Memorandum. Re-Review of Triethanolamine, Diethanolamine, and Ethanolamine

Transcription

1 Memorandum To: From: CIR Expert Panel Members and Liaisons Monice M. Fiume MMF Senior Scientific Analyst/Writer Date: November 18, 2010 Subject: Re-Review of Triethanolamine, Diethanolamine, and Ethanolamine In 1983, the Expert Panel published a safety assessment on triethanolamine (TEA), diethanolamine (DEA), and monoethanolamine (MEA; now named ethanolamine) with the conclusion that TEA, DEA, and MEA are safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of ethanolamines should not exceed 5%. MEA should only be used in products that do not contain N-nitrosating agents. Since that 1983 assessment, new data have been published, including an NTP report on TEA that shows some evidence of carcinogenic activity in female mice and on DEA that shows clear evidence of carcinogenic activity in male and female mice. Subsequent to these studies, several follow-up studies to identify a mode of action have appeared in the literature. While DEA, carcinogenicity in mice, and mode of action in mice have been discussed at a number of Panel meetings, and industry has made presentations on this subject, the question of a re-review was never formally raised. Therefore, this rereview package t was prepared for the Panel as a procedural follow-through. While these three ingredients were grouped together in the original assessment, continuing to keep them together may not be the best approach. So, three separate re-review document are being presented. If the Panel decides that any of these ingredients should be re-opened, you have the option to re-open one, two, or all three of the ingredients, and the report can be split or kept as one document. As a reminder of the Panel s discussions at past meetings, the minutes from the June 1999, June 2008, and June 2009 meetings are being provided, as well as a synopsis of the presentation given by Dr. William Stott, who represented the Alkanolamines Panel of the American Chemistry Council, regarding a proposed mechanism of action for carcinogenicity and its relevance to humans. In addition to those minutes, information/correspondence previously submitted to the CIR, as well as Dr.Andersen s previous memos to the Panel regarding issues involving DEA, also are being provided.

2 Dialkyl- and dialkanolamines (including DEA) and their salts are on the European Union (EU) list of substances which must not form part of the composition of cosmetic products, due to concern over their readiness for nitrosamine formation. Dialkanolamines are also completely prohibited for use in cosmetic formulations in Canada. Monoalkylamines, monoalkanolamines (including MEA) and their salts are allowed for use with restrictions by the EU, as are trialkylamine, trialkanolamines (including TEA), and their salts. The CIR has issued a number of reports on TEA-, DEA-, and MEA-containing ingredients. A list of these ingredients is attached, with the conclusion reached by the CIR, to give you an indication of the impact this report has. (The number of TEA-, DEA-, and MEA-containing ingredients not yet reviewed is quite extensive.) As a final note, you will notice that the format used for these re-review documents looks different than what you are accustomed to. The CIR is in the process of updating the presentation of data. For example, rather than breaking Animal Toxicology section into Acute, Short-Term, Subchronic, and Chronic studies, this information will be presented under Acute (Single Dose) Toxicity and Repeated Dose Toxicity. Feedback on the new format is appreciated

3 TEA-CONTAINING INGREDIENTS REVIEWED BY THE CIR TEA Dodecylbenzenesulfonate safe as used when formulated to be non-irritating (Discussion stated that TEA was previously reviewed and found TEA Tridecylbenzenesulfonate safe as used) Final Report, 2008 JACT 12(3) 1993 (orig.) TEA-EDTA safe as used (TEA was not mentioned in Discussion) IJT 21(S2) 2002 TEA Lactate safe for use in cosmetic products at concentrations 10%, at final formulation ph > 3.5, when formulated to avoid increasing sun sensitivity or when directions for use include daily use of sun protection; safe for use in salon products at concentrations 30%, at final formulation ph 2 3.0, in products designed for brief, discontinuous use followed by thorough rinsing from the skin, when applied by trained professionals, and when application is accompanied by directions for daily use of sun protection (TEA was not mentioned in Discussion) IJT 17(S1) 1998 TEA Lauryl Sulfate TEA Salicylate TEA Stearate can be used without significant irritation at a final concentration thereof not exceeding 10.5%; greater concentrations may cause irritation, especially if allowed to remain in contact with the skin for significant periods of time (TEA was not mentioned in Discussion) safe as used when formulated to avoid skin irritation and when formulated to avoid increasing the skin's sun sensitivity, or, when increased sun sensitivity would be expected, directions for use include the daily use of sun protection (TEA was not mentioned in Discussion) safe as used in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing; in products intended for prolonged contact with the skin, the concentration of TEA Stearate should not exceed 15% in formulation; should not be used in products under conditions resulting in N-nitrosation reactions (Discussion states TEA conclusion) JACT 1(4) 1982 IJT 22 (S3) 2003 JACT 14(3) 1995 TEA Cocoyl Hydrolyzed Collagen safe as used (TEA was not mentioned in Discussion) JACT 2(7) 1983 (not reopened in 2005) Cocamide DEA DEA Dodecylbenzenesulfonate Isostearamide DEA Myristamide DEA Stearamide DEA Lauramide DEA Linoleamide DEA Oleamide DEA (Cocamide DEA. originally) DEA-CONTAINING INGREDIENTS REVIEWED BY THE CIR safe as used in rinse-off products and safe at concentrations 10% in leave-on cosmetic products.; should not be used as an ingredient in cosmetic products in which N-nitroso compounds are formed (original Discussion addressed nitrosation, but not levels of free DEA) safe as used when formulated to be non-irritating (Discussion stated that DEA was previously reviewed and found safe as used) safe for use in rinse-off products; in leave-on products, these ingredients are safe for use at concentrations that will limit the release of free ethanolamines to 5%, but with a maximum use concentration of 40%; these ingredients should not be used in cosmetic products in which N-nitroso compounds may be formed (Discussion addressed DEA) safe as used; should not be used as ingredients in cosmetic products containing nitrosating agents (original Discussion addressed nitrosation, but not levels of free DEA) JACT 15(6) 1996 JACT 5(5) 1986 (orig.) Final Report, 2008 JACT 12(3) 1993 (orig.) Final Report, 1995 JACT 5(5) 1986

4 Acetamide MEA Cocamide MEA Isostearamide MEA Myristamide MEA Stearamide MEA MEA Salicylate MEA-CONTAINING INGREDIENTS REVIEWED BY THE CIR safe at 7.5% in leave-on products and safe as used in rinse-off products; cosmetic formulations containing Acetamide MEA should not contain nitrosating agents or significant amounts of free acetamide (Discussion addressed nitrosation) safe as used in rinse-off cosmetic products and safe at concentrations up to 10% in leave-on products; should not be used as an ingredient in cosmetic products containing N-nitrosating agents, or in product formulations intended to be aerosolized (Discussion refers to cocamide MEA) safe for use in rinse-off products; in leave-on products, these ingredients are safe for use at concentrations that will limit the release of free ethanolamines to 5%, but with a maximum use concentration of 17%; these ingredients should not be used in cosmetic products in which N-nitroso compounds may be formed (Discussion addressed MEA) safe as used when formulated to avoid skin irritation and when formulated to avoid increasing the skin's sun sensitivity, or, when increased sun sensitivity would be expected, directions for use include the daily use of sun protection (MEA was not mentioned in Discussion) JACT 12(3) 1983 (not reopened in 2008) IJT 18(S2) 1999 Final Report, 1995 IJT 22(S3)

5 TEA, DEA, MEA HISTORY Original Report: In 1983, the Expert Panel determined that these ingredients were safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of ethanolamines should not exceed 5%. Ethanolamine (MEA) should be used only in rinse-off products. Triethanolamine (TEA) and diethanolamine (DEA) should not be used in products containing N-nitrosating agents. June 1999: discussed NTP carcinogenicity results; presentations were made by Dr. Lehman- McKeeman and Dr. Stott June 2008: presentation was made by Dr. Stott; Acetamide MEA was discussed, with reference to the MEA, DEA, TEA report June 2009: discussed DEA carcinogenicity; mentioned that the DEA report was not be reopened December 2010: formal rereview package was presented to the Panel

6 SEARCH STRATEGY TEA/DEA/MEA TOXLINE PUBMED EU Jan DEA not to be used & choline & carcinogen* choline & deficiency & 38 human & cancer TEA & carcinogen* & choline 5 2 MEA Jan 25, OR (downloaded 58) (1980-current) restrictions restrictions UPDATED SEARCH May 31, 2010 ( OR OR ) AND (REPRODUCTI* OR TERATOGEN*) 142 (Toxline); 41 (DART) (DART) ( OR OR ) AND (DEVELOPMENT* OR FETOTOX*) 378 (Toxline); 47 ( OR OR ) AND TOX* ( OR OR ) AND (GENOTOX* OR MUTAGEN* OR CLASTOGEN*) 286 Toxline); 7 (DART); 9 (CCRIS) ( OR OR ) AND (SENSITIZA* OR SENSITIZE* OR SENSITIS* OR IRRIT*) 306 (Toxline); 6 (DART) ( OR OR ) AND (METBOLI* OR ABSORB* OR ABSORP* OR DISTRIBUT* OR EXCRET*) 403 (Toxline); 18 (DART) AND CARCINOGEN* AND CHOLINE - 0 Total Download (most duplicates removed): 1218 UPDATED SEARCH Sept 21, 2010 last 12 mos OR OR hits/1 useful

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23 Re-Review of Triethanolamine Introduction... 1 Chemistry... 1 N-Nitrosodiethanolamine Formation... 1 Use... 2 Cosmetic... 2 Non-Cosmetic... 2 Toxicokinetics... 3 Absorption, Distribution, Metabolism, Excretion... 3 Dermal... 3 Oral... 4 Other... 4 Toxicological Studies... 4 Acute Toxicity... 4 Dermal... 4 Oral... 4 Other... 5 Repeated Dose Toxicity... 5 Dermal... 5 Oral... 5 Inhalation... 6 Reproductive and Developmental Studies... 6 Dermal... 6 Oral... 6 Genotoxicity... 7 In Vitro... 7 In Vivo... 7 Carcinogenicity... 7 Dermal... 7 Oral... 8 Irritation and Sensitization... 8 Irritation... 8 Skin... 8 Mucosal... 9 Sensitization... 9 Provocative Testing Phototoxicity/Photoallergenicity Clinical Use Case Studies Miscellaneous Studies N-Nitrosodiethanolamine Formation Possible Mode of Action for Carcinogenic Effects Risk Assessment Exposure Assessment Tables Table 1. Definition and Structure Table 2. Physical and Chemical Properties Table 3. Current and historical frequency and concentration of use of TEA according to duration and type of exposure References... 14

24 0.3% was found in TEA samples. 3 N-Nitrosodiethanolamine Formation INTRODUCTION Triethanolamine (TEA), an ingredient that functions as a fragrance ingredient, surfactant emulsifying agent, and ph adjuster in cosmetic products, has previously been reviewed by the CIR Expert Panel. In 1983, the Expert Panel concluded that TEA is safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of TEA should not exceed 5%. TEA should not be used with products containing N-nitrosating agents. In the 1983 assessment, data demonstrated that TEA was a mild skin and eye irritant, and that irritation increased with increasing ingredient concentration. This current document contains summaries of data obtained from a search of published literature, as well as summary information from the original safety assessment CHEMISTRY TEA is an amino alcohol. The structure and synonyms of TEA are given in Table 1, and chemical and physical properties are given in Table 2. TEA is produced commercially by aminating ethylene oxide with ammonia. The replacement of three hydrogens of ammonia with ethanol groups produces TEA. TEA contains small amounts of diethanolamine (DEA) and ethanolamine (MEA). TEA is reactive and bifunctional, combining the properties of alcohols and amines. The reaction of ethanolamines and sulfuric acid produces sulfates. TEA can act as an antioxidant against the autoxidation of fats of both animal and vegetable origin. TEA can react with nitrite or oxides of nitrogen to form N-nitrosodiethanolamine (NDELA). The optimum ph for nitrosamine formation is between 1 and 6; however the rate of NDELA formation in the ph range of 4-9 is four to six times greater in the presence of formaldehyde. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 TEA is produced by reacting 3 moles of ethylene oxide with 1 mole of ammonia; additional ethylene oxide will continue to react to produce higher ethylene oxide adducts of TEA. 2 Typically, ethylene oxide is reacted with ammonia in a batch process to produce a crude mixture of approximately one-third each MEA, DEA, and TEA. The crude is later separated by distillation. Based on unpublished survey data collected by the Food and Drug Administration (FDA), a DEA impurity level of Nitrosamines are compounds containing the R 1 R 2 N-NO functional group. Nitrosation is the process of converting organic compounds (e.g., alkyl amines) into nitroso derivatives (e.g., nitrosamines) by reaction with nitrosating agents. These agents include nitrous acid (HNO 2 ), oxides of nitrogen (e.g., nitrites), and other compounds capable of generating a nitrosonium ion, NO +. The formation of a specific nitrosamine, N-nitrosodiethanolamine (NDELA), from reaction of TEA with nitrite was examined in vitro and in vivo. 4 The TEA used in these studies had an impurity content of 0.4% DEA. In an aqueous (aq.) matrix, approximately 3% TEA converted to NDELA at a ph of 4.0 in the presence of acetic acid. At the same ph, in the presence of sulfuric or hydrochloric acid, only about 1% of the TEA was nitrosated. At ph 7, the greatest nitrosation to NDELA, 0.5%, occurred in the presence of sulfuric acid. No conversion of TEA to NDELA was detected at ph 2 or 10. In nutrient broth cultures (neutral ph), 0.08% and 0.68% of the TEA was nitrosated to NDELA in a diluted (high cecal inoculum) and full-strength (low cecal inoculum) media. (The percent nitrosation was determined using values that were corrected for DEA impurity-related NDELA formation.) 1

