and non-genotoxic results; in vivo studies have shown that, overall, caffeine is not genotoxic (IARC, 1991; OECD, 2002a,b). Cardiovascular Effects Caffeine is a stimulant, and has been studied for its physiological and behavioral effects. Caffeine increases heart rate and blood pressure, increases diuresis, increases locomotion and alertness, and decreases sleepiness. Clinical studies indicate that doses <450 mg do 2

381 not increase the risk or severity of cardiac arrhythmia, while acute doses of 150 mg caffeine may decrease heart rate (James, 1991; Green et al., 1996; Myer, 1998). Studies evaluating effects of caffeine on cardiovascular health or serum cholesterol levels have not provided a consistent adverse effect with caffeine consumption (Nawrot et al., 2003). Reproductive and Developmental Toxicity Studies that evaluated the potential teratogenicity of caffeine have been discussed in a review (Christian and Brent, 2001). In animal studies, caffeine administration to pregnant animals has been found to induce teratogenicity and toxic effects on the development of the fetuses only at doses that also caused toxic effects in the dams (Christian and Brent, 2001). Christian and Brent (2001) concluded that studies reporting positive teratogenic effects were administering large doses of caffeine that were far greater than human consumption and were providing the caffeine via gavage, which results in much higher peak serum caffeine levels and therefore overstates toxicity potential. Neither rodents or humans could attain such peak exposures by consuming solutions of caffeine over several hours, the usual mode of human caffeine consumption. There have been questions raised on whether high intake of coffee or caffeine may increase the risk of miscarriage. Caffeine crosses the placenta and increases maternal catecholamine levels (Goldstein and Warren, 1962). The American College of Obstetricians and Gynecologists (2010) have concluded the following: Moderate caffeine consumption (less than 200 mg per day) does not appear to be a major contributing factor in miscarriage or preterm birth. The relationship of caffeine to growth restriction remains undetermined. A final conclusion cannot be made at this time as to whether there is a correlation between high caffeine intake and miscarriage. Conclusions Based on animal and clinical studies, the long history of caffeine use, moderate daily consumption of caffeine does not appear to cause an irreversible adverse effect on human health. Drinking Water Guidance Value Nawrot et al. (2003) concluded in their review of the effects of caffeine on human health that for the healthy adult population, moderate daily caffeine intake at a dose level up to 400 mg/day (equivalent to 6 mg/kg body weight/day in a 65-kg person) is not associated with adverse effects such as general toxicity, cardiovascular effects, effects on bone status and calcium balance (with consumption of adequate calcium), changes in adult behavior, increased incidence of cancer and effects on male fertility. It was indicated that habitual daily use of caffeine at greater than mg/day ( mg/kg) (4-7 cups of coffee or 7-9 cups of tea) could be considered a health risk. For women, caffeine intake greater than 400 mg/day (6.7 mg/kg) may increase the risk of detrusor instability (unstable bladder) development in women (Nawrot et al., 2003). 3

382 More recently, the American College of Obstetricians and Gynecologists (2010) have concluded that moderate caffeine consumption (<200 mg/day) does not appear to be a major contributing factor in miscarriage or preterm birth. Thus, the acceptable daily intake of caffeine will be set at 200 mg/person/day for the derivation of a drinking water guidance value. Assuming that humans consume 2 liters of water a day, the drinking water guidance value for caffeine is determined to be 100 mg/l. References American College of Obstetricans and Gynecologists (2010). Moderate caffeine consumption during pregnancy. Committee Opinion No Obstet. Gynecol. 116: Beach, C. A., Bianchine, J. R. and Gerber, N. (1984). The excretion of caffeine in the semen of men: pharmacokinetics and comparison of the concentrations in blood and semen. J. Clin. Pharmacol. 24: Bonati, M., Latini, R., Tognoni, G., Young, J. F. and Garattini, S. (1984). Interspecies comparison of in vivo caffeine pharmacokinetics in man, monkey, rabbit, rat, and mouse. Drug Metabolism Reviews 15: Carrillo, J. A. and Benitez, J. (2000) Clinically significant pharmacokinetic interactions between dietary caffeine and medications. Clinical Pharmacokinetics 39: Clarke, R.J. (1987). Coffee Technology. In Quality Control in the Food Industry, 2 nd Edition (Herschdoefer, S.H., ed.), pp , Academic Press, London; cited in IARC (1991). Christian, M.S., and Brent, R.L. (2001). Teratogen update: evaluation of the reproductive and developmental risks of caffeine. Teratology 64: Collomp, K., Anselme, F., Audran, M., Gay, J. P., Chanal, J. L. and Prefaut, C. (1991). Effects of moderate exercise on the pharmacokinetics of caffeine. Eur. J. Clin. Pharmacol. 40: Dreisbach, R. H. (1974). Handbook of Poisoning: Diagnosis and Treatment. Lange Medical Publications, Los Altos, CA. (cited in IOM, 2004). FDA (2012) Caffeine Intake by the U.S. Population, U.S. Food and Drug Administration, NFOIAElectronicReadingRoom/UCM pdf 4