25 30%, with some reporting 100% TEA. 13 Non-Cosmetic USE Cosmetic TEA functions in cosmetics as a fragrance ingredient, surfactant emulsifying agent, and ph adjuster. 5 In 1981, according to data provided through the Food and Drug Administration (FDA) Voluntary Cosmetic Registration Program (VCRP), TEA was used in 2757 formulations; 2235 of those products were leave-on formulations, and 2419 involved dermal exposure. Products containing TEA were used at concentrations of 0.1% to >50%, with most formulations containing 5% TEA. 1 VCRP data obtained in 2010 report that TEA is used in 4015 formulations; 3208 of those products are leave-on formulations, and 3304 involve dermal exposure. 6 According to data submitted by industry in response to a survey conducted by the Personal Care Products Council (Council), TEA is used at concentrations of %. 7 In leave-on products, the reported use concentrations range from %; the 6% leave-on use is reported for face and neck creams, lotion, and powders. The complete current and historical use data for TEA are shown in Table 3. The number of leave-on and dermal contact uses have increased substantially since the original review, and the maximum concentration of use has decreased. Also, the number of non-coloring hair formulations and formulations involving mucous membrane exposure increased greatly since TEA is used in aerosolized products, and effects on the lungs that may be induced by aerosolized products containing this ingredient are of concern. The aerosol properties that determine deposition in the respiratory system are particle size and density. The parameter most closely associated with deposition is the aerodynamic diameter, d a, defined as the diameter of a sphere of unit density possessing the same terminal settling velocity as the particle in question. In humans, particles with an aerodynamic diameter of 10µm are inhalable. Particles with a d a from µm settle in the upper respiratory tract and particles with a d a < 0.1 µm settle in the lower respiratory tract. 8,9 Particle diameters of µm and 80 µm have been reported for anhydrous hair sprays and pump hairsprays, respectively. 10 In practice, aerosols should have at least 99% of their particle diameters in the µm range, and the mean particle diameter in a typical aerosol spray has been reported as ~38 µm. 11 Therefore, most aerosol particles are deposited in the nasopharyngeal region and are not inhalable. The melting point of TEA is very close to room temperature. Accordingly, depending on storage and application conditions, aerosolized TEA may be a liquid/vapor, instead of a particle. TEA is listed by the European Commission (EC) in Annex III Part 1: the list of substances which cosmetic products must not contain, except subject to the restrictions and conditions laid down. 12 Trialkylamine, trialkanolamines, and their salts, including TEA, are allowed at a maximum concentration of 2.5% in non rinse-off products. In non-rinse-off and other products, the following limitations apply for TEA: do not use with nitrosating systems; minimum allowable purity is 99%; maximum allowable secondary amine content is 0.5% in raw material; maximum allowable nitrosamine content is 50 µg/kg; and must be kept in nitrite-free containers. According to data obtained from Health Canada, some leave-on type products reportedly use TEA as high 10 and TEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes, mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate for resins, plasticizers, and rubber chemicals. It is used as a lubricant in the textile industry, as a humectant and softening agent for hides, as an alkalizing agent and surfactant in pharmaceuticals, as an absorbent for acid gases, and in organic syntheses. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 2

26 TEA has uses as an indirect food additive (21 CFR ). 14 TEA is also used as a rust inhibitor in water-based metalworking fluids. 15 TOXICOKINETICS Absorption, Distribution, Metabolism, Excretion Dermal [ 14 C]TEA (purity not specified), 2000 mg/kg, was applied to a 2.0 cm 2 area of skin on the back of mice, and the dosing area was not protected. 16 Peak radioactivity occurred in the blood 2 h after application, with a half-life of absorption and elimination of 10.2 min and 18.6 h, respectively. When compared to a dose of 1000 mg/kg [ 14 C]TEA in acetone, which was applied to a 1.0 cm 2 area, the concentrations of radioactivity in the blood were 5-fold greater with the 2000 mg/kg dose. (Additionally, dermal application of 1000 mg/kg in acetone resulted in approximately 1000-fold higher 14 C blood radioactivity levels than found in mice dosed intravenously (i.v.) with 1.0 mg/kg.) Another group of mice was also dosed dermally with 2000 mg/kg [ 14 C]TEA in water; 200 µl, ph 7.0, was applied using a glass ring to protect the dosing site. The blood concentrations of radioactivity of mice dosed with aq. TEA were then compared to those dosed with neat TEA, and similar results were found. The blood kinetics and absorption, distribution, metabolism, and excretion (ADME) of [ 14 C]TEA were determined following dermal application of 2000 mg/kg neat [ 14 C]TEA without occlusion to male C3H/HeJ mice. 17 (Non-radiolabeled TEA was 99.6% pure; radiochemical purity was 98.6%.) TEA was extensively and rapidly absorbed following a single open application of 2000 ml/kg neat [ 14 C]TEA. The majority of the radioactivity, 49-62% of the total dose (~58-72% of the absorbed dose), was excreted in the urine, primarily as unmetabolized TEA. DEA and MEA were not detected in the urine. Approximately 18-28% of the total dose (~20-32% of the absorbed dose) was excreted in the feces. The amount of radioactivity remaining in the body after 48 h ranged from %, and the amount recovered at the application site ranged from % for the open applications and 6-11% for the occluded applications. A study was performed to compare the toxicokinetics of the dermal absorption of [ 14 C]TEA in C3H/HeJ mice to F344 rats. 18 At 48 h after dermal application of 1000 mg/kg TEA to mice, approximately 60% of the radioactivity was recovered in the urine and 20% in the feces. Less than 10% of the radioactivity was recovered at the application site. In the urine, 95% of the recovered radioactivity was as unchanged TEA. [ 14 C]TEA was absorbed more slowly and less extensively in rats. (Additional details, including purity and detailed results for rats, were not provided.) The National Toxicology Program (NTP) examined the ADME of TEA following dermal administration to B6C3F 1 mice and F344 rats. 19 With mice, groups of 4 females were given a single dose of 79 or 1120 mg/kg [ 14 C]TEA in acetone; the dose contained µci, with the appropriate amount of non-labeled TEA in a volume of 190 µl/dose. (Radiochemical purity of [ 14 C]TEA was 97%; the purity of non-labeled TEA was confirmed, but the purity was not stated.) The dose was applied to a 1.44 cm 2 area of clipped skin, and a non-occlusive cover was used. Approximately 60-80% of the dose was abssorbed, and absorption increased with increasing dose. In the urine % and 48-56% of the dose was recovered after 24 and 72 h, respectively, and TEA was excreted mostly unchanged. Approximately 5-9 and 8-13% of the dose was recovered in the feces at the same time periods. With rats, groups of 4 females were given a single dermal dose of 68 or 276 mg/kg [ 14 C]TEA in acetone; the dose contained 65 µci, with the appropriate amount of non-labeled TEA in a volume of 190 µl/dose. The dose was applied to a 12 cm 2 area of clipped skin, and a non-occlusive cover was used. Only 19-28% of the dose was absorbed over 72 h; absorption increased with increasing dose, but not significantly. In the urine, 13-24% of the dose was recovered in 72 h as mostly un- 3

27 changed TEA. The amount recovered in the feces after 72 h was <0.25%. Very little radioactivity, <1%, was present in the tissues; a number of tissues had elevated concentrations of radiolabel relative to blood. Oral TEA (purity not specified) was administered orally as a single dose or as a repeated dose for 5-6 days. 20 (Dosing details were not described.) At 24 h after administration of the single dose, the excretion ratio of unchanged dose in the urine and feces was 53 and 20% of the dose, respectively. With repeated administration, the excretion ratio per day remained constant. Gender did not affect the ratios. TEA glucuronide was detected, but in a very small amount. (Actual concentration not specified.) TEA was rapidly absorbed in the gastrointestinal tract, and excreted mostly in the urine in unchanged form. Other A group of 27 male C3H/HeJ mice was given an i.v. injection of 1 mg/kg [ 14 C]TEA as an aq. solution (0.5 mg/ml), and the dose volume was 2 ml/kg. 17 (Non-radiolabeled TEA was 99.6% pure; radiochemical purity was 98.6%.) Radioactivity in the blood declined in a biphasic, exponential manner for 24 h, with a relatively rapid initial phase of [ 14 C] elimination, followed by a slower terminal phase. The majority of the radioactivity, approximately 69%, was excreted in the urine, primarily as unmetabolized TEA. TEA and MEA were not detected in the urine. Some of the radioactivity, ~17%, was excreted in the feces. The average amount of radioactivity recovered in the tissues was 3.1%. The NTP examined the ADME of TEA following i.v. administration to B6C3F 1 mice and F344 rats. 19 Groups of 4 female mice and 4 female rats were given a single i.v. dose of 3 mg/kg [ 14 C]TEA in isotonic saline. For mice, the dose contained 6 µci, with the appropriate amount of non-labeled TEA, for a dosing volume of 2 ml/kg. (Radiochemical purity of [ 14 C]TEA was 97%; the purity of non-labeled TEA was confirmed, but the purity was not stated.) At 24 h, 26 and 14% of the dose was excreted in the urine and feces, respectively, while at 72 h, these values were 62 and 28%, respectively. TEA was excreted mostly unchanged. Less than 0.5%, was detected in expired carbon dioxide. A number of tissues contained higher concentrations of TEA equivalents relative to blood. For rats, the dose contained 47 µci, with the appropriate amount of non-labeled TEA, for a dosing volume of 1 ml/kg. Much more of the radioactivity was excreted in the urine for rats compared to mice, and excretion was more rapid. Approximately 90% of the dose was recovered in the urine in 24 h, and 98% in 72 h, mostly as unchanged TEA. Like mice, <0.5%, was detected in expired carbon dioxide. Only 0.9% of the radioactivity was detected in the tissues after 72 h. TOXICOLOGICAL STUDIES Acute Toxicity Dermal The acute dermal toxicity of TEA was examined using groups of 6 rabbits. Undiluted TEA, 91.8 and 88.1% active, was applied to the intact and abraded skin of 3 rabbits under a 24 h occlusive patch. The exposure to actual TEA was 2 g/kg. None of the animals died. Mild erythema and edema were reported at 24 h. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Oral The acute oral toxicity of TEA was determined using guinea pigs and rats. In guinea pigs, undiluted TEA has an LD 50 of 8 g/kg, and the LD 50 of TEA in a gum arabic solution was between 1.4 and 7.0 g/kg. Using rats, the oral LD 50 of undiluted TEA ranged from 4.19 g/kg g/kg. The purity ranged from 78.6% TEA (with 8.6% DEA and 1.7% MEA) to unspecified high purity. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 4

28 Other The i.p. LD 50 of TEA was 1.45 g/kg for mice. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Repeated Dose Toxicity Dermal A closed patch continuous exposure test was performed using 10 guinea pigs in which commercial and high purity TEA, 8 g/kg, was applied daily 5 days/wk. All guinea pigs died by the 17 th application; adrenal, pulmonary, hepatic, and renal damage were observed. In a 13-wk study, 1 mg/kg of a hair dye formulation containing % or 1.5% TEA was applied to the backs of 12 rabbits for 1 h, twice weekly. The test site skin was abraded for half of the animals. No systemic toxicity was observed, and there was no histomorphologic evidence of toxicity. In a 6-mos study in which TEA was applied caudally to rats for 1 h/day, 5 days/wk, no toxic effects were observed with a 6.5% solution. However, using a 13% solution, changes (not specified) were seen in liver and central nervous system function. The addition of 1.4 mg/l TEA to the drinking water of the rats dosed dermally with 13% TEA did not increase the toxic effects. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 In a 2-wk study, undiluted TEA (purity not specified) was applied dermally to B6C3F 1 mice at doses of g/kg and to F344 rats at doses of g/kg, 5 days/wk. 21 Chronic active necrotizing inflammation of the skin at the application site occurred at a greater frequency and severity in rats than in mice. No renal or hepatic lesions were detected with either species. In a 2 wk and a 13-wk dermal study with up to 100% TEA (99.3% pure) in acetone using male and female C3H/HeJ mice, no toxicity or irritation was observed. 22 The only treatment-related effect was a mild thickening of the epidermis. In a 13-wk NTP dermal study using male and female B6C3F 1 mice, application of mg/kg bw TEA in acetone or 4000 mg/kg neat resulted in decreased mean body weights and body weight gains for some male mice. 23 (Purity of TEA was 99%. Functional group titration indicated <0.4% MEA or DEA.) Clinically, irritation was observed for the highest dose group, while microscopically, inflammation was observed for this dose group and acanthosis was noted for all dose groups, with severity increasing with dose. Absolute kidney and liver weights of males and females of the 4000 mg/kg group and relative kidney to body weights of males dosed with 1000 mg/kg were increased compared to controls. Absolute and relative spleen weights were also significantly increased in high dose females compared to controls. In a 13-wk dermal study using male and female F344/N rats, application of mg/kg bw TEA (99% pure) in acetone or 2000 mg/kg neat, resulted in significant decreases of mean body weights and body weight gains in the high dose animals. 23 Functional group titration indicated <0.4% MEA or DEA present.) Clinically, irritation was observed at the application site, and microscopic lesions included acanthosis and inflammation. Kidney weights of males and females dosed with 500 mg/kg were increased compared to controls, and dosed females, but not males, had greater incidences of nephropathy, as compared to controls. Oral Oral studies were conducted in which groups of 8-20 rats were dosed with g/kg/day TEA for 60 days to 6 mos, and groups of 8 guinea pigs were dosed with g/kg/day TEA for 60 or 120 doses. Repeated oral ingestion of TEA produced evidence of hepatic and renal damage. Some deaths occurred in groups of rats fed 0.17 g/kg/day TEA. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Male and female B6C3F 1 mice and F344 rats were given drinking water containing 0-8% TEA (purity not specified) for 14 days. 24,25 Male and female dose high dose mice, and male and female rats given 4% TEA, had decreased body 5

29 weights. All but one of the high dose rats were euthanized early due to severe dehydration. No treatment-related changes were observed in mice given 4% TEA or rats given 2% TEA in the drinking water. Inhalation In a 5-day dose-range finding inhalation study, 5 male and 5 female Wistar rats were exposed, nose only, to target concentrations of mg/m 3 TEA (98.9% pure) for 6 h. 26 Concentration-dependent laryngeal inflammation and edema were observed at microscopic examination, and the no observed adverse effect concentration (NOAEC) was 100 mg/m 3. The full, 28 day/20 exposure study used target concentrations of 0, 20, 100, and 500 mg/m 3, and the mass median aerodynamic diameter (MMAD) was µm. A functional observational battery was conducted using 7 rats/sex/group. Minimal to moderate focal inflammation in the submucosa of the larynx was observed; effects were concentration-dependent. No systemic toxicity was observed, and there were no effects on organ weights. There were no indications of neurotoxicological effects. Based on the results of this study, the 90-day NOAEC for local irritation was calculated to be 4.7 mg/m 3. In a 14-day inhalation study, B6C3F 1 mice and F344 rats were exposed to mg/m 3 TEA (purity not specified) 6h/day, 5 days/wk, for 2 wks. 27,28 Female mice and male and female rats of the high dose group had decreased body weights, and male mice of the high dose group had increased kidney weights. Increased kidney weights in rats dosed with 500 mg/m 3, and decreased thymus and heart weights in mice at all doses, were not clearly associated with TEA. The only histopathologic observation was a minimal acute inflammation of the laryngeal submucosa in both mice and rats; however, this occurred sporadically and there was no dose-response associated with this lesion. In a 28-day inhalation study, groups of 10 male and 10 female Wistar rats were exposed to 0.02, 0.1, and 0.5 mg/l TEA (purity not specified) as an aerosol, 6 h/day, 5 days/wk, according to the Organisation for Economic Co-operation and Development (OECD) guideline Exposure was head and nose, and he particle size was µm. The only clinically significant observation was a slight irritation of the upper respiratory tract in the high dose group. The no-observed effect level (NOEL) for systemic toxicity was >0.5 mg/l, and the NOEL for upper respiratory tract irritation was 0.02 mg/l. REPRODUCTIVE AND DEVELOPMENTAL STUDIES Dermal Hair dyes containing % or 1.5% TEA were applied topically to the shaved skin of groups of 20 gravid rats on days, 1, 4, 7, 10, 13, 16, and 19 of gestation, and the rats were killed on day 20 of gestation. No developmental or reproductive effects were observed. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 TEA, 0.5 g/kg in acetone (purity not stated), was applied dermally to clipped skin on the back of male and female F344 rats. 30 A volume of 1.8 ml/kg was applied daily for 10 wks prior to mating, during mating, and through gestation and lactation. No effect on mating or fertility or offspring growth or survival was observed. A similar study was performed in which Swiss CD-1 mice were given daily applications of 2 g/kg TEA at a volume of 3.6 ml/kg. 31 No adverse reproductive effects were observed. Oral A Chernoff-Kavlock teratogenicity screening test was performed using mated female CD-1 mice, in which the animals were dosed orally by gavage with 1125 mg/kg TEA on days 6-15 of gestation. 32 (It was stated that the TEA was the purest grade commercially available.) No adverse reproductive effects were observed. 6

30 GENOTOXICITY In Vitro Undiluted TEA, at concentrations of 100 mg/plate, was not mutagenic in Salmonella typhimurium with or without metabolic activation. TEA with sodium nitrite, but not TEA alone, was mutagenic in Bacillus subtilis without metabolic activation. NDELA, which is not mutagenic in B. subtilis without metabolic activation, was found in the mixture. In an unscheduled DNA synthesis test in which primary rat hepatocyte cultures were exposed to 10-8 to 10-1 M TEA and [ 3 H]thymidine, simultaneously, TEA did not appear to cause DNA-damage-inducible repair. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 TEA, in distilled water or dimethylsulfoxide, was not mutagenic to Escherichia coli 33 or S. typhimurium, with or without metabolic activation, at doses of 0-20,000 µg/plate TEA (88.2% purity) did not cause gene conversion in Saccharomyces cerevisiae. 34 TEA was negative in a rec assay at doses of µg/disk. No induction of sister chromatid exchanges occurred in Chinese hamster ovary (CHO) cells at µg/ml without metabolic activation and 0-10,100 µg/ml with metabolic activation, 36 and chromosomal aberrations were not induced in cultured rat liver cells 34 or at doses of µg/ml in cultured Chinese hamster cells. 33,36 TEA, µg/ml, was negative in a cell transformation assay using hamster embryo cells. 33 In Vivo A mouse peripheral blood micronucleus test was performed using samples collected from mice that were dosed dermally for 90-days with 0-4 g/kg TEA in an NTP study. 37 Results were negative in both male and female mice. CARCINOGENICITY Dermal In a series of 3 experiments using a total of 560 CBA x C 57 Bl 6 male mice, the carcinogenic effects of 99%+ pure TEA and 80%+ industrial grade TEA and the cocarcinogenic effect of TEA and syntanol DC-10 (not defined) were examined over a mo timeframe. TEA was not carcinogenic or cocarcinogenic.. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 The first carcinogenicity study of TEA using B6C3F 1 mice performed by the NTP was deemed inadequate due to a Helicobacter hepaticus infection. 23 Therefore, a second 2-yr study was performed in which TEA in acetone was applied dermally at doses of mg/kg to male B6C3F 1 mice and at doses of mg/kg to female B6C3F 1 mice. 38 (Purity of TEA was 99+%. Using high-performance liquid chromatography/mass spectrometry, 0.491% DEA was detected as an impurity. A slight increase in DEA was seen in acetone and ethanol solutions after 11 days of storage; the dose formulations were prepared approximately every 2 wks.) The body weights of high dose males were decreased compared to controls during wks and at the end of the study. Dermal irritation increased with increasing dose, and was more severe in males than in females. At necropsy, treatment-related epidermal hyperplasia, suppurative inflammation, and ulceration and dermal chronic inflammation occurred at the application site in most test groups, and the incidence and severity increased with increasing dose. Lesions were found, and it was concluded that there was equivocal evidence of carcinogenic activity of TEA in male mice, based on the occurrence of liver hemangiosarcoma, and some evidence of carcinogenic activity in female mice, based on increased incidences of hepatocellular adenoma. In a 2-yr NTP dermal carcinogenicity study using F344/N rats, TEA in acetone was applied at doses of mg/kg in acetone to males and at doses of mg/kg to females. 23 (Purity of TEA was 99%. Functional group titration indicated <0.4% MEA or DEA.) Irritation was observed at the application site, and frequency increased with increasing dose. At the interim necropsy, the absolute and relative kidney weights of high dose females were significantly greater than the controls. Microscopically, at the site of application, dermal lesions, including acanthosis, inflammation, and/or ulcera- 7

31 tion, were observed. It was concluded that there was equivocal evidence of carcinogenic activity in male rats, based on a marginal increase in the incidences of renal tubule cell adenoma, and there was no evidence of carcinogenic activity in female rats. The carcinogenic potential of TEA was evaluated using a Tg AC transgenic mouse model. 39 Groups of female homozygous mice were dosed dermally with 3-30 mg TEA/mouse in acetone, 5x/wk for 20 wks. TEA was inactive in Tg AC mice. Oral Groups of 40 male and 40 female ICR-JCL mice were fed a diet containing 0.01, 0.03, or 0.3% TEA throughout their lifetime. The malignant tumor incidence was 2.8, 27, and 36% for females, respectively, and 2.9, 9.1, and 3.6% for males, respectively. Treated females had a much higher incidence of thymic and non-thymic tumors in lymphoid tissues than treated males. Survival was similar for treated and control animals. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 The oral carcinogenic potential of TEA was examined by administering 1 and 2% TEA in drinking water to male and female B6C3F 1 mice for 82 wks. 40 (DEA was present as an impurity at 1.9 %.) Body weights of male mice of the 2% group were decreased during wks 1-20 when compared to controls. No significant changes in organ weights were observed. No dose-related increase of the incidence of any tumors was observed in the treated groups, and there was no evidence of carcinogenic potential of TEA upon oral administration. Drinking water containing 1 or 2% TEA was given to male and female F344 rats for 2 yrs. 41 (DEA was present as an impurity at 1.9%.) From wk 69 on, the dose concentrations for females were reduced by half because of associated nephrotoxicity. A dose-related decrease in body weight gain was reported for male and female test group rats, and a dose-dependent increase in mortality, starting at wk 60, was observed. Absolute and relative kidney-to-body weights were significantly increased in males and females, and the increase was dose-related. Severe chronic nephropathy was statistically significantly increased in males of the high dose group and females of both dose groups. No treatment-related effects were found in the liver. There was no increase in the incidence of any tumors in the treated groups compared to controls when using the Chisquare test. Since increased nephrotoxicity appeared to affect the lifespan of the treated animals, especially the females, an age-adjusted statistical analysis was performed on the incidences of main tumors or tumor groups for males and females, and a positive trend was noted in the occurrence of hepatic tumors (neoplastic nodule/hepatocellular carcinoma) in males and of uterine endometrial sarcomas and renal-cell adenomas in females. The researchers stated that, because these tumors have been observed spontaneously in F344 rats, and since their incidences in the control group was lower than that of historical controls, the occurrence of the tumors may not be attributable to TEA. Instead, increased incidence of renal tumors in the high-dose group may have been associated with renal damage. The researchers concluded that TEA was toxic to the kidneys, especially in females, but it was not carcinogenic to F344 rats. Skin IRRITATION AND SENSITIZATION Irritation Non-Human The primary skin irritation potential of undiluted TEA was determined using rabbits. After 10 open applications of 0.1 ml to rabbit ears and 10 unoccluded applications to the intact skin of the abdomen, and 3 semi-occluded 24- h applications to abraded skin, it was concluded that TEA was slightly to moderately irritating, and prolonged or repeated exposure may be irritating. Twenty-four h occluded patch tests using groups of 8 male rabbits were performed in 22 laboratories; the primary irritation score ranged from 0-5.5/24, and the total score for all 22 laboratories was 27.3/400. In a preliminary study, occlusive dermal applications of % aq. TEA to pairs of 8

32 guinea pigs resulted in one erythematic reaction to undiluted TEA, and in another preliminary study, no irritation was observed when 5, 10, or 25% TEA was applied to the backs of guinea pigs. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 The irritancy potential of TEA (purity not specified) was evaluated in an ear swelling test using female BALB/c mice. 42 A significant increase in irritancy was observed with 25 and 50% TEA compared to the vehicle (4:1 acetone/olive oil) group. Human Clinical studies were performed with formulations containing TEA. In a few studies on formulations containing % TEA, the researchers concluded that no irritation was observed, while short-lived acute irritation was reported for formulations containing % TEA. However, according to the Expert Panel s interpretation of the results of a number of other studies, formulations containing % TEA were irritating. In clinical provocative testing using 5-10 hyper reactors, 100% TEA produced an irritant reaction on nonscarified skin, 10% TEA in ethanol was a marked irritant on scarified skin, while 5% in ethanol was a slight irritant on scarified skin. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 A patch test with TEA (purity not specified) was performed on 20 subjects, and erythema and transepidermal water loss (TEWL) were measured, and the contents of suction blister fluids (SBF) were evaluated for primary proinflammatory mediators. 43 Aq. TEA, 0-100%, was applied occlusively for 24 h; µl, concentrated to 20 µl with drying, were applied. The percent of non-responders to 100% TEA was 80%; those that did respond had weak and non-uniform erythema. The incidence was below or about that found with the solvent controls. For the challenge phase, 765 µmol/cm 2 TEA was applied occlusively to 12 subjects for 6-24 h. No increase in TEWL or change in eicosanoid profile of the SBF was observed. TEA was a non-irritant. Mucosal Non-Human The ocular irritation potential of ml undiluted TEA was evaluated in a number of studies using rabbits. With high concentrations and long contact time, TEA may be irritating to rabbit eyes. Using rabbits, 10% aq. TEA produced essentially no irritation with or without rinsing. A formulation containing 12.6% TEA, 0.1 ml, was evaluated in a study using 6 rhesus monkeys. Slit lamp examination revealed some corneal effects in 2 monkeys at 24 h and slight positive fluorescein staining in one monkey at 72 h. The vaginal irritation potential of a spermicidal formulation containing 1.92% TEA was evaluated by placing 0.5 ml of the ointment inside the vaginas of 6 rats for 3 days. The formulation was a non-irritant. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Sensitization In Vitro The sensitization potential of TEA was evaluated in a local lymph node assay (LLNA) performed with groups of 5 BALB/c mice. 42 This study was performed in conjunction with the ear-swelling test described previously. Lymphocyte proliferation increased with dose, but the increases were not statistically significant. TEA was not identified as a sensitizer in the LLNA. Non-Human TEA was not a sensitizer to guinea pigs when 20 guinea pigs were given dermal applications of undiluted TEA 1x/wk for 3 wks, followed by challenge applications 14 and 21 days after dosing. No sensitization was seen when four lots of TEA were evaluated using groups of 20 guinea pigs; induction applications were applied for up to 6 h, 1x/wk, for 3 wks, and the challenge was performed after 14 days. One of the studies used undiluted TEA during induction, while the other 3 studies used 50% TEA at induction. All four studies used challenge patches with 90% TEA. No sensitization was observed in a similar study in which induction patches contained a 25% active TEA solution, and a challenge patch with the 25% solution was applied after 1 wk of non-treatment. 9

33 From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 The hypersensitivity of mice to TEA (99+% pure) was determined. 44 TEA, in an acetone:olive oil mixture (4:1) at concentrations of 3%, 10%, or 30%, was applied daily for 5 consecutive days to groups of 8 female B6C3F 1 mice, and the animals were challenged 7 days later with a 30% solution. For some animals, dermabrasion, as well as intradermal injections of Freund s complete adjuvant (FCA), was used. There were no treatment-related effects on survival or body weights. There were no statistically significant or dose-related hypersensitivity responses to TEA observed with a radioisotopic method or in an ear swelling test, with or without FCA. Results were negative in three maximization studies examining the sensitization potential of TEA. 15 In the first test, performed using Pirbright-White guinea pigs, induction consisted of intradermal injections of 2% TEA (98.9% pure) in isotonic saline and epicutaneous application of undiluted TEA, and challenge used 10% TEA in isotonic saline. In the second test using 20 Dunkin-Hartley guinea pigs, intradermal and epicutaneous inductions used 1.5% technical grade TEA and 25% technical grade TEA with 10% sodium lauryl sulfate pre-treatment, respectively, and challenge doses consisted of 1, 5, and 10% technical and analytical grade TEA. The third study used 15 animals and the same induction protocol (grade of TEA not specified); 2/15 reacted to 10% TEA after 1, but not 3, days. Human In patch tests in which TEA was tested at 5%, 2% aq, or 5% in petrolatum, 9/479, 23/500, and 2/100 subjects had positive reactions for contact dermatitis. The Expert Panel interpreted these findings as sensitizing. In a patch test with 64 subjects in which 0.5 ml of 1% TEA (containing 88.6% TEA and 6% DEA) was used, the test solution was not sensitizing. The majority of formulations containing % TEA were not sensitizing, and a formulation containing 20.04% TEA, tested on 26 subjects, was not considered sensitizing when it produced 2 slight reactions upon challenge. However, according to the interpretation of the Expert Panel, there were a few cosmetic formulations containing 2.1 and 2.4% TEA that the Panel determined to be sensitizing. In other studies with cosmetic formulations containing 2.1% TEA, the researchers concluded that reactions observed at challenge were probably due to skin fatigue. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Provocative Testing A group of 737 patients was patch tested with 6 different emulsifiers, including 2.5% TEA (purity not specified) in petrolatum. 45 The patch tests were performed according to International Contact Dermatitis Research Group (ICDRG) recommendations. A total of 39 patients had positive reactions to the emulsifiers, and 20 of those patients, 5 males and 15 females, had positive reactions to TEA. There were 106 (0.1%) irritant reactions reported. The results were clinically relevant in 7 patients. Many of the patients allergic to TEA were also allergic to other ingredients. Over a 15-yr period, provocative patch testing using TEA was performed on 85,098 subjects. 15 There were 323 (0.3%) positive reactions to TEA, and most of the reactions (289; 0.3%) were weak positives. The researchers stated that occupational exposure was not a risk factor for TEA contact allergy. Phototoxicity/Photoallergenicity Non-Human A formulation containing 1% TEA was applied to the stripped skin of 6 guinea pigs, and each animal was then exposed to ultraviolet A (UVA) light for 2 h. No erythema or edema was observed. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Human There were no phototoxicity or photosensitization reactions with a number of formulations containing % TEA, nor were there any reactions with a formulation containing 20.04% TEA. However, in one study with a formulation containing 4.2% TEA, the Expert Panel felt that the formulation was either mildly phototoxic or there 10