383 Green, P. J., Kirby, R. and Suls, J. (1996) The effects of caffeine on blood pressure and heart rate: a review. Annals o f Behavioral Medicine 18: (cited in Nawrot et al., 2003). Goldstein, A., and Warren, R. (1962). Passage of caffeine into human gonadal and fetal tissue. Biochem. Pharmacol. 11: Hodgman, M. J. (1998) Caffeine. In; Encyclopedia of Toxicology. (P. Wexler, Ed.). Academic Press, San Diego, CA. p IARC (1991). Coffee, Tea, Mate, Methylxanthines and Methylglyoxal, Volume 51, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, International Agency for Research on Cancer (IARC), Lyon, France. IOM (2004). Caffeine for the Sustainment of Mental Task Performance. Formulations for Military Operations. Institute of Medicine (IOM), pp , National Academy Press, Washington, D.C. James, J. E. (1991) Cardiovascular system. In; Caffeine and Health. (J. E. James, Ed.). Academic Press, London. p (cited in Nawrot et al., 2003). Kaplan, G. B., Greenblatt, D. J., Ehrenberg, B. L., Goddard, J. E., Cotreau, M. M., Harmatz, J. S. and Shader, R. I. (1997). Dose-dependent pharmacokinetics and psychomotor effects of caffeine in humans. J. Clin. Pharmacol. 37: (cited in Mandel, 2002). Maier, H.G. (1981). Kaffee, pp , 63, Paul Parey, Berlin; cited in IARC (1991). Mandel, H. G. (2002.) Update on caffeine consumption, disposition and action. Food Chem. Toxicol. 40: Mrvos, R.M., Reilly, P. E., Dean, B. S. and Krenzelok, E.P. (1989). Massive caffeine ingestion resulting in death. Vet. Human Toxicol. 3: Myers, M. G. (1998). Cardiovascular effects of caffeine. James, J. E. International Life Sciences Institute (ILSI), (cited in Nawrot et al., 2003). Nawrot, P., Jordan, S., Eastwood, J., Rotstein, J., Hugenholtz, A., and Feeley, M. (2003). Effects of caffeine on human health. Food Additives and Contaminants 20: OECD (2002a). SIDS Initial Assessment Report for Caffeine (CAS No ), UNEP Publications. OECD (2002b). IUCLID Data Set for Caffeine (CAS No ), UNEP Publications. 5

384 Toxicity Profile Crystalline Silica, Cristobalite (CAS No ) Crystalline Silica, Quartz (CAS No ) Silica is an off-white granule that occurs naturally in various crystalline and amorphous or other non-crystalline forms. Crystalline silica is characterized by silicon dioxide (SiO2) molecules oriented in fixed, periodic patterns to form stable crystals. The primary crystalline form of silica is quartz. Other crystalline forms of silica include cristobalite, tripoli and tridymite. Particle size is a key determinate of silica toxicity, since toxicity is restricted to particles that are small enough to be deposited into the target regions of the respiratory tract. Oral Exposure No oral studies were located; however, crystalline silica is not expected to exhibit toxicity by the oral route. Although absorption studies were not found for crystalline silica, kinetic studies on amorphous silica show no absorption from the gastrointestinal tract. Dermal Exposure No dermal studies were located; however, crystalline silica is not expected to exhibit toxicity by the dermal route. Inhalation Exposure See attached OECD-SIDS Initial Targeted Assessment Profile on Quartz and Cristobalite, SIAM 32, April 2011.