34 was UV enhancement of an irritant response. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 CLINICAL USE Case Studies Eczema of the face of 2 female patients was exacerbated by a cream that contained TEA. 46 Patch testing was performed using the ICDRG standard series, a cosmetic battery, the TEA-containing cream, and TEA at 5%, 2%, and 1% in petrolatum (pet.). Both patients reacted to the TEA-containing cream (+ reaction) and to 5% TEA pet. (++ reaction). One patient reacted to 2% TEA (+ reaction), and neither reacted to 1% TEA pet. Patch tests were negative for all other compounds. In a control group of 50 subjects, patch testing with 5% TEA pet. was negative. Over a 4-yr period, the incidences of positive patch test reactions to the same TEA-containing cream were 69/171 patients in one clinic and 49/191 in another. 47 It was hypothesized that the difference between the clinics was due to differences in sampling methods; the first clinic tested only those patients that had recently used the TEA-containing cream or who had suspected reactions. In follow-up patch testing with a total of 54 subjects from the 2 clinics, 15 of which were controls, 19 subjects had a positive allergic response to the TEA-containing cream, 40 had a positive irritant response, and 13 had negative responses. With % TEA, 6 subjects had a positive response. However, with 5-20% TEA stearate, 8/8 patients and 15/15 controls had a positive irritant response. (TEA stearate was tested because it was demonstrated that TEA stearate was formed from the combination of TEA and stearic acid in formulation.) The researchers postulated the reactions were irritant reactions to TEA stearate. (The amount of TEA stearate present in formulation was estimated to be 4.8%, and 6/23 subjects patch tested with 5% TEA stearate had irritant reactions.) Two cases of occupational asthma in metal workers exposed to cutting fluid containing TEA were reported. 48 Exposure to TEA at temperatures higher than that of ambient air was a common feature. MISCELLANEOUS STUDIES N-Nitrosodiethanolamine Formation In vivo, female B6C3F1 mice were dosed dermally or orally with 1000 mg/kg TEA, in conjunction with oral exposure to sodium nitrite. 4 Following 7 days of dermal dosing, no NDELA was detected in the blood, ingesta, or urine of test, vehicle control, or sodium nitrite control mice. (The limits of detection for the blood, ingesta, and urine were 0.001, 0.006, and 0.47 µg/ml, respectively.) With a single oral dose, the concentrations of NDELA found in the blood and ingesta of mice 2 h post-dosing were ± µg/g and ± µg/g, respectively. Possible Mode of Action for Carcinogenic Effects It has been reported that choline deficiency induces liver cancer in rodents; therefore, the potential of TEA to cause choline deficiency in the liver of female B6C3F 1 mice was investigated as a mode of tumorigenesis. 49 Female mice were dosed dermally with unoccluded applications of mg/kg/day TEA in acetone, 5 days/wk for 3 wks, and female CDF rats were dosed in a similar manner with 250 mg/kg/day TEA. (Purity of TEA was 99+%; DEA impurity levels were 0.04 and 0.45%.) No clinical signs of toxicity were noted for mice or rats. Phosphocholine and betaine levels were statistically significantly decreased in the high dose mice, and choline levels were decreased in these mice. The decrease in phosphocholine levels was variable, but dose-related. (More pronounced effects were observed when the TEA having 0.45% DEA impurity was used.) In rats, no changes in choline or its metabolites were noted. The potential of TEA to inhibit the uptake of [ 3 H]choline by CHO cells was also investigated, and a dose-related decrease was observed. The researchers concluded that TEA may cause liver tumors in mice via a choline-depletion mode of action, and this effect is likely caused by the inhibi- 11

35 tion of choline uptake by the cells. The researchers stated that, while DEA impurity may contribute to choline depletion, a choline-deficiency mode of tumorigenesis appears to be a property of TEA, exclusive of any DEA impurity. RISK ASSESSMENT Exposure Assessment The dermal exposure of consumers to TEA was estimated assuming a presence of 2.5% TEA in cosmetic products (based on the limit set by the EC) and that all TEA is unreacted. 50 Using an EC algorithm method, the dermal exposure of consumers to an eye make-up powder is mg/kg bw/day and to a body lotion is 6.25 mg/kg bw/day. Using a DERMAL program method, the dermal potential dose rate for a bar soap containing 2.5% TEA is mg/day. According to an evaluation of TEA by the International Agency for Research on Cancer (IARC) Working Group, there is inadequate evidence in humans, as well as in animals, for the carcinogenicity of TEA. 51 The overall evaluation of the IARC is that TEA is not classifiable as to its carcinogenicity to humans (Group 3). 12

36 TABLES Table 1. Definition and Structure Ingredient Definition Synonyms Function(s) in Cosmetics Formula/Structure CAS No. Triethanolamine HO The product of the ammonolysis of three stoichiometric equivalents of ethylene oxide 2,2,2 -nitrilotris[ethanol] trolamine TEA fragrance ingredient surfactant-emulsifying agent ph adjuster HO N OH Table 2. Physical and Chemical Properties Property Value Reference Physical Form clear viscous liquid 1 Color colorless to pale yellow 19 Odor Ammonical 1 Molecular Weight Density/Specific Gravity (20 C) 1 Viscosity C 18 Vapor Pressure <0.01 mm 20 C 19 Vapor Density 5.14 mmhg 18 Melting Point 21.6 C 19 Boiling Point mm Hg 19 Water Solubility miscible in water 19 Other Solubility insoluble in benzene, ether, and petroleum distillates 1 miscible with methanol or acetone; sparingly soluble in hydrocarbon solvents; readily 18 forms salts with organic and inorganic acids log K ow 20 C 50 Disassociation Constant pka 25 C 26 Table 3. Current and historical frequency and concentration of use of TEA according to duration and type of exposure # of Uses Conc. of Use (%) data year ,52 Totals >50* Duration of Use Leave-On >50* Rinse Off Exposure Type Eye Area Possible Ingestion Inhalation Dermal Contact >50* Deodorant (underarm) Hair - Non-Coloring Hair-Coloring Nail Mucous Membrane Bath Products Baby Products * - included unreported concentration of use 13

37 REFERENCES 1. Elder RE (ed). Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. J Am Coll Toxicol. 1983;2:(7). 2. Dow Chemical Company. The alkanolamines handbook Midland, MI: The Dow Chemical Company.Secondary reference in Knaak et al Kraeling MEK, Yourick JJ, and Bronaugh RL. In vitro human skin penetration of diethanolamine. Food Chem Toxicol. 2004;42: Saghir SA, Brzak A, Markham DA, Bartels MJ, and Stott WT. Investigation of the formation of N-nitrosodiethanolamine in B6C3F1 mice following topical administration of triethanolamine. REg Toxicol Pharmacol. 2005;43: Gottschalck T.E. and Bailey, J. E. eds. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal Care Products Council, Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Washington, DC: FDA.Updated May Personal Care Products Council. Concentration of use data - submitted by industry in response to a Council survey Unpublished data submitted by the Council. 8. James AC, Stahlhofen W, Rudolf G, and et al. Annex D. Deposition of inhaled particles. Annals of the ICRP. 1994;24:(1-3): Oberdorster G, Oberdorster E, and Oberdorster J. An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 2005;113:(7): Bowers D. Unpublished information on hair spray particle sizes provided at the September 9, 1999 CIR Expert Panel meeting Johnson MA. The influence of particle size. Spray Technology and Marketing. 2004;Nov European Commission.European Commission Enterprise and Industry. Cosmetics - Cosing. Annex III/1,62 - Trialkylamines, trialkanolamines and their salts Accessed Heatlh Canada.Cosmetic Ingredient Hotlist Accessed Food and Drug Administration.Everything Added to Food in the United States (EAFUS) Accessed Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine. Data analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60: Waechter JM and Rick DL. Triethanolamine: Pharmacokinetics in C3H/HeJ mice and Fischer-344 rats following dermal administration Midland. MI: Dow Chemical Co.Secondary reference in Knaak et al Stott WT, Waechter JM, Rick DL, and Mendrala AL. Absorption, distribution, metabolism and excretion of intravenously and dermally administered triethanolamine in mice. Food Chem Toxicol. 2000;38: Triethanolamine Chapter: 7.6. Ethel Browning's Toxicity and Metabolism of Industrial Solvents. Buhler DR and Reed DJ. Amsterdam/New York/Oxford: Elsevier. 19. National Toxicology Program. NTP Technical report on the toxicology and carcinogenesis studies of triethanolamine (CAS No ) in B6C3F 1 mice. (Dermal study). NTP TR Kohri N, Matsuda T, Umeniwa K, Miyazaki K, and Arita T. Development of assay method in biological fluids and biological fate of triethanolamine. Yakuzaigaku. 1982;42:(4):

38 21. Melnick R and Hejtmancki M Mezza L Ryan M persing R Peters A. Comparative effects of triethanolamine (TEA) and diethanolamine (DEA) in short-term dermal studies. (Secondary reference in Melnick and Tomaszewski 1990). The Toxiologist. 1988;8: DePass LR, Fowler EH, and Leung H-W. Subchronic dermal toxicity study of triethanolamine in C3H/HeJ mice. Fd Chem Toxicol. 1995;33:(8): National Toxicology Program. Toxicology and carcinogenesis studies of triethanolamine (CAS No ) in F344/N rats and B6C3F 1 mice. (Dermal studies.) TR No Hejtmancik M, Mezza L, Peters AC, and Athey PM. The repeated dose dosed water study of triethanolamine (CAS No ) in Fischer-344 rats Columbus OH: Columbus Division Laboratories.Unpublished data summarized in Knaak et al Hejtmancik M, Mezza L, Peters A, and Athey PM. The repeated dose dosed water study of triethanolamine (CAS No ) in B6C3F 1 mice Unpublished data summarized in Knaak et al. 1997: Columbus Division Laboratories.Unpublished data summarized in Knaak et al Gamer AO, Rossbacher R, Kaufmann W, and van Ravenzwaay B. The inhalation toxicity of di- and triethanolamine upon repeated exposure. Food Chem Toxicol. 2008;46: Mosberg A, McNEill D, Hejtmancik M, Persing RL, and Peters A. The repeated dsoe inhalation study of triethanolamine (CAS No ) in Fischer-344 rats Columbus OH: Battelle Columbus Division Laboratories.Unpublished data summarized in Knaak et al Mosberg A, McNEill D, Hejtmancik M, Persing RL, and Peters A. The repeated dose inhalation study of triethanolamine (CAS No ) in B6C3F 1 mice Columbus OH: Battelle Columbus Division Laboratories.Unpublished data summarized in Knaak et al BASF AG. Twenty-eight day inhalation study of TEA using Wistar rats. Unpublished report, Project No. 40I0711/ OECD SIDS Initial Assessment Report for SIAM 3 (Triethanolamine) Secondary reference in OECD SIDS Initial Assessment Report for SIAM Battelle Columbus Laboratories. Mating trial dermal study of triethanolamine (CAS No ) in Fischer 344 rats. Final report (NIH Cotnract No. N01-ES-45068). Secondary reference in NTP Battelle Columbus Laboratories. Mating trial dermal study of triethanolamine (CAS No ) in Swiss CD-1 mice. Final report (NIH Cotnract No. N01-ES-45068). Secondary reference in NTP Secondary reference in NTP TR Environmental Protection Agency.High Production Volume (HPV) Challenge Program Revised Test Plan and Robust Summaries for Polyphosphoric Acid Esters of Triethanolamine, Sodium Salts Inoue K, Sunakawa T, Okamoto K, and Tanaka Y. Mutagenicity tests and in vitro transformation assays on triethanolamine. Mutat Res. 1982;101: Dean BJ, Brooks TM, Hodson-Walker G, and Hutson DH. Genetic toxicology testing of 41 industrial chemicals. Mutat Res. 1985;153: Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, and Zeiger E. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environ Mutagen. 1986;8:(Suppl 7): Galloway SM, Armstrong MJ, Reuben, Colman, Brown B, Cannon C, Bloom AD, Nakamura F, Ahmed M, Duk S, Rimpo J, Margolin BH, Resnick MA, Anderson B, and Zeiger E. Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environ Mol Mutagen. 1987;10:(Suppl 10): Witt KL, Knapton A, Wehr CM, Hook GJ, Mirsalis J, Shelby MD, and MacGregor JT. Micronucleated erythrocyte frequency in peripheral blood of B6C3F 1 mice from short-term. prechronic, and chronic studies of the NTP carcinogenesis bioassay program. Environ Mol Mutagen. 2000;36: National Toxicology Program. Toxicology and carcinogenesis studies of triethanolamine (CAS NO ) in B6C3F 1 mice. (Dermal study.) NTP TR

39 39. Tennant RW, French JE, and Spalding JW. Identifying chemical carcinogens and assessing potential risk in short-term bioassays using transgenic mouse models. Environ Health Perspect. 1995;103: Konishi, Y., Denda, A., Uchida, K., Emi, Y., Ura, H., Yokose, Y., Shiraiwa, K., and Tsutsumi, M. Chronic toxicity carcinogenicity studies of triethanolamine in B6C3F1, mice. Fundam.Appl Toxicol. 1992;18:(1): Maekawa, A., Onodera, H., Tanigawa, H., Furuta, K., Kanno, J., Matsuoka, C., Ogiu, T., and Hayashi, Y. Lack of carcinogenicity of triethanolamine in F344 rats. J Toxicol Environ Health. 1986;19:(3): Anderson SE, Brown KK, Butterworth LF, Fedorowicz A, Jackson LG, Frasch HF, Beezhold D, Munson AE, and Meade BJ. Evaluation of irritancy and sensitization potential of metalworking fluid mixtures and components. J Immunotoxicol. 2009;6:(1): Müller-Decker K, Heinzelmann T, Fürstenberger G, Kecskes A, Lehmann W-D, and Marks F. Arachidonic acid metabolism in primary irritant dermatitis produced by patch testing of human skin with surfactants. Toxicol Appl Pharmacol. 1998;153: National Toxicology Program.Abstract for IMM The immunotoxicity of triethanolamine (CAS No ) Accessed Tosti A, Morelli R, and Bardazzi F. Prevalence and sources of sensitization to emulsifiers: a clinical study. Contact Derm. 1990;23: Jones SK and Kennedy TC. Contact dermatitis from triethanolamine in E45 cream. Contact Derm. 1988;19:(3): Batten TL, Wakeel RA, Douglas WS, Evans C, White MI, Moody R, and Ormerod AD. Contact dermatitis from the old formula E45 cream. Contact Derm. 1994;30: Savonius B, Keskinen H, Tupperainen M, and Kanerva L. Occupational asthma caused by ethanolamines. Allergy. 1994;49: Stott WT, Radtke BJ, LinscombeVA, Mar MH, and Zeisel SH. Evaluation of the potential of triethanolamine to alter hepatic choline levels in female B6C3F1 mice. Toxicol Sci. 2004;79:(2): Organisation for Economic Co-operation and Development. SIDS Initial Assessment Report for SIAM 3 (Triethanolamine) International Agency for Research on Cancer (IARC). Triethanolamine. IARC Monogr Eval.Carcinog Risks Hum. 2000;77: Personal Care Products Council. Updated concentration of use of triethanolamine Unpublished data submitted by the Council (3 pp). 16

40 Re-Review of Diethanolamine Introduction... 1 Chemistry... 1 Use... 1 Cosmetic... 1 Non-Cosmetic... 2 Toxicokinetics... 2 Absorption, Distribution, Metabolism and Excretion... 2 In-Vitro... 2 Dermal... 3 Non-Human... 3 Human... 4 Oral... 4 Non-Human... 4 Other... 5 Non-Human... 5 Percutaneous Absorption... 6 Non-Human... 6 Human... 6 Toxicological Studies... 7 Acute (Single Dose) Toxicity... 7 Oral... 7 Other... 7 Repeated Dose Toxicity... 7 Dermal... 7 Oral... 8 Inhalation... 9 Reproductive and developmental studies Dermal Oral Inhalation Genotoxicity In Vitro Carcinogenicity Dermal Irritation and Sensitization Irritation Skin Mucosal Sensitization Provocative Testing Miscellaneous Studies N-Nitrosodiethanolamine Formation Immunotoxicity Inhibition of Choline Uptake Species Selective Effect on DNA Synthesis Effect on Hippocampal Neurogenesis and Apoptosis Effect on Cell Proliferation and Apoptosis in the Liver Possible Mode of Action for Carcinogenic Effects Risk Assessment Tables Table 1. Definition and Structure Table 2. Physical and Chemical Properties Table 3. Current and historical frequency and concentration of use according to duration and type of exposure Table 4. Percutaneous penetration study details (Study 1) Table 5. Percutaneous penetration study details (Study 2) References... 20

41 INTRODUCTION Diethanolamine (DEA), an ingredient that functions in cosmetics as a ph adjuster, has previously been reviewed by the CIR Expert Panel. 1 In 1983, the Expert Panel concluded that DEA is safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of DEA should not exceed 5%. DEA should not be used with products containing N- nitrosating agents. In that review, it was shown that DEA was a mild skin and eye irritant, and that irritation increased with increasing ingredient concentration. Since that time, DEA has been discussed at a number of Panel meetings. It was first discussed at the June 1999 meeting, and then at the meetings in June 2008 and The Panel has also heard industry presentations on DEA. However, no formal action was taken regarding a re-review of DEA. A substantial number of published documents have become available since the 1983 report was issued. The Panel should now formally decide if the new information, which included a carcinogenicity study performed by the National Toxicology Program (NTP) warrants a re-review DEA, or does it confirm the original conclusion. To help with your decision, this document contains summaries of data obtained from a search of published literature, as well as summary information from the original safety assessment. CHEMISTRY DEA is an amino alcohol. DEA is produced commercially by aminating ethylene oxide with ammonia. The replacement of two hydrogens of ammonia with ethanol groups produces DEA. DEA contains small amounts of triethanolamine (TEA) and ethanolamine (MEA). DEA is reactive and bifunctional, combining the properties of alcohols and amines. At temperatures of C, DEA will react with fatty acids to form ethanolamides. The reaction of ethanolamines and sulfuric acid produces sulfates and, under anhydrous conditions, DEA may react with carbon dioxide to form carbamates. DEA can act as an antioxidant in the autoxidation of fats of both animal and vegetable origin. DEA can react with nitrite or oxides of nitrogen to form N-nitrosodiethanolamine (NDELA). The optimum ph for nitrosamine formation is between 1 and 6; however the rate of NDELA formation in the ph range of 4-9 is four to six times greater in the presence of formaldehyde. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 The structure and synonyms of DEA are given in Table 1, and chemical and physical properties are given in Table 2. DEA is produced by reacting 2 moles of ethylene oxide with 1 mole of ammonia. 2 Typically, ethylene oxide is reacted with ammonia in a batch process to produce a crude mixture of approximately one-third each MEA, DEA, and TEA. The crude is later separated by distillation. Dow Chemical Company reports that DEA is commercially available with a minimum purity of 99.3%, containing 0.45% max. DEA and 0.25% max TEA. 3 DEA is structurally similar to choline and ethanolamine. 4 USE Cosmetic DEA functions in cosmetics as a ph adjuster. 5 In 1981, according to data provided through the Food and Drug Administration (FDA) Voluntary Cosmetic Registration Program (VCRP), DEA was used in 18 formulations, and all but one of those products were rinse-off formulations. 1 Twelve uses were in hair coloring products. Products containing DEA were used at concentrations of 5%. VCRP data obtained recently indicate that DEA is used in 30 formulations; 15 are leave-on formulations and 15 are rinse-off, and 13 uses are in non-coloring hair formulations. 6 According to data submitted by 1

42 industry in response to a survey conducted by the Personal Care Products Council (Council), DEA is used at concentrations of %. 7 The highest concentration used in leave-on products is 0.06% in moisturizing products. The complete current and historical use data for DEA are shown in Table 3. Reportedly, DEA per se is rarely if ever used in personal care products. 8 The potential for exposure to DEA exists from the use of alkanolamides of DEA (which are condensation products of DEA and fatty acids). DEA is present in aerosolized products, and potential effects on the lungs of aerosolized products containing this ingredient are of concern. The aerosol properties that determine deposition in the respiratory system are particle size and density. The parameter most closely associated with deposition is the aerodynamic diameter, d a, defined as the diameter of a sphere of unit density possessing the same terminal settling velocity as the particle in question. In humans, particles with an aerodynamic diameter of 10µm are inhalable. Particles with a d a from µm settle in the upper respiratory tract and particles with a d a < 0.1 µm settle in the lower respiratory tract. 9,10 Particle diameters of µm and 80 µm have been reported for anhydrous hair sprays and pump hairsprays, respectively. 11 In practice, aerosols should have at least 99% of their particle diameters in the µm range and the mean particle diameter in a typical aerosol spray has been reported as ~38 µm. 12 Therefore, most aerosol particles are deposited in the nasopharyngeal region and are not inhalable. According to the opinion of the Scientific Committee on Consumer Safety of the European Commission, dialkyland dialkanolamines and their salts are on the list of substances which must not form part of the composition of cosmetic products. 13 According to the Commission s opinion paper, all amines occur only in the form of their salts in all cosmetic products. This is because all amines are alkaline compounds which are always neutralized by an acid component to produce their salts. There is concern about the potential for nitrosamine formation; in principle, secondary amines are potential precursors of nitrosamines. In Canada, DEA is completely prohibited as per the Cosmetic Hotlist; Health Canada prohibits the use of dialkanolamines, including DEA. 14 This prohibition is based on the EU prohibition in the Cosmetics Regulation, Annex II. The use of DEA in product formulations in Canada is being investigated because of reported use at concentrations of 3%. (Health Canada, personal communication). Non-Cosmetic DEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes, mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate for resins, plasticizers, and rubber chemicals. DEA is used as lubricants in the textile industry, as humectants and softening agents for hides, as alkalizing agents and surfactants in pharmaceuticals, as absorbents for acid gases, and in organic syntheses. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 DEA has uses as an indirect food additive (21 CFR ). 15 TOXICOKINETICS Absorption, Distribution, Metabolism and Excretion In-Vitro Three full thickness skin preparations from CD rats, CD-1 mice, New Zealand White (NZW) rabbits, and 6 from female mammoplasty patients were used to compare the dermal penetration of DEA through the skin of different species. 16 [ 14 C]DEA (96.5% purity; sp. act mci/mmol) was applied to the skin sample undiluted, or as an aq. solution, at a dose of 20 mg/cm 2. Dose volumes of 35 µl undiluted [ 14 C]MEA or 95 µl of the aq. solution (37% w/w) were applied to the exposed surface of the skin (1.77 cm 2 ). These volumes maintained an infinite dose during the 6 h exposure period. Skin sample 2

43 integrity was confirmed with the use of a reference chemical, [ 14 C]ethanol. With undiluted DEA, the cumulative dose absorbed was 0.04% in rat skin, 1.30% in mouse skin, 0.02% in rabbit skin, and 0.08% in human skin. With aq. DEA, an increase in the cumulative dose absorbed was seen in all species: 0.56% in rat skin, 6.68% in mouse skin, 2.81% in rabbit skin, and 0.23% in human skin. Penetration of undiluted and aq. DEA was greater through mouse skin than any other skin sample. With undiluted DEA, penetration was similar for rat, rabbit, and human skin samples. Penetration of DEA was greater for an aqueous solution than for undiluted DEA. The researchers hypothesized that this may be attributable to elevated skin hydration caused by the application of the aqueous solution to the skin. Human liver slices were incubated with 1 mm [ 14 C]DEA (>97% purity). 17 After 4 and 12 h, 11 and 29% of the DEA, respectively, absorbed into the liver slices. The radioactivity was comprised mostly of DEA (85-97%); four other metabolites were present at low concentrations. The liver slices were fractionated into aqueous, organic, and pellet fractions. The aqueous-extractable radioactivity was comprised primarily of DEA, with up to four other components. DEA-derived radioactivity in the organic extracts was predominately (>90%) comprised of phospholipids containing non-methylated headgroups. DEA was readily absorbed and incorporated into ceramides, forming mostly ceramide-phosphdea, and it was slowly methylated therein. Dermal Non-Human A single dose of 81.1 mg/kg DEA in 15 µl ethanol was applied to a 1 cm 2 area of the intrascapular region of male B6C3F 1 mice. 18 After 48 h, 58.1% of the dose was recovered in tissues or excreta; 28.3% of the dose was excreted in the urine, 4.4% in the feces, and 2.2% was present in the skin at the site of application. A single occluded dose of [ 14 C]DEA was applied to a 19.5 cm 2 area on the back of rats for 6 h, and the treated site of 50% of the animals was rinsed. 19 In the animals that were not rinsed, 80% of the dose was found in the wrappings, and 3.6% found in the skin. In rinsed animals, rinsates from the wrappings contained 58% of the dose, and from the skin contained 26% of the dose. Unrinsed animals absorbed 1.4% of the dose, while those that were rinsed absorbed 0.64%. The majority of the radioactivity was found in the carcass, liver, and kidneys. The researchers also conducted a repeated-dose percutaneous-absorption study in which as a pre-exposure, 1500 mg/kg/day non-radiolabeled DEA was applied occlusively under a 2 in x 2 in gauze square to the backs of rats 6 h/day for 3 or 6 days. [ 14 C]DEA, 1500 mg/kg/day, was then applied for 3-6 consecutive days under occlusion; each dose was left in contact with the skin for 48 h. Totals of 21 and 41% of the dose were absorbed by the animals dosed for 3 and 6 days, respectively. The majority of the recovered radioactivity was in the wrapping. The carcass, liver, and kidneys contained most of the radioactivity in the animals. Totals of 4.3 and 13% of the radioactivity were recovered in the urine of animals dosed with non-radiolabeled DEA for 3 and 6 days, respectively. Pre-exposure to DEA resulted in a 1.5- to 3.0-fold increase in the absorption rate. Groups of 4-5 male Fischer 344 rats were used to evaluate the absorption, distribution, metabolism, and excretion (ADME) of DEA (99% purity) following dermal administration. 20 The dose, which contained 6-20 µci radiolabel, unlabeled DEA, and ethanol, for a total dose volume of 25 µl per dose, was applied to a 2 cm 2 area of skin under a non-occlusive covering. Absorption increased significantly with increasing dose. After 48 h, only 2.9% of the radioactivity was absorbed following dermal application of 2.1 mg/kg, while 10.5 and 16.2% of the radioactivity was absorbed with doses of 7.6 and 27.5 mg/kg, respectively. Of the amount absorbed, 1.2, 4.3, and 4.5% of the radioactivity was recovered at the dose site 3

44 following application of 2.1, 7.6, and 27.5 mg/kg [ 14 C]DEA, respectively. The greatest tissue/blood ratios were found in the liver and lung. Groups of 4-5 male B6C3F 1 mice were used in dermal studies of the ADME of DEA. The total volume for mice was 15 µl/dose, and the dose was applied to a 1 cm 2 area of skin using a non-occlusive covering. Absorption through mouse skin was greater than through rat skin. At 48 h after dermal application of 8 and 23 mg/kg [ 14 C]DEA, 26.8 and 33.8% of the dose was absorbed in the mice. A statistically significant increase in the amount absorbed after dosing with 81 mg/kg, 58.1%, was observed. The amount of radioactivity found at the site of application was only 4.0, 3.1,and 2.2% of the dose following application of 8, 23, and 81 mg/kg [ 14 C]DEA, respectively, and the amount excreted in the urine 48 h after application was 7.5%, 10.4, and 16.4%, respectively. Again, the tissue/blood ratio was greatest in the liver and kidneys. DEA, 80 mg/kg bw in acetone, was applied to a 2 cm 2 area on the backs 7 female C57BL/6 mice for 11 days. 21 DEA and its methylated metabolites accumulated in the liver and plasma of mice. Also, a statistically significantly decrease in hepatic concentrations of choline and its metabolites were reported. Human Three female subjects applied a lotion containing 1.8 mg DEA/g lotion to their entire body 2x/day for 1 mo. 21 Blood samples were collected 1 day prior to the start of dosing, at 1 wk, and at 1 mo. Two of the subjects completed 1 mo of application, while the third completed 3 wks. Application of the DEA-containing lotion for 1 mo resulted in increased concentrations of DEA and dimethyldea in plasma. However, when compared to mice that were dosed with 80 mg/kg bw/day DEA for 11 days, the human absorption rates were 100- to 200- times less than those found in mice. Oral Non-Human Four male Fischer 344 rats were dosed orally by gavage with 7 mg/kg aq. [ 14 C]DEA to examine the ADME of DEA (>97% purity). 17 The amount excreted in the urine at 24 and 48 h was 9 and 22%, respectively, and the amount in the feces was 1.6 and 2.4%, respectively. At 48 h after dosing, 27% of the dose accumulated in the liver, 5% in the kidneys, and 0.32, 0.27, 0.19, and 0.18% in the spleen, brain, heart, and blood, respectively. As measured in the liver and the brain, 87-89% of the radioactivity distributed into the aq. phase, and 70-80% of that total radioactivity was as unchanged DEA. In the liver, the remaining radioactivity was distributed between three methylated metabolite fractions, while in the brain, only one minor, non-methylated metabolite was present. With repeat oral dosing, DEA continued to accumulate in tissues, reaching steady states at 4-8 wks. The percent of the dose measured in the blood, brain, and liver decreased during 8 weeks of repeated oral dose of 7 mg/kg/day when compared to the single dose post-48-h values. Again, DEA was the major radioactive component. Using high-performance liquid chromatography (HPLC), almost all of the organic-extractable hepatic radioactivity eluted with the PC fraction, with greater than 95% of the material in the form of phospholipids containing an N,N-dimethyl-DEA headgroup. In the brain, the entire organic-extractable radioactivity eluted in the PE fraction, and it was almost entirely comprised of DEA-containing headgroups. According to the researchers, the results of this study demonstrated that DEA is O-phosphorylated and N-methylated, and that these metabolites are incorporated as the polar headgroups in aberrant phospholipids. The researchers felt this was evidence that DEA and MEA, a naturally-occurring alkanolamine, share common biochemical pathways of transformation. Retention and bioaccumulation of DEA-derived radioactivity was attributed partly to aberrant phospholipids being incorporated into tissues, most probably in cell membranes. 4