385 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

386 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

387 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10

388 SIAM 32, April 2011 CAN (relying on death certificates instead of relying on repeated x-rays, which were lacking for each miner, may have underestimated the number of cases) and the fact that the conversion of dust counts to gravimetric measurements may not be accurate based on the estimation of 13% silica content in the respirable dust (although based on a relatively large number of samples (n = 82) collected in two different surveys, there was broad range of content in these samples (1% to 48%, SD = 9), and the percentage of respirable quartz may have differed in earlier years, but data were lacking for these years). Two other human studies have identified similar LOAECs based on the critical endpoint of radiographic confirmed silicosis. A LOAEC of mg/m 3 (mean exposure) was identified in a cross sectional study of South African gold miners and a LOAEC of mg/m 3 (mean exposure) was derived in a mining community population-based random sample survey in Colorado. Carcinogenicity studies Studies on animals Experimental studies conducted in rats have shown clear and consistent increases in lung tumours after chronic inhalation exposure. In the nine rat studies identified, five were inhalation studies and four were intratracheal instillation studies. All studies except one inhalation study showed increased incidence of lung tumours. For the inhalation studies with treatment related tumours, concentrations ranged from 1 to 50 mg/m 3 (1, 6, and 30 mg/m 3 of DQ-12 quartz; 12 and 50 mg/m 3 of Min-U-Sil 5 quartz) and duration of exposure ranged from 29 days to 2 years. In the inhalation study with no treatment-related tumours, exposure was 60 mg/m 3 of Sikron F300 quartz for 13 weeks. The following is a description of the neoplastic results in the study also identified as the critical study for non-neoplastic results. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. In the exposed group, 18 animals developed tumours (12 in females, 5 in males), as opposed to 3 and 2 for the control and positive control groups respectively. The majority (10/18) of the tumours observed were adenocarcinomas. The mean mass of particles in the lungs at the end of the exposure period was 0.91 mg/lung. For the intratracheal instillation studies, doses ranged from 4 to 57 mg/kg-bw (based on 7, 12 or 20 mg/animal of Min-U-Sil (5) quartz or 20 mg/animal of novaculite quartz). Exposure regimes were diverse and included single instillation with observation for up to two years, to weekly instillation for 10 weeks. It is noteworthy that the single intratracheal administration of a 95% pure quartz of silicotic granulomas after 3 weeks and lung tumours after 11 months. The most common tumours reported across the long term rat studies were adenocarcinomas, however other tumours such as squamous-cell carcinoma, alveolar carcinoma and bronchiole-alveolar adenoma were also reported, and all animals that developed tumours also showed some degree of fibrosis. Of particular interest is the intratracheal instillation study, investigating the sequence of pathological events leading to lung tumors. An unspecified number of rats/sex/dose received a single intratracheal instillation of various crystalline silica dusts or ferric oxide, allowing direct administration into the bronchial tree. The doses were 12 or 20 mg of Min-U-Sil 5 quartz (MQZ), 12 mg of hydrofluoric-acid-etched Min-U-Sil 5 quartz (HFMQZ), 12 mg of cristobalite, 12 mg of tridymite and 12 mg of ferric oxide suspended in saline. All groups were observed until six months post exposure, except for both MQZ groups, the HFMQZ and the ferric oxide group which were observed up until 17 months post-exposure. Interim sacrifices were conducted at 6, 11 and 17 months. The rat lungs showed a clear progression of effects. The sequence of pathological events were, an initial inflammatory response leading to a marked hyperplasia and hypertrophy of alveolar cells after one month, and at six months hyperplasia was evident but no lung tumours were observed. In this study, lung tumours were observed starting at the 11 month sacrifice with a 17% and 42% incidence in males and females (based on 3/18 males and 8/19 females), respectively, and at 17 months incidences were 32% and 59% (based on 6/19 males and 10/17 females, respectively). No lung tumours were found in ferric oxide treated rats. Similar studies have also been conducted in hamsters and mice. Although treated mice and hamsters showed treatment related signs (inflammation or fibrosis), no tumours were observed in hamsters. No increase in the incidence of lung tumours was seen in mice treated with quartz; however silicotic granulomas and lymphoid cuffing around airways but no fibrosis were seen in the lungs of quartz-treated mice. Human epidemiology data There is an extensive dataset of human studies investigating the link between crystalline silica exposure and cancer. IARC (1997) identified over 50 epidemiological studies based on occupational exposure to dust containing respirable crystalline silica. Main industry sectors from which the human data is derived include gold mines, foundries, granite/stone industry, pottery workers and refractory brick workers. From the least confounded studies, it was noted that lung cancer tended to increase with the following parameters: cumulative This document may only be reproduced integrally. 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389 SIAM 32, April 2011 CAN exposure; duration of exposure; peak intensity of exposure; presence of radiographically defined silicosis; and length of follow-up time from date of silicosis diagnostic. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). Since the 1997 IARC report, a large number of epidemiological studies have been published, with the more recent studies being updates from supplementary follow-up of results from previously assessed case-control studies cohorts, new results based on refined exposure assessments or adjustment for confounders or metaanalyses of the pooled data from these epidemiology studies In a meta-analysis of the data from 10 cohort studies of gold, tin and tungsten miners, granite workers, industrial sand, diatomaceous earth and pottery workers with quantitative exposure estimates for crystalline silica were pooled in order to analyse the risk related to lung cancer. The pooled cohort standardized mortality ratio (SMR, against national rates) [See Appendix 1 for definition] was 1.2 (Confidence Interval [CI] ). The results from the case-control analyses show a statistically significant trend with duration of exposure (odds ratios (ORs) [See Appendix 1] by quintile of cumulative exposure increased from 1.0 to 1.6 [CIs of 0.85 to 2.1] and by quintile of average concentration increased from 1.0 to 1.7 [CIs of 1.1 to 2.3]), supporting the importance of the increasing lung burden of silica in the occurrence of cancer. Overall, the authors concluded that the results support the carcinogenicity conclusion presented by IARC (1997). To investigate the link between crystalline silica, silicosis and lung cancer, epidemiological data published between 1966 and 2001 were gathered. Over 50 studies were selected and pooled according to type of study and the parameter being linked to lung cancer (i.e. silica exposure, presence of silicosis in subjects). The quality of study, adjustment for confounding factors, co-exposure to other carcinogens and availability of a more recent analysis of a same cohort were taken into consideration in the final selection of the studies. Analysis of the relationship between exposure to silica and lung cancer included 17 cohort and 13 case-control studies. For the analysis of lung cancer versus silicosis, 11 cohort and 5 case-control studies were selected. The third analysis included 6 cohort and 2 case-control studies to evaluate the risk of lung cancer in non-silicotics. A random effect model was used to conduct each meta-analysis. Pooled cancer risk ratios (RRs) were 1.32 (CI ) for crystalline silica exposure, 2.37 (CI ) for individuals exposed to silica with confirmed silicosis (Silicotics) and 0.96 (CI ) for individuals with no evidence of silicosis (non-silicotics) with confirmed exposure to silica, supporting the general observation that silicosis has a stronger temporal relationship with crystalline silica exposure and furthermore support the view that a human silicotic response could be a preliminary stage in the development of cancer. A more recent meta-analysis included 28 cohort, 15 case-control and two proportionate mortality ratio (PMR) [See Appendix 1] studies from a variety of occupational settings conducted between 1996 and Risk ratios (RRs) were calculated based on type of study and silicosis status using fixed and random effect models (results presented here are from the random model). RRs for all cohort studies was 1.34 (CI ), and were 1.69 (CI ) for silicotics, 1.25 (CI ) for those with undefined silicosis status and 1.19 (CI ) for non-silicotics. In the case-control studies, the general RR was 1.41 (CI ), and the same sub-groups as mentioned above resulted in RRs of 3.27 (CI ), 1.41 (CI ) and 0.97 (CI ), respectively. The proportionate mortality ratio for the last two studies was 1.24 (CI ). The authors noted that the association between lung cancer and exposure to crystalline silica was more consistent for silicotics, i.e., those diagnosed with silicosis and RR values split into type of occupational settings in which participants worked. Based upon the above three meta-analysis studies and the epidemiology studies discussed in IARC (1997), the following can be concluded. Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. Cancer risk is often more significant in workers exposed to crystalline silica over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles. There have been reports of tumours outside of the lungs in persons with high silica exposure; however, these reports are sparse and the data inconsistent and have not been unequivocally linked to exposure to either one of the crystalline forms (quartz or cristobalite). Some of the reported locations are: oesophagus, stomach, liver, skin and bone. Sufficient epidemiological or toxicological data do not currently exist for quantitative assessment of the exposure-response relationship on these other tissues or organs. Genotoxicity Potential genotoxicity has been assessed in multiple in vitro and in vivo assays. Table 1 below gives a brief summary of the positive results observed in each type of assay. Table 1. Summary of positive results over total number of results for each assay and each category (all studies This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 5 of 10

390 SIAM 32, April 2011 CAN conducted with crystalline silica: quartz, except where indicated). Assay In vitro Animal data Human data Positives/Total In vivo In vitro In vivo d Rec Assay 0/1 0/1 In vitro DNA strand break 1/1 2/2 5/5 b 1/1 6/6 3/3 Sister chromatid exchange 0/1 0/1 c 1/1 0/2 1/1 Micronucleus 2/3 0/1 2/2 b 1/1 4/5 1/2 Chromosome aberration 0/1 1/1 0/1 1/1 Aneuploidy/polyploidy 0/3 0/3 Cell transformation 4/4 4/4 Hprt mutation 1/2 2/2 a 1/1 b 2/3 2/2 Oxidative DNA damage 4/5 2/2 2/2 4/5 DNA binding 1/1 1/1 p53 activation 0/1 0/1 a. one assay conducted with crystalline silica: cristobalite c. crystalline silica: tridymite d. crystalline silica dust (subtypes not provided). In vivo All the in vivo human genotoxicity studies are based on three independent studies that used blood samples from workers from diverse occupational settings with confirmed exposures to crystalline silica dust; however, quantification of exposure was not provided. After stratification by smoking status, sister chromatid exchange remained statistically significant in both smokers and non-smokers although the frequency was higher in smokers. For the chromosome aberration assay conducted as part of the same study (blood samples from workers in the stone crusher industry), the increased frequency was no longer significant after stratification. In the DNA damage study of foundry and pottery workers and the micronucleus assay of workers involved in sandblasting and related jobs, results were positive when compared to controls. However, in both studies, smoking status influenced the results, contributing to the increased DNA damage observed since results were greater in smokers versus non-smokers, and the frequency of micronuclei in nasal epithelial cells was higher in smokers (p=0.002) but when using peripheral blood lymphocytes did not differ statistically between smokers and non-smokers who were similarly exposed to silica dust, The role of in situ generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been well established in the following types of DNA damage: small scale insertions, DNA base pair deletion, base modification, chromosomal change/loss, microsatellite instability, DNA strand break, 8- hydroxydeoxyguanosine (8-OHdG) mutation and point mutations. ROS and RNS generation is postulated to be (a part of) the DNA damage mechanism for crystalline silica. Studies are described below to support this hypothesis. DNA was exposed in vitro to various crystalline silica dusts, to H 2 O 2, or to both. Results show that DNA damage was limited when dust or H 2 O 2 were administered alone but increased with the co-exposure. When the reactive oxygen scavenger, dimethylsulfoxide, was added to the test system, DNA strand break was inhibited, data supporting the viewpoint that it is the presence of radicals generated in response to quartz and cristobalite that causes the DNA damage and not quartz or cristobalite themselves. Hprt mutation assays in rat alveolar epithelial cells, both in vitro and in vivo, were positive in response to quartz. The positive results in vivo were seen only in the presence of significant inflammatory responses in the treated animals. Also, in a parallel in vitro experiment, rat alveolar epithelial cells were incubated with the bronchoalveolar lavage fluid from the rats exposed to quartz. Both macrophage and neutrophil enriched lavage cells induced mutation in the exposed alveolar epithelial cells. Addition of catalase (an enzyme which inactivates H 2 O 2 ) before incubation inhibited the increase in hprt mutation. Rats were exposed to either crystalline or amorphous silica in a manner to induce the same level of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 6 of 10