45 Groups of 3-5 male Fischer 344 rats were used to evaluate the ADME of a single oral administration of [ 14 C]DEA (99% purity). 20 The dose administered contained from µci radiolabeled, unlabeled DEA, and water to achieve a target dose of 5 ml/kg bw. Gastrointestinal absorption was nearly complete after 200 mg/kg DEA. After 48 h, 22% of the dose was excreted in the urine and 2.4% in the feces, primarily as unchanged DEA. Radioactivity was not detected in carbon dioxide. Excretion was mostly unchanged DEA. A total of 57% of the radioactivity was found in the body 48 h after dosing with 7 mg/kg [ 14 C]DEA. Disposition of radioactivity was greatest in the liver (27.3%), muscle (16.3%), skin (5.1%), and kidneys (5%); only 0.2% of the radioactivity was found in the blood after 48 h. Dose did not affect distribution in the tissues The researchers then evaluated the ADME of DEA upon repeat oral exposure. Four rats were dosed orally with 7 mg/kg/day [ 14 C]DEA for 5 days. Approximately 40% of the total dose was excreted during dosing. At 48 h after the last dose, a total of 42% of the radioactivity was found in the tissues, with the greatest distribution in the liver (18.2%), muscle (12.4%), kidneys (5.56%), and skin (4.18%). To further investigate DEA bioaccumulation, rats were dosed with 7 mg/kg/day DEA, 5 days/wk, for 2, 4, or 8 wks. During the last week of each dosing period, 69, 79, and 92% of the dose was excreted, respectively. After 8 wks of dosing, most of the radioactivity was recovered as unchanged DEA; however there were also significant amounts of poorly retained metabolites, N-methylDEA, and another metabolite that was tentatively identified as a quaternized lactone. The % dose recovered in the liver after 2, 4, and 8 wks of dosing was 12.3, 7.9, and 4.12%, respectively. It was estimated that steady-state for bioaccumulation occurred after 4 wks of repeat dosing, except for the blood, which continued to bioaccumulate DEA throughout dosing. Clearance of bioaccumulated DEA appears to be a first-order process, with a whole body elimination half-life of ~6 days. Other Non-Human Rats (number not specified) were given a single i.v. dose of 7.5 mg/kg [ 14 C]DEA. 22 After 48 h, 28 and 1% of the dose was excreted in the urine and feces, respectively. The remainder was found in the tissues, with the highest concentrations in the liver and kidneys. (Additional details not provided.) Groups of Sprague-Dawley rats (number per group not specified) were given an i.v. dose of 10 or 100 mg/kg DEA in physiological saline. 19 Animals were killed 96 h after dosing. Peak blood concentrations appeared 5 min after dosing. Elimination from the blood was biphasic. Totals of 25 and 36% of the dose were excreted in the urine with 10 and 100 mg/kg DEA, respectively. Groups of 3-5 male Fischer 344 rats were used to evaluate the ADME [ 14 C]DEA (99% purity) in phosphate-buffered saline after intravenous (i.v.) administration. 20 After 48 h, 28% of the dose was excreted in the urine, 0.6% in the feces, and only 0.2% in carbon dioxide. Using HPLC, it was determined that most of the radioactivity in the urine was present as unchanged DEA. A total of 54% of the dose was found in the body 48 h after dosing with 7 mg/kg [ 14 C]DEA, and as with oral dosing, the greatest disposition was found in the liver (27%), muscle (15%), skin (4.5%), and kidneys (4%). Only 0.2% of the radioactivity was found in the blood after 48 h. Groups of 5 female Sprague-Dawley rats were dosed i.v., via the cannulated jugular vein, with 10 or 100 mg/kg [ 14 C]DEA (97.4% purity); the dose volume was 2 ml/kg, and each rat received ~4.2 µci 14 C. 23 Blood samples were taken at various intervals up to 84 h after dosing. The peak concentrations of radioactivity in both the plasma and the red blood cells were observed 5 min after dosing. Clearance of radioactivity from the plasma was calculated to be approximately 50 and 93 ml/h/kg for the low and high dose, respectively. In blood, these values were 84 and 242 ml/h/kg, respectively Urine and feces were collected for 96 h after dosing. During this time, the major route of excretion was urinary; 25 and 36% of the dose was recovered in the urine. Urinary excretion was rapid at the high dose level, with 23% of the dose 5

46 recovered in the first 12 h. Only 8.5% of the dose was recovered during this time frame with the low dose. The majority of the radioactivity was recovered in the carcass; 35 and 28% for the low and high dose, respectively. In the tissues at 96 h after dosing with 10 and 100 mg/kg DEA, approximately 21 and 17% of the dose was recovered in the liver, 7 and 5% in the kidneys, and 5 and 5% in the skin, respectively. The researchers stated that because the majority of the administered radioactivity was recovered in the tissues, particularly in the liver and kidneys, this indicated a propensity for bioaccumulation. There was some evidence that the bioaccumulation was dose-dependent. Percutaneous Absorption Non-Human [ 14 C]DEA was applied to a 2 cm 2 area of the intrascapular region of male F344/N rats at a dose of 2.1, 7.6, or 27.5 mg/kg bw. 18,22 After 48 h, 2.9, 10.5, or 16.2% of the dose, respectively, was absorbed. It was shown that a 10-fold increase in the concentration of DEA resulted in a 450-fold increase in the rate of absorption. [ 14 C]DEA was applied to a 1.0 cm 2 area of the back of 4 B6C3F 1 mice, and the site was protected. 22 Approximately 59% of the dose was absorbed, and the absorption rate was 19.9 µg/cm 2 /h. The in vitro absorption of 2 mg/cm 2 [14C]DEA was determined using fuzzy rat skin. 24 A total of 1.4% of the applied dose was applied over 24 h, with 1.9% of the dose remaining in both the stratum corneum and viable dermis/epidermis. Values were similar at 72 h. Human The percutaneous absorption of cosmetic formulations spiked with [ 14 C]DEA (95-99% purity) was examined using viable and non-viable human skin. 25 Details regarding the product types used, the relevant ingredient compositions, the doses applied, absorption of DEA, and retention in the skin are given in Table 4. Very little DEA was found in the receptor fluid, with only 0.1% of the applied dose recovered in the receptor fluid of the shampoo and hair dye formulations. While the amount absorbed was similar for these two product types, the distribution and localization were different, with most of the DEA penetrating from the shampoos being localized in the stratum corneum, and that from the hair dyes being found in deeper epidermal and dermal layers. With the lotion, % of the dose recovered in the receptor fluid. Approximately 65% of the penetrated DEA from the TEA lotion formulation was found in the lower layers of the skin. DEA did not appear to be covalently bound to the skin, and extended times before analysis did not result in any statistically significant difference compared to the values obtained after 24 h. Repeat application studies with viable skin did not produce a significant change of dose absorbed into receptor fluid. However, with non-viable skin, absorption into the receptor fluid from the lotion increased each day, from 0.6% on the first day to 2.6% on the third day; testing showed that the skin barrier did not remain intact for the entire 72 h. Using a shampoo and a lotion formulation, non-viable skin gave penetration values similar to viable skin. The researchers concluded that most of the DEA that penetrated was not available for systemic absorption. Another study examining the percutaneous penetration of cosmetic formulations spiked with radiolabeled and unlabeled DEA was performed mimicking simulated use conditions using fresh and frozen human skin samples. 26 Details regarding the product types used, the relevant ingredient compositions, the doses applied, absorption of DEA, and retention in the skin are given in Table 5. Very little DEA penetrated the skin; % of the applied dose of the shampoo formulations penetrated, % of the applied dose of the hair dye formulations, and 0.508% of the bubble bath formulation penetrated the skin. With a moisturizer formulation, 0.605% of the applied dose penetrated fresh skin, while 0.456% penetrated frozen skin. The researchers also examined the penetration of a simple aq. 1% solution of DEA through fresh and frozen human skin samples. The cumulative 24 h percutaneous absorption was approximately 5-fold greater in frozen skin compared to 6

47 fresh skin, i.e., 8.87 µg/cm 2 compared to 1.73 µg/cm 2. The 24-h cumulative penetration values represented and 0.086% of the dose for frozen and fresh skin, respectively. The amounts of DEA remaining on and in the skin were 1.68 and 1.14% of the applied dose recovered from the frozen and fresh skin samples, respectively. The researchers further investigated the distribution of [ 14 C]DEA between aqueous and lipid fractions of viable skin strata. The radioactivity on the skin samples from 2 donors (n=12) was determined after a 24 h exposure. At 24 h, the cumulative permeation value was 0.405% of the applied dose for one donor (abdominal skin) and 0.067% for the other (breast skin). (The researchers stated that it may be significant that one sample was abdominal skin and the other was breast skin.) Tape-strip profiles for both donors appeared to indicate that the DEA had become evenly distributed throughout the stratum corneum. The majority of DEA was recovered in aqueous extract, as opposed to organic extract, of the epidermal and dermal tissue, which suggested that the material was in the free state and not associated with the lipid fraction. Oral Other TOXICOLOGICAL STUDIES Acute (Single Dose) Toxicity The acute oral toxicity of DEA was determined using guinea pigs and rats. Using groups of 2-3 guinea pigs, all guinea pigs survived with dosing of 1.0 g/kg, but none survived dosing with 3.0 g/kg DEA gum arabic solution. Using groups of 5 rats, the oral LD 50 of undiluted DEA ml/kg, and for a group of 6 rats, the LD 50 of DEA in water was 1.82 g/kg. Using rats, 20% aq. DEA had an acute LD 50 of g/kg, based on results of testing performed over a 10 yr time period. With groups of 10 rats, a hair preparation containing 1.6% DEA had an LD 50 of 14.1 g/kg when diluted and 12.9 ml/kg when undiluted. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 The intraperitoneal (i.p.) LD 50 of DEA was 2.3 g/kg for mice. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Repeated Dose Toxicity Dermal In a 13-wk study, 1 mg/kg of a hair dye formulation containing 2.0% DEA was applied to the backs of 12 rabbits for 1 h, twice weekly. The test site skin was abraded for half of the animals. No systemic toxicity was observed, and there was no histomorphologic evidence of toxicity From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 DEA was applied dermally to groups of 5 male and 5 female B6C3F1 mice 5x/wk for 2 wks at doses of mg/kg bw in 95% ethanol. 27 All of the male and 3 of the female high-dose test animals died during the study. Ulceration, irritation, and crusting were observed at the application site of male mice of the 1250 and 2500 mg/kg groups and females of the 2500 mg/kg group. Microscopically, moderate to marked epidermal ulceration and inflammation were observed in these animals. Ulcerative necrosis extended into the underlying dermis. Minimally severe acanthosis, without inflammation, was seen in the 160, 320, and 630 mg/kg dose groups. Absolute and relative liver weights increased in a dose-dependent manner in males and females. The LOAEL was 160 mg/kg bw. Repeated dermal exposure of DEA (99.6% purity) in 96% ethanol was applied to the shaved backs of the following groups of B6C3F 1 mice for the following time periods: 8 males and 8 females were dosed daily with 0 or 160 mg/kg bw (dose volume 2.13 ml/kg bw) for 1 wk, followed by a 3-wk recovery period; groups of 10 males were dosed 5 days/wk with 0 7

48 or 160 mg/kg bw for 1, 4, or 13 wks; and groups of 8 males were dosed 5 days/wk with mg/kg for 1 or 13 wks. 28 With the last dosing scheme, application of 630 and 1250 mg/kg DEA was discontinued, and the animals killed, after 1 wk due to severe skin lesions; the high dose was then 160 mg/kg. In the other animals, including controls, some erythema and/or focal crust formation was observed and attributed to the procedure and/or the vehicle, but not to DEA. No DEA-related deaths were observed. Body weights were not affected by dosing. Statistically significant increases in liver weights were seen in mice dosed with 10 mg/kg bw/day DEA for 1 or more weeks. Microscopically in the liver, eosinophilia was found in animals dosed with 40 mg/kg DEA for 1 or more weeks, and hepatocellular giant cells were seen in animals dosed with 160 mg/kg for 13 wks. Unoccluded dermal applications of mg/ml DEA (purity >99%) in acetone were applied to the backs of 10 male and 10 female B6C3F 1 mice, 5 days/wk for 13 wks, at doses of mg/kg bw. 29 (The area of the dose site was not provided.) Two male mice and 4 female mice dosed with 1250 mg/kg DEA were killed in moribund condition. Final mean body weights of male mice dosed with 1250 mg/kg were statistically significantly decreased compared to controls. Clinical signs of toxicity, observed in males and females dosed with 630 and 1250 mg/kg DEA, were irritation, crust formation, and thickening at the application site. Ulceration and inflammation were observed in the 630 and 1250 mg/kg dose groups. Dose-dependent increases in absolute and relative liver weights, associated with hepatocellular cytological changes, were observed, and hepatocellular necrosis was seen in males dosed with mg/kg DEA. Absolute kidney weights were statistically significantly increased in males and females of all test groups and relative kidney weights were increased in males of all test groups and females dosed with 630 or 1250 mg/kg DEA; nephropathy was not found. Heart weights were increased in high dose males and females, and degeneration was reported. Unoccluded dermal applications of mg/ml DEA (purity >99%) in 95% ethanol were applied to the backs of 10 male and 10 female F344/N rats, 5 days/wk for 13 wks, at doses of mg/kg bw. 30 (The area of the dose site was not provided.) One male and 2 females given 500 mg/kg died or was killed in moribund condition during the study. Clinical signs of toxicity, observed in males and females given ppm DEA, were irritation and crusting at the application site. Final mean body weights were statistically significantly decreased in males dosed with 250 and 500 mg/kg and females dosed with mg/kg DEA. Increases in absolute and relative kidney weights were observed with increased incidence of renal lesions. Increases in absolute and relative liver weights were not accompanied by an increase in hepatic lesions. Demyelination in the medulla oblongata was seen in all high dose animals and 7 females given 250 mg/kg DEA; this lesion was minimal in severity. Oral Oral studies were conducted in which neonatal rats were dosed with 1-3 mm/kg/day DEA, as a neutralized salt, on days 5-15 after birth, male rats were dosed with 4 mg/ml neutralized DEA in drinking water for 7 wks, and rats were fed g/kg/day DEA in feed for 90 days. Repeated oral ingestion of DEA produced evidence of hepatic and renal damage. Deaths occurred in the 7 wk and 90 day studies. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Female CD-1 mice, 3 per group, were dosed orally, by gavage, with mg/kg bw DEA in distilled water for 7 days. 31 One animal of the 100 mg/kg group died, and the death was considered test article-related. (Two deaths in the 1000 mg/kg group were attributed to gavage error.) In a 13-wk study, 10 male and 10 female B6C3F 1 mice were dosed orally, by gavage, 5 times/wk with mg/kg DEA in deionized water. 32 Two males of the high dose group died during the study. Body weights and body weight gains of 8