391 SIAM 32, April 2011 CAN inflammation in the lungs. The inflammatory response was assessed by measuring the proportion of neutrophils in the bronchiolar lavage fluid. The actual concentrations were 3 and 50 mg/m 3 for crystalline and amorphous silica respectively. The animals were exposed for 13 weeks. Hprt mutation frequency was measured in the alveolar epithelial cells at the end of the exposure period. Mutation frequency was greatly increased only in the crystalline silica treated rats; no treatment related increase was found in the rats treated with the amorphous form. In an 8-OHdG assay conducted to monitor DNA damage by reactive oxygen species, female rats were exposed to 0, 0.3, 1.5 and 7.5 mg/animal of quartz via intratracheal instillation. Effects were observed 90 days postexposure. A clear dose-response relationship was identified between quartz exposure and various inflammation markers (differential cell count, protein, lung surfactant lipids and tumour necrosis factor alpha). Inflammation was present starting at the lowest dose. However, 8-OHdG showed a statistically significant increase starting at 1.5 mg/animal only. Similarly, in another study, 8-OHdG and DNA strand breaks were observed at concentrations of or above 10 ug/m 3 in rat lung epithelial cells. In the aim of investigating the role of ROS in lung carcinogenesis, rat lung epithelial cells were incubated with polymorphonuclear (PMN) leukocytes (involved in the inflammatory process and responsible for the release of certain ROS) or hydrogen peroxide. Statistically significant increases in 8-OHdG were observed in the presence of PMN or hydrogen peroxide in a dose-response manner. In a series of experiments which used in vitro stimulation of macrophages with crystalline silica and in vivo intranasal instillation of crystalline silica in mice, it was demonstrated that that the chronic fibrosis seen in a murine model of silicosis in vivo is dependent on the presence of adaptor molecule ASC and Nalp3 inflammazome. These data support a potential mode of action whereby silica triggers cellular responses that in turn activate alveolar macrophages, resulting in an inflammatory response and silicosis. In mice deficient in Nalp3 inflammazome, the development of inflammation and collagen deposition was significantly reduced compared with normal mice 3 months after the initial intranasal instillation of silica. Analysis of Lung Tumour Data The weight of evidence for both rats and humans indicates that fibrotic and silicotic lesions in the lung result from inhalation exposure to crystalline silica and that lung cancer is secondary to those lesions in the lung. Although the mechanism of induction for the lung tumours has not been fully elucidated, there is sufficient supportive mode of action evidence from the data presented to demonstrate that a threshold approach to risk assessment is appropriate based on an understanding of the key events in the pathogenesis of crystalline silica induced lung tumours. The body of evidence include the following: In experimental studies, all rats that developed tumours also showed fibrosis. Adenocarcinomas, the most common type of tumour identified in rats, are commonly associated with fibrosis and deeply scarred lung tissue. Experimental rat studies showed a clear progression of the effects from initially mild inflammation, followed by fibrosis over-time, leading eventually to lung tumours. Tumours are not present in all treated species dosed in the same way. The tumours, both in rats and humans, are concentrated in the lungs only, although other organs are indirectly exposed. In human studies, cancer risk is often more significant in workers exposed over a 20 year period or to higher cumulative exposure levels; however a consistent finding is that the onset of silicosis, requires a smaller lag period than that for the appearance of tumours. Similarly, cancer risk is often more significantly associated at higher quintiles of exposure compared to the lower quintiles Lung cancer rates are higher in workers confirmed to have silicosis versus similarly exposed workers that do not have silicosis. For genotoxicity, in vitro results were mostly mixed and in vivo results were mostly positive. However, the vast majority of the positive genotoxicity assay results can be explained by the generation of reactive oxygen species, as demonstrated experimentally, where ROS scavenging prevents the genotoxicity. In vivo, macrophage deficient mice (macrophages produce ROS in response to crystalline silica) do not develop silicosis nor do they develop tumours and the Nalp3 inflammazome, a key player in the macrophage initiated inflammatory response, is required for the development of pulmonary fibrosis after This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 7 of 10

392 SIAM 32, April 2011 CAN inhalation of silica. Though inhalation exposure to crystalline silica in multiple occupational settings is clear, the increase in risk, based on the several recent meta-analyses of the multiple human epidemiological studies, remains low. It is worth noting that where aggressive engineering controls have been made to reduce silica dust levels in the workplace (Sweden), silicosis has been eliminated. By corollary, existing exposures outside of the workplace in such areas do not pose a risk for silicosis to the general population. The respirable forms of quartz and cristobalite possess properties indicating a hazard for human health (repeated dose toxicity, carcinogenicity and genotoxicity). The mode of action in the lungs involves irritation, inflammation and reactive species formation, leading to silicosis, and eventually to tumour formation. Exposure Uses -oxide minerals, including quartz are ubiquitous in the natural environment. In particular, as a component of sand, quartz may find use in a diverse array of applications. Several high volume uses include, but are not limited to, the use of sand as a filling material for the construction of roads and in general building activities, the use of sand and gravel aggregates as abrasives on roads in winter and the use of fly-ash, which may contain 4-14% quartz and 0.5-1% cristobalite, as a cement additive. These abrasives when used on winter roads are usually mixed with road salts and may be sand only, stone dust, sand and gravel aggregates, or pre-treated sand. They are used mainly by rural municipalities or in areas where cold temperatures diminish the efficiency of salts for de-icing. Quantities of abrasives used in Canada were kg, kg, and kg for 2007, 2008 and 2009, respectively. Industrial sand, high purity silica sand products with closely controlled sizing are expected to contain quartz and cristobalite, include lump silica (2-3mm up to 15 cm or more), silica sand (75-3mm) and silica flour certain types of pulverizers (eg. ball mills and tube mills). Silica sand may be used in the manufacture of glass and glass fibres, silicate chemicals and silicon carbide, the hydraulic fracturing of wells, foundry moulding, and for sandblasting. Silica flour may find use in the ceramics and cement industries, as a filler and extender in rubber and coatings, and as an abrasive in soaps. capacities. Quartz is a natural component of these clays, and consequently, it may be present in cat litter products. High pu -quartz is a piezoelectric material, which means that application of a voltage induces a distortion in the crystal shape and vice versa. This ability to interconvert electrical and mechanical energy has led to the use of quartz crystals in electronic devices requiring precise timing control, for example telephones, radios, watches and computers. According to a survey conducted in Canada, quartz and cristobalite are also used in abrasives, adsorbents, filter products (diatomaceous earth), grout and cement. These substances reportedly also find use as fillers, which add bulk and improve wear resistance, in paints and coatings, adhesives, sealants, polymer films, caulking, epoxy resins and silicones. Also, quartz is listed as an ingredient in 60 cosmetic products in Canada. The types of products include anti-wrinkle preparation, eye and face makeup, lipstick, hair dyes, shampoos and grooming products, as well as skin cleansers, moisturizers and tanning preparations. Natural Sources In Canada, quartz naturally occurs in many types of rock formations. Those with high silicon dioxide content (95% SiO 2 or more) include vein and massive intrusion bodies, quartz pebbles, silica sand, sandstone and quartzite. Sandstone is a sedimentary rock mostly composed of quartz grains cemented by a bonding material such as clay, calcite or iron oxide. Quartzite is a hard, compact, metamorphosed sandstone made of grains of quartz firmly bonded with a siliceous cement. Mineral aggregates (e.g., sand and gravel) have variable silicon dioxide content. Quartz is also found as crystals, aggregates or discrete particles in certain igneous rocks (e.g., granites and pegmatites), soils, sediments, air and surface water. This omnipresence is consistent with the fact that silicon is the second most abundant chemical element on Earth. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 8 of 10

393 SIAM 32, April 2011 CAN Cristobalite is naturally produced in the ashes of volcanic eruptions, and by combustion metamorphism which is a local phenomenon of spontaneous combustion of naturally occurring substances such as bituminous rocks, coal or oil. It may be found in cavities in volcanic rocks and in thermally metamorphosed sandstones and may also be a transient stage in the diagenesis of diatomaceous shale with the result that soils made of these geologic formations may be rich in cristobalite. Unlike quartz, the natural occurrence of cristobalite is limited to specific geographic regions and mineral types. Anthropogenic Sources Natural quartz is isolated from ore via beneficiation, which involves milling or grinding the material into particles that are separated into desired mineral and waste. The materials obtained are either used directly or further purified. In Canada, in 2006, kg of pure quartz were mined, and kg of sand and gravel aggregates were produced. The proportion of quartz in silica sand deposits and gravel aggregates will vary from one site to another. Cristobalite can form from silica melts during the preparation of silica glass; quartz is not obtained from melts but is manufactured at elevated temperature and pressure via a hydrothermal process. Cristobalite also forms during the calcination 2 of diatomaceous earth. Human Exposure Estimate Ambient air The exposure assessment is focussed on respirable quartz and cristobalite, which in ambient air comprises a component of total particulate matter (PM). In Canada, data on the concentrations of silicon in PM was available and used as a surrogate for quartz and cristobalite. This approach is conservative because the measured silicon includes all silicon-containing substances and therefore represents the upper limit for quartz and cristobalite in ambient air. 3 of silicon in PM with (dichot)) (the total particulate matter with aerodynamic radii le 10 ) is obtained by adding these values) in Canada. In 2009, as part of the NAPS Program, silicon concentrations were determined on over 1600 samples of PM 2.5 (dichot) and over 1500 samples of PM 10 (dichot) at 24 urban locations across Canada. An estimate of exposure to quartz and cristobalite can be obtained by assuming that all the silicon in the PM is represented stoichiometrically as SiO 2, and multiplying the reported concentration of silicon by 2.14 to obtain a value for silica. The intake of respirable quartz and cristobalite by the general population of Canada is estimated using a range covering the lowest 50 th percentile SiO 2 concentration in PM 10, measured in 3 ) to the highest 50 th percentile concentration in PM 10 3 ). The 50 th percentile SiO 2 concentrations ranged from 0.1 to 2.1 µg/m 3 across the survey sites; the top of this range is quite close to the average of the maximum values for the 24 sites (3.7 µg/m 3 ). The outdoor data were used to represent the indoor levels, because information on indoor silicon concentrations was not available, and the range of PM 10 measured indoors is generally lower than the outdoor range. Thus, this approach conservatively overestimates indoor exposure in homes. The highest exposure group based on these calculations is children ages 0.5 to 4 years with an estimated daily -bw per day; the estimated daily intake decreases with age due to changes in the ratio of inhalation rates to body weights; the daily intake of adults, years old, is estimated to range from 0.03 to bw per day. Consumer Products Exposure to respirable quartz from the use of cosmetic products, which contained quartz as an ingredient, was considered low because they are not formulated for spray application, the loose powders were reported to contain less than 0.1% quartz, and in these products the substance is not expected to be associated with other components of the formulation and not available in a free form. For consumer Do It Yourself (DIY) activities around the home, the highest mean breathing zone concentration of particles from sanding dry wall (median cut- 3, was used to derive an upper- 2 Calcinations: Heat treating a substance, but without fusion, to bring about change in its physical or chemical constitution. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 9 of 10