49 high dose males were reduced. No clinical signs of toxicity were noted. Treatment-related renal lesions were found, but details were not provided. Groups of 10 male albino rats were given feed containing mg/kg bw/day DEA (99% purity) for 32 days. 33 Nine of the 10 high dose animals died during the study. All test animals had decreased hemoglobin and hematocrits, with an increased white blood cell count. The relative liver weights of animals fed 0.01 and 0.1% DEA, and the absolute liver weights of animals of the 0.1% group, were increased. In a repeat 30-day study using the same procedure and dose levels, 7 of the 10 high dose animals died, and the remaining 3 high-dose animals killed, prior to study termination; body weights and feed consumption were significantly reduced in this group. Again, hemoglobin and hematocrit were reduced in the 0.1% group, and the hemoglobin value was reduced in the 0.1% group. Groups of 10 male and 10 female B6C3F 1 mice were given 0-10,000 ppm DEA (>99% purity) in the drinking water for 13 wks. 29 The ph of the solution was adjusted to 7.4. All males and females of the 5000 and 10,000 ppm groups and 3 females of the 2500 ppm group died during the study. Body weight gains were decreased in males of the 2500 ppm group and females of the 1250 and 2500 ppm groups. No significant gross effects were noted at necropsy. Statistically significant, dose-dependent increases in absolute and relative liver weights were observed, with hepatocellular cytological alterations and necrosis in animals given 2500 pm DEA. Absolute and relative kidney weights also increased dose-dependently, with statistically significant increases in mice given 1250 or 2500 ppm DEA, and neuropathy in all male test groups and female test groups given 2500 or 5000 ppm DEA. Heart weights were increased in female mice given 2500 ppm DEA, and relative heart weights were increased in males of the 2500 ppm group and females of the 1250 and 2500 ppm groups. Groups of 10 male F344/N rats were given ppm and 10 female F344/N rats were given ppm DEA in the drinking water for 13 wks. 30 Two males of the high dose group died during the study. The following dose-related effects were observed: decreases in body weight gains in males and females; hematological effects; increases in kidney weights accompanied by renal lesions; and increases in relative liver weights in males and females. The brain and spinal cord were also identified as targets of DEA toxicity. Inhalation Short-term inhalation of 200 ppm DEA vapor or 1400 ppm DEA aerosols produced respiratory difficulties and some deaths in male rats. Inhalation of 25 ppm for 216 continuous hours resulted in increased liver and kidney weights, while exposure of male rats to 6 ppm DEA following a workday schedule for 13 wks caused decreased growth rate, increased lung and kidney weights, and some deaths. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 In a 2-wk dose-range finding inhalation study, 10 male and 10 female Wistar rats were exposed, nose only, to target concentrations of mg/m 3 DEA (>99% pure) for 6 h/day, 5 days/wk. 34 Mass median aerodynamic diameter (MMAD) was µm. Test article-related effects were not seen with 100 or 200 mg/m 3 DEA. With 400 mg/m 3 DEA, decreased body weights and body weigh gains were seen in males and relative and absolute liver weights were seen in females. Microscopically, no effects were seen in the respiratory tract; the larynx was not examined. Based on the results of the dose-range finding study, target concentrations of 0, 15, 150, and 400 mg/m 3 DEA were used in a 90-day study, in which groups of 13 male and 13 female rats were exposed via inhalation to 65 exposures, 6-h/day 5 days/wk. The MMAD was µm. A functional observational battery was conducted using 10 rats/gender/group. Body weight gains were reduced in males of the high dose group. Some statistically significant effects on clinical chemistry values were seen in the mid and high dose group. Males and females of the high dose group had statistically significant increases in blood content in the urine, and males of the mid and high dose groups excreted significantly elevated amounts of renal tubu- 9

50 lar epithelial cells. Relative liver weights were statistically significantly increased in males and females of the high dose group and females of the mid-dose group, and relative kidney weights were statistically significantly increased in males and females of the mid and high dose groups. Focal squamous metaplasia of the ventral laryngeal epithelium was observed in test animals of all groups, and a concentration-dependent increase in laryngeal squamous hyperplasia, and in the incidence and severity of local inflammation of the larynx and trachea, were observed. There were no indications of neurotoxicological effects. In a third study, test groups of 10 male and 10 female Wistar rats were exposed to target concentrations of 0, 1.5, 3, and 8 mg/m 3 DEA using the same dosing schedule as above, and recovery groups of 10 females were exposed to 3 or 8 mg/m 3, with a post-exposure period of 3 mos. No dose-related clinical signs were observed. Liver weights of test, but not recovery, females dosed with 8 mg/m 3 were statistically significantly increased. Laryngeal effects were similar to those described above. No microscopic changes were observed in the upper respiratory tract of recovery animals. The 90-day no observed effect concentration (NOAEC) was determined to be 1.5 mg/m 3 DEA. In inhalation studies, Sprague-Dawley rats, Hartley guinea pigs and Beagle dogs (number per species and sex not specified) were each exposed to 0.5 ppm DEA for 6 h/day, 5days/wk, for a total of 45 exposures. 33 All animals survived until study termination. There were no clinical signs of toxicity, and no evidence of irritation. No gross or microscopic lesions were observed at necropsy. REPRODUCTIVE AND DEVELOPMENTAL STUDIES Dermal Hair dyes containing up to 2% DEA were applied topically to the shaved skin of groups of 20 gravid rats on days, 1, 4, 7, 10, 13, 16, and 19 of gestation, and the rats were killed on day 20 of gestation. No developmental or reproductive effects were observed. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 A reproductive and developmental toxicity study was performed in which mg/kg DEA (98.5% purity) in ethanol was applied to a 2 cm 2 area on the back of 15 male C57BL/6 mice, 15 per group, for 4 wks. 35 (It was not stated whether the test area was covered.) These males were then mated with untreated females. In the parental male mice, sperm motility was significantly decreased in a dose-dependent manner. In male pups, a significant decrease in epididymis weight was seen in the 80 mg/kg group at postnatal day (PND) 21, and reductions in male reproductive weights of high-dose pups was seen at PND 70. There were no significant differences in skeletal formation, and differences in growth and development parameters were not significant. Doses of mg/kg DEA (98.5% purity) in ethanol were applied to a 2 cm 2 area on the backs of groups of 10 gravid female C57BL/6 mice on day 6 of gestation through PND 21. The body weights of male and female pups of the high dose group were statistically significantly decreased compared to controls. No specific differences in organ weights were observed in pups of the test groups as compared to controls. There were no significant differences in skeletal formation. Some dose-dependent effects on growth and developmental parameters were noted. Sperm motility was decreased in male pups, but this result was not statistically significant. CD rats and NZW rabbits were used to evaluate the potential of DEA ( 99.4% purity) to produce developmental toxicity with dermal exposure. 36 Groups of 25 gravid CD rats were dosed dermally with mg/kg/day DEA in deionized water, 6 h/day, on days 6-15 of gestation, under an occlusive covering. Dosing volume was 4 ml/kg/day. Control animals were dosed with vehicle only. Teratogenic effects were not seen at any dose. Dermal application of 500 mg/kg DEA 10

51 resulted in alterations of maternal hematological parameters, but did not affect embryonal/fetal development. Other signs of maternal toxicity were seen at this dose and with 1500 mg/kg/day. The NOEL for embryonal/fetal toxicity was estimated to be 380 mg/kg/day, which incorporates an adjustment for the 10-24% deficit in expected dose that occurred on days of gestation. Groups of 15 mated rabbits were dosed dermally with mg/kg/day DEA in deionized water, 6 h/day, on days 6-18 of gestation, under an occlusive covering. Dosing volume was 2 ml/kg/day, and controls were dosed with vehicle only. Dermal administration of 350 mg/kg/day DEA produced severe skin irritation in rabbits, and signs of maternal toxicity were observed at this dose. No developmental toxicity was observed, and there was no evidence of teratogenicity at any dose. The NOEL for maternal toxicity of DEA in rabbits was 35 mg/kg/day, and the embryonal/fetal NOEL was 350 mg/kg/day DEA. Oral In a Chernoff-Kavlock screening test, groups of 4 gravid CD-1 mice were dosed orally, by gavage, with mg/kg bw DEA in distilled water on days 6-15 of gestation. 31 Two, 3, and 4/4 animals of the 720, 1370, and 2605 mg/kg dose groups died during the study. Rough hair coats were observed at all dose levels. Group of 50 female gravid CD-1 female mice were dosed orally, by gavage, with 0 or 450 mg/kg bw DEA in distilled water on days 6-15 of gestation. No animals died during the study. The reproductive index and average number of live litters on day 0 were not affected by dosing, but the average number of live litters on day 3 was reduced. Mean body weights and body weight gains of pups were also decreased on PND 3. Gravid Sprague-Dawley rats were dosed orally, by gavage, with mg/kg/day DEA on days 6-15 of gestation. 37 Maternal mortality was observed at doses of mg/kg/day. At doses of 50 and 200 mg/kg/day, no differences in gross developmental endpoints were found between test and control animals. The NOEL for embryonal/fetal toxicity was 200 mg/kg/day DEA. However, maternal weight gains were significantly reduced at that dose. (Additional details were not provided.) Groups of 12 gravid female Sprague-Dawley rats were dosed orally, by gavage, with mg/kg bw/day DEA (>98% purity) distilled water on days 6-19 of gestation, while controls were dosed with vehicle only. 38 Dosing volume was 5 ml/kg. Surviving dams and pups were killed on PND 21. All females dosed with 300 mg/kg DEA were killed prior to study termination due to excessive toxicity. Toxicity was also observed for one dam dosed with 200 mg/kg, and only 5 dams of the 250 mg/kg group delivered live litters and survived until study termination. The calculated LD 50 was 218 mg/kg bw/day. No significant maternal or developmental toxicity was seen with 50 mg/kg bw/day DEA. Signs of maternal and developmental toxicity were seen at doses of 125 mg/kg bw/day, including reduced maternal weight gains, increased kidney weights in dams, increased post-implantation and postnatal mortality, and reduced live pup weights. The NOAEL for maternal toxicity and teratogenicity was 0.05 mg/l, and the LOAEL for these parameters were 125 mg/kg/day. Inhalation In a range-finding study, groups of 10 gravid Wistar rats were exposed, nose-only, to target concentrations of mg /l DEA, 6 h/day, on days 6-15 of gestation. 39 (DEA purity was >98.7%.) All animals survived until study termination. Relative liver weights were increased in animals of the 0.2 mg/l group, and absolute and relative liver weights were increased in animals of the 0.4 mg/l group. No treatment-related effects were observed with 0.1 mg/l DEA. Groups of 25 gravid Wistar rats were exposed, nose-only, to target concentrations of mg DEA aerosol/l air, 6 h/day, on days 6-15 of gestation. 40,41 (DEA purity >98.7%; vehicle not specified.) Maternal toxicity, as indicated by vaginal hemorrhage, was seen at the highest dose level. No treatment-related malformations were observed at 0.2 mg/m 3. 11

52 The NOAEC for both maternal and developmental toxicity was 0.05 mg/l, and the NOAEC for teratogenicity was >0.2 mg/l, which was the highest dose tested in this study. GENOTOXICITY In Vitro DEA, with and without metabolic activation using liver preparations from rats induced with a polychlorinated biphenyl mixture, was not mutagenic to Salmonella typhimurium TA100 or TA1535. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 With or without metabolic activation, DEA was not mutagenic in Ames test using Salmonella typhimurium TA98, TA100, TA1535, or TA1537, was negative in a mouse lymphoma assay, and did not induce sister chromatid exchanges or chromosomal aberrations in a Chinese hamster ovary cell cytogenetic assay. 18 DEA was not clastogenic in a mouse micronucleus test. Positive results were reported in an in vitro assay for induction of DNA single-strand breaks in isolated hepatocytes for rats, hamsters (both at 25 µmol/tube), and pigs ( 12.5 µmol/tube). CARCINOGENICITY Dermal The National Toxicology Program (NTP) evaluated the carcinogenic potential of DEA (>99% purity) in ethanol using B6C3F 1 mice and F344/N rats. 18 Groups of 50 male and 50 female mice were dosed dermally with mg/kg/day, 5 days/wk, for 103 wks. Survival of dosed females, but not males, was significantly decreased in a dose-dependent manner. Mean body weights of test animals were decreased at various intervals throughout the study. In male mice, the incidences of hepatocellular adenoma and of hepatocellular adenoma and carcinoma (combined) in all dose groups, and the incidence of hepatocellular carcinoma and hepatoblastoma in the 80 and 160 mg/kg group, were statistically significantly increased compared to controls. In female mice, the incidence of hepatocellular neoplasms was significantly increased. Male mice also had a dose-related increase in the incidences of renal tubule hyperplasia and renal tubule adenoma or carcinoma (combined), and an increase in the incidence of renal tubule adenoma. In male and female mice, incidences of thyroid gland follicular cell hyperplasia were increased. Hyperkeratosis, acanthosis, and exudate were treatment-related changes observed at the application site. It was concluded that there was clear evidence of carcinogenic activity of DEA in male and female B6C3F 1 mice based on increased incidences of liver neoplasms in males and females and increased incidences of renal tubule neoplasms in males. Groups of 50 male rats were dosed dermally with 0-64 mg/kg bw DEA, and groups of 50 female rats with 0-32 mg/kg bw, 5 days/wk for 103 wks. The only treatment-related clinical finding was irritation at the application site. Minimal to mild non-neoplastic lesions were found at the site of application of dosed males and females. The incidence and severity of nephropathy in dosed females, but not males, was significantly greater in the treated groups compared to controls. There was no evidence of carcinogenic activity of DEA in male or female F344/rats. The carcinogenic potential of DEA was evaluated using a Tg AC transgenic mouse model. 42,43 Groups of female homozygous mice were dosed dermally with 0-20 mg DEA/mouse in 95% ethanol, 5x/wk for 20 wks. DEA was inactive in Tg AC mice. 12

53 IRRITATION AND SENSITIZATION Irritation Skin Non-Human The primary skin irritation potential of DEA was determined using rabbits. Undiluted DEA, applied to an unspecified number of rabbits using 10 open applications of 0.1 ml to the ears and 10 semi-occluded applications to the abdomen, was moderately irritating. No irritation was observed with 10% aq. DEA following the same protocol. Using groups of 6 rabbits, application of 30 and 50% DEA using semiocclusive patches to intact and abraded skin produced essentially no irritation. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Human In a study in which an undiluted formulation containing 1.6% DEA was applied to the back of 12 female subjects for 23 h/day for 21 days, the formulation was considered an experimental cumulative irritant. No irritation was reported during the induction phase of sensitization studies for a formulation containing 2% DEA, tested as a 10% solution in distilled water (165 subjects), for a formulation containing 2.7% DEA, tested undiluted (100 subjects), or for a formulation containing 1.6% DEA, tested undiluted (25 subjects). From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Mucosal Non-Human The ocular irritation potential of % DEA was evaluated using rabbits. As a 30% aq. solution, DEA was essentially non-irritating, while a 50% aq. solution was a severe irritant. Instillation of 0.02 ml undiluted DEA produced severe injury to rabbit eyes. A hair preparation containing 1.6% DEA had a maximum avg. irritation score of 0.7/110 for rinsed and unrinsed eyes. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Sensitization In Vitro DEA had an EC3 value of 40% in a mouse local lymph node assay, resulting in a categorization of weak potency of for skin sensitization. 44 Non-Human In a maximization study using 15 Dunkin-Hartley guinea pigs, intradermal and epicutaneous induction used 1% and 17.6% aq. DEA, respectively, after 10% sodium lauryl sulfate pre-treatment. 45 At challenge with 0.7, 3.5, or 7% DEA, 1/15 animals reacted to the lowest and highest challenge concentrations after 2, but not 3 days. In a second maximization test using 20 Himalayan spotted guinea pigs, the intradermal induction, epicutaneous induction, and epicutaneous challenge concentrations were 5%, 75%, and 25% DEA in physiological saline, respectively. 45,46 Freund s complete adjuvant (FCA) was used at intradermal induction. Two animals had mild erythema at day 1, and one animals had mild erythema at day 2. DEA was not a sensitizer. A maximization study was performed on a test substance (known as FORAFAC 1203) containing 2% DEA using a treatment group of 10 male and 10 female guinea pigs. 47 (The type of product was not specified; it also contained 40% water, 30% butoxydiglycol, 11% fluorinated copolymer, and a few other ingredients with concentrations of 8%). The intradermal induction, which included the use of 50% FCA, used a concentration of 5% in sterile saline, and the topical induction and challenge used undiluted product. A control group of 5 males and 5 females was treated with vehicle. The test substance containing 2% DEA induced delayed hypersensitivity in 100% of the test animals. No reactions were observed in the controls. 13

54 Human Formulations containing 1.6 and 2.7% DEA, tested undiluted, and a formulations containing 2% DEA, tested at a 10% solution in distilled water, were not sensitizing in clinical studies. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Provocative Testing Over a 15-yr period, provocative patch testing using DEA was performed on 8791 patients. 45 There were 157 (1.8%) positive reactions to DEA, and most of the reactions (129; 1.4%) were weak positives. There were 17 (0.2%) irritant reactions reported. Cosensitization was reported; 77% of the patients that reacted to DEA also tested positive to MEA. Occupational sensitization was reported; of 7112 male patients, 1.0% that did not work in the metal industry had positive reactions to DEA, as opposed to 3.1 and 7.5% of those working in the metal industry and those exposed to water-based metalworking fluids, respectively. MISCELLANEOUS STUDIES In male albino rats, following repeated oral administration of 320 mg/kg/day in drinking water of radiolabeled DEA, a decrease was seen in the amount of injected MEA and choline incorporated in the liver and the kidneys after 1, 2, and 3 wks as compared to 0 wks. MEA and choline phospholipid derivatives were synthesized faster and in greater amounts, and were catabolized faster than DEA phospholipid derivatives. This was not seen with a single 250 mg/kg injection of DEA. The effect of DEA on the mitochondrial function was also investigated using male albino rats. Administration of neutralized DEA in the drinking water at doses of 490 mg/kg/day for 3 days or of 160 mg/kg/day for 1 wk produced alterations of hepatic mitochondrial function. Oral and i.p. administration of DEA my affect, directly or indirectly, the serum enzyme levels, isozyme patterns, and concentrations of some amino acids and urea in the male rat liver and kidney. Repeated DEA administration in the drinking water increased male rat hepatic mitochondrial ATPase and altered mitochondrial structure and function. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 N-Nitrosodiethanolamine Formation The formation of NDELA upon dermal dosing with DEA (99.7% purity), with and without supplemental oral sodium nitrite, was determined in male B6C3F 1 mice. 48 Groups of 5-6 mice were dosed orally with aq. DEA or dermally with DEA in acetone. DEA, 160 mg/kg/day, was applied for 7 days/wk for 2 wks. One group of mice dosed dermally was allowed access to the application site, while the other was not. Studies were performed both with and without ~40 mg/kg/day supplemental sodium nitrite in drinking water. NDELA was not found in the urine or blood of mice dosed with DEA without nitrite or in the blood or gastric contents of those given supplemental nitrite with DEA. NDELA formation from DEA (>99% purity) and nitrite was also examined in another study. 49 Female B6C3F 1 mice were dosed dermally or orally with 4 mg/kg DEA, in conjunction with oral exposure to sodium nitrite. Following 7 days of dermal dosing, no NDELA was detected in the blood, ingesta, or urine of test, vehicle control, or sodium nitrite control mice. (The limits of detection for the blood, ingesta, and urine were 0.001, 0.006, and 0.47 µg/ml, respectively.) With a single oral dose, NDELA was formed in all of the animals; the amount of NDELA detected in the blood and ingesta of mice 2 h post-dosing were ± µg/g and ± µg/g, respectively. Immunotoxicity Groups of 48 female F344 female rats were dosed orally, by gavage, with mg/kg bw DEA in distilled water for 14 days. 50 Rats exposed to 10 and 200 mg/kg DEA had significant decreases in body weight and/or body weight gains. Liver and kidney weights were increased in a dose-dependent manner. Exposure to DEA did not alter the number of B-cells, 14

55 T-cells, or T-cell subsets. The proliferative response to allogenic cells was increased in a dose-dependent manner. Natural killer cell response and cytotoxicity of resident macrophages were decreased in DEA-treated animals. Inhibition of Choline Uptake The ability of DEA to alter cellular choline levels was examined in a number of studies. In the study described previously in which B6C3F 1 mice were dosed orally and dermally with 160 mg/kg/day DEA in conjunction with oral sodium nitrite, a pronounced decrease in choline and its metabolites was observed in the livers. 48 The smallest decreases were seen in the mice that were dosed dermally and not allowed access to the test site, and the greatest increase was seen in the mice dosed orally. In Syrian hamster embryo (SHE) cells, DEA inhibited choline uptake at concentrations 50 µg/ml, reaching a maximum 80% inhibition at µg/ml. 4 DEA also reduced phosphatidylcholine in the phospholipds, was incorporated into SHE lipids, and transformed SHE cells in a concentration-dependent manner. Excess choline blocked these biochemical effects and inhibited cell transformation, and the researchers hypothesized there is a relationship between the effect of DEA on intracellular choline availability and utilization and its ability to transform cells. In a study performed to test the hypothesis that DEA treatment could produce biochemical changes consistent with choline deficiency in mice, it was found that DEA treatment caused a number of biochemical changes consistent with choline deficiency in mice. 51 Hepatic concentrations of S- adenosymethionine (SAM) decreased. Biochemical changes were seen without fatty livers, an observation often associated with choline deficiency. To study the dose-response, reversibility and strain-dependence of DEA effects, B6C3F 1 mice were dosed dermally with DEA in ethanol for 4 wks. 51 Control animals were either not dosed or dosed with ethanol only. The pattern of changes observed in choline metabolites after DEA treatment was very similar to that observed in choline-deficient mice, and the NOEL for DEA-induced changes in choline homeostasis was 10 mg/kg/day. Fatty livers were not observed. (Lehman- McKeeman hypothesized that the lack of fatty livers is the result of an age-dependence mechanism. 52 ) The reactions were dose-dependent, strain-dependent, and reversible. Dermal application of 95% ethanol decreased hepatic betaine levels, suggesting that used of ethanol as a vehicle for dermal application of DEA could exacerbate the biochemical effects of DEA. Species Selective Effect on DNA Synthesis In studies examining species selectivity of effects caused by DEA, increases in DNA synthesis were observed in mouse and rat, but not human, hepatocytes following treatment with DEA. 53 Additionally, when the hepatocytes were incubated in medium containing reduced choline, DNA synthesis was increased in mouse and rat hepatocytes, but not human hepatocytes. Conversely, choline supplementation reduced DEA-induced DNA synthesis in mouse and rat hepatocytes. Effect on Hippocampal Neurogenesis and Apoptosis The effect of DEA on neurogenesis was investigated using C57BL/6 mice. 54 DEA, mg/kg bw in ethanol, was applied to a 2 cm 2 area on the backs of gravid female mice, 6 per group, on days 7-17 of gestation. A dose-related decrease in litter size was observed at doses >80 mg/kg, and the decrease was statistically significant at doses of mg/kg bw/day. The livers of the maternal mice were analyzed on day 17 of gestation, and hepatic concentrations of choline and its metabolites were statistically significantly decreased. In the fetal brain, treatment with 80 mg/kg bw/day DEA diminished the proportion of cells that were in the mitotic phase to 50% of controls. The number of apoptotic cells of the hippocampal area was >70% higher in fetuses of DEA-treated mice compared to controls. The researchers stated that the effect observed in the mouse fetal brain after administration of DEA was likely to be secondary to diminished choline levels. The researchers also hypothesized that a potential mechanism for the effect of choline deficiency, and maybe DEA, on progenitor cell proliferation and apoptosis involves abnormal methylation of promoter regions of genes. 15

56 The doses used in the above study were based on expected concentrations of 1-25% DEA in cosmetic formulations. However, based on comments from the Cosmetic, Toiletry, and Fragrance Association (now known as the Personal Care Products Council) indicating the over-estimation of the amount of DEA contained in consumer products, 8 the researchers tested lower doses to establish a dose-response relationship. Groups of 7 mice were dosed dermally with 0-80 mg/kg bw DEA (purity >99.5%) as described previously, with the exception that acetone was used as the vehicle. While the results reported in the earlier study with 80 mg/kg bw DEA were confirmed, no differences were seen between treated and control groups with <80 mg/kg bw. In a study to identify the potential mechanism for the alterations described above, mouse neural precursor cells were treated in vitro with DEA. 55 Cells exposed to 3 mm DEA had less cell proliferation at 48 h and had increased apoptosis at 72 h. DEA treatment decreased choline uptake into the cells, resulting in diminished choline and phosphocholine. A three-fold increase in choline concentration prevented the effects of DEA exposure on cell proliferation and apoptosis; intracellular phosphocholine levels remained low. The researcher hypothesized that DEA interferes with choline transport and choline phosphorylation in neural precursor cells. Additionally, it was suggested that DEA acts by altering intracellular choline availability. Effect on Cell Proliferation and Apoptosis in the Liver To determine whether repeated dermal administration of DEA induced cell proliferation and/or apoptosis in the livers of mice, male and female B6C3F1/Crl mice were dosed dermally with 0 and 160 mg/kg bw DEA (99.6% purity) in 96% ethanol for 1 wk followed by a 3-wk recovery period, and male mice were exposed dermally to 0 or 160 mg/kg for 1, 4, or 13 wks. 28 A dose response relationship was examined with application of mg/kg DEA to male mice for 1 or 13 wks. After 1 wk of dosing, increased cell proliferation in the liver was observed in males and females; this effect was reversible. Repeated application of 10 mg/kg DEA to male B6C3F 1 mice caused increased liver cell proliferation. DEA had no effect on the number of apoptotic cells in the liver. Possible Mode of Action for Carcinogenic Effects A non-genotoxic mode of tumorigenic action is indicated, because DEA is not mutagenic or clastogenic. Choline deficiency has been shown to increase spontaneous carcinogenesis in rodents, and choline deficiency may promote liver tumor formation. 4 Since disposition data indicate that DEA is less readily absorbed across rat skin than mouse skin, resulting in lower blood and tissue levels of DEA in rats than in mice, it is suggested that, in rats, the levels of DEA that occur are not high enough to markedly alter choline homeostasis. If true, species differences observed in tumor susceptibility could be a function of the internal dose of DEA. Alternatively, species differences in tumor susceptibility may explain the increased incidence of hepatocarcinogenesis in B6C3F 1 mice compared to rats exposed to DEA.. Additionally, rats and mice are reported to be much more susceptible to choline deficiency than humans. 56 As further support for this mode of action, a species-selective inhibition of gap junctional intercellular communication by DEA in mouse and rat, but not human, hepatocytes with medium containing reduced choline concentrations provided additional support that the mechanism for DEA-induced carcinogenicity involves cellular choline deficiency. 57 Also, Bachman et al. have hypothesized the DEA-induced choline deficiency leads to altered DNA methylation patterns, which facilitates tumorigenesis. 58 Two other hypotheses as to the mode of action of DEA carcinogenicity as possible alternatives to the intracellular choline deficiency hypothesis have been proposed. 52 One involves the nitrosation of DEA to NDELA and the other the formation of DEA-containing phospholipids. The researcher did not find these likely for the following reasons. Regarding the first alternate hypothesis, nitrosation to NDELA; NDELA was not detected in mouse plasma or urine after cotreatment of 16

57 mice with DEA and nitrite. In regards to the second, altered phospholipids; while DEA has been shown to be incorporated into phospholipids, without qualitative or quantitative differences between rats and mice, carcinogenic effects are seen only in mice, making it unlikely that incorporation of DEA into the phospholipids is a major determinant of carcinogenic response. Leung et al reviewed the information available and also felt that choline deficiency is the mechanism