394 SIAM 32, April 2011 CAN -bw per event. Quartz is used to formulate a large number of paints and coatings. To estimate potential inhalation exposure to quartz from these products, the spray painting of wall paints with an airless spray gun was considered appropriate as a conservative scenario. Exposure to respirable paint particles was estimated using data from controlled a laboratory study in which walls of poorly ventilated test rooms were painted by professional painters using an airless sprayer to apply interior latex paint. The maximum concentration of 13% quartz in paint in Canada was used to estimate exposure. The upper-bound estimate of exposure to quartz, based on the maximum concentration of 13% quartz in paint in the Canadian market and the maximum concentration of respirable paint particles measured in these controlled studies when recommended personal protective -bw per event. Inhalation of ambient air containing quartz and cristobalite is the dominant pathway of chronic exposure (excluding that from DIY activities) for the general population. Because SiO 2 makes up only approximately 5% of PM 10, silicon concentrations (expressed as SiO 2 ) measured in the Canadian NAPS survey of 24 urban locations were considered most relevant to the estimation of exposure by the general population. Quartz and cristobalite comprise only a portion of the total SiO 2 in PM 10, therefore, the use of the total silicon concentration to represent the upper bound crystalline silica concentrations results in an overestimation of exposure. Appendix 1: Definitions of Epidemiological Terms in the ITAP SMR (Standardized Mortality Ratio): The ratio (x 100) of observed to expected deaths in a study population. Expected deaths are calculated by applying a set of standard age-specific mortality rates to the age distribution of the study population. Standardized ratios are only useful for comparisons. They have no intrinsic meaning. OR (Odds Ratio): In epidemiological case-control studies, a relative measure of disease occurrence. The odds in favour of a particular disease occurring in an exposed group are divided by the odds in favour of its occurring in an unexposed group. If the condition being studied is rare, the odds ratio is a close approximation to the relative risk. RR (Risk Ratio): The probability of the occurrence of a disease in a group that has been exposed to some environmental, medicinal, microbial, or toxic influence, relative to its probability in a randomly selected population. PMR (Proportionate Mortality Ratio): Proportionate mortality is the proportion of deaths in a specified population over a period of time attributable to different causes. Each cause is expressed as a percentage of all deaths, and the sum of the causes must add to 100%. These proportions are not mortality rates, since the denominator is all deaths, not the population in which the deaths occurred. Thus, proportionate mortality ratio is a measure of the frequency of occurrence of the proportionate mortality in a defined population during a specified interval of time. This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 10 of 10

395 SIAM 32, April 2011 CAN INITIAL TARGETED ASSESSMENT PROFILE (Human Health) CAS No. Quartz: CAS RN Cristobalite: CAS RN Chemical Name Quartz and Cristobalite Molecular Formula: SiO 2 Structural Formula Unit Cell: Trigonal symmetry Unit Cell: Tetragonal symmetry Quartz: CAS RN Cristobalite: CAS RN SUMM ARY CONCLUSIONS OF THE TARGETED ASSESSM ENT NOTE: The present assessment is targeted to address the following human health endpoints: repeated dose toxicity and carcinogenicity via the inhalation route of exposure, and genotoxicity. It cannot be considered as a full SIDS Initial Assessment. Summary information on exposure is also reported here. Other endpoints for human health and the environment included in the Canadian screening assessment but have not been presented to OECD member countries, and thus are not included in this profile. The final screening assessment will be published under the responsibility of the Government of Canada. Rationale for Targeting the Assessment The Government of Canada "categorized" or prioritized all 23,000 chemical substances on its Domestic Substances List (DSL) from 1999 to September 2006, as required by its Canadian Environmental Protection Act, 1999 (CEPA 1999). Additional details may be found at Using information from Canadian industry, academic research and other countries, Government of Canada scientists applied a set of rigorous tools to the 23,000 chemical substances on the DSL. They were categorized to identify those that were: inherently toxic to humans or to the environment and that might be persistent and/or bioaccumulative; and substances to which people might have greatest potential for exposure. During this priority-setting exercise, distinct approaches were taken for identifying substances of likely concern for human health and the environment, and subsequent assessment activities may have focused on either human health or ecological endpoints. Through categorization, the Government of Canada has identified approximately 4,000 of the 23,000 This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 1 of 10

396 SIAM 32, April 2011 CAN chemical substances on the DSL as priorities for further assessment, research and/or measures to control their use or release. Quartz and cristobalite were identified at that time, applying the categorization criteria, as high priorities for human health risk because they were considered to pose greatest potential for exposure and their respirable forms are classified by the International Agency for Research on Cancer as carcinogenic to humans (quartz and cristobalite) and by the National Toxicology Program as known human carcinogens (crystalline silica). These substances did not meet the ecological categorization criteria for bioaccumulation potential or inherent toxicity to aquatic organisms. Under the Canadian legislation a determination of whether one or more of the criteria of the CANADIAN ENVIRONMENTAL PROTECTION ACT (CEPA) 1999, section 64 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposures in the general environment. For humans, this includes, but is not limited to, exposures from ambient and indoor air, drinking water, foodstuffs, and the use of consumer products. A conclusion under CEPA 1999 on the substances in the Chemicals Management Plan (CMP) Challenge is not relevant to, nor does it preclude, an assessment against the hazard criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System [WHMIS] for products intended for workplace use. Physical-chemical properties The silicon dioxide group represents a polymorphic category containing a large number of forms identical in composition but with different atomic arrangements which afford different chemical properties. There are two sub-categories within this group: crystalline silica, to which the present substances quartz and cristobalite belong, and non-crystalline or amorphous silica. The key distinction between these sub-categories is that in crystalline substances, the building blocks are arranged in regular, repeating 3-dimensional pattern having long range order, whereas amorphous materials do not display long range order. In all forms of silica, (crystalline and non-crystalline), the silicon atom is tetrahedral and bound to four neighbouring oxygen atoms. Quartz and cristobalite are solid at room temperature, existing normally as colourless or white crystals. The melting points for quartz and cristobalite are o C and o C, respectively and for both compounds, the boiling point is 2230 o C. Even though no experimental data were available, their vapour pressure ow (octanol-water partition coefficient) and log K oc (organic carbon-water partition coefficient) are not applicable to these substances. The densities range from kg/m 3 for quartz and kg/m 3 for cristobalite. The very similar physico-chemical properties of quartz and cristobalite reflect their closely related crystal forms. The solubility of crystalline silicates decreases as a function of silica tetrahedral packing density and long-range crystal order. For example, cristobalite has a more open framework structure than quartz and its density is lower, therefore, its solubility is higher. The water solubility of these minerals is also function of temperature, ph, particle size, and the presence of a disrupted surface layer. This may explain the variability of solubility values reported by many authors. The most probable solubility value for quartz is approximately 3.8 mg Si/L, or 6.4 mg/l expressed as the SiO 2 species, while the solubility of cristobalite is approximately 8.7 mg Si/L, or 18 mg SiO 2 /L. The kinetics of dissolution of these substances is slow due to the high activation energy required to hydrolyse the Si-O-Si bond. Human Health Targeted Endpoints The majority of the studies described here have been reviewed by the International Agency for Research on Cancer (IARC 1997). However, additional data relevant to the screening assessment were identified up to August Repeated dose toxicity/non-neoplastic effects (development of silicosis). Studies on animals Significant short-term and subchronic studies have demonstrated adverse effects in the lungs, while one of the 6 studies showed effects on the spleen in mice. Rats were exposed to 0, 10 or 100 mg/m 3 of cristobalite via inhalation for 6 hours/day during 3 days. Animals were observed 3 months after exposure. Elevated levels of granulocytes and elevated markers of cytotoxicity from the lung lavage fluid were noted in all exposed groups. Another study of similar duration (9 days) conducted in mice also identified a LOAEC of 10 mg/m 3. Effects observed included minimal interstitial thickening, accumulations of mononuclear cells and slight lymphoid tissue hypertrophy in the lungs. In a 4-week inhalation study, female rats were exposed to 0, 0.1, 1 or 10 mg/m 3 of quartz 6 hours/day, 5 days/week. Bronchoalveolar lavage fluid was evaluated at 1, 8, and 24 weeks after exposure. Elevated levels of This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 2 of 10

397 SIAM 32, April 2011 CAN granulocytes and signific - -glu]) were observed at 1 mg/m 3 -glu were only significant at 24 weeks after exposure. A LOAEC of 1 mg/m 3 was identified at 24 weeks. Male rats (4 animals per dose) were exposed to 0 or 3 mg/m 3 of cristobalite via inhalation for 6 hours/day, 5 days/week during 13 weeks. Pulmonary inflammation and fibrosis were observed in the exposed group at the end of treatment. When mice were similarly exposed to 5 mg/m 3 of quartz for 6 hours/day, 5 days/week for 15 or 27 weeks, the authors observed increased spleen weight and formation of plaque in the spleen. In two separate studies, in which rats or hamsters were exposed to quartz via inhalation for at least 6 months, LOAECs of 2 and 3 mg/m 3 were identified, respectively. All the effects observed were related to inflammation and fibrosis of the lung tissue. Several chronic studies investigated exposure of the respirable forms (i.e. accumulated via inhalation in the lung tissues) of quartz and cristobalite to rats, mice and hamsters. The following is a description of the study in which the lowest non-neoplastic LOAEC was determined. Groups of 50 rats/sex were exposed 6 hr/day, 5 days/week for 24 months to filtered air or 1 mg/m 3 of DQ-12 quartz, containing 74% of respirable quartz, through whole-body inhalation. An additional 50 rats/sex were exposed to 5 mg/m 3 of titanium dioxide as positive controls. The mean mass of particle at the end of the exposure period was 0.91 mg/lung. The LOAEC identified was 0.74 mg/m 3 (adjusted for 74% respirable quartz) based on lipoproteinosis, multifocal, inflammatory cell infiltrate and alveolar hyperplasia. Human epidemiology data In humans, the lowest observed adverse effect level was identified in a U.S. cohort study. The study was conducted on 3330 gold miners (all are males), who had an average of 9 years underground exposure during the period 1940 to The cohort was followed up through Silicosis 1 was identified through death certificates or chest X-rays. A job-exposure matrix together with work history was used to estimate individual exposure. The total silica content in the respirable dust in the mine was estimated at 13% and the median crystalline silica exposure was 0.05 mg/m 3. In this sub population of miners, 170 cases of silicosis were identified. The best predictor for risk of silicosis was cumulative exposure, which varied from less than 1% for a 0.5 mg/m 3 -year exposure to 68-84% when exposed to more than 4 mg/m 3 -year (based on the average daily dust exposure during the workday each year and summed over time for each miner). The main limitations identified by the authors include the limited number of radiographic surveys, the potential bias from death certificates 1 Silicosis: Lung disease caused by inhalation of crystalline silica dust, and resulting in inflammation and scarring in forms of nodular lesions in the upper lobes of the lungs. By definition, clinically or pathologically diagnosed silicosis implies prior exposure to silica (Silicotics). It does not follow that a history of exposure to silica necessarily Figure 1. Silicotic nodule characterised by a central zone of hyalinised collagen with a whorled appearance and peripheral dust-containing macrophages (Rees and Murray, 2007). This document may only be reproduced integrally. The conclusions in this document are intended to be mutually supportive, and should be understood and interpreted together. Page 3 of 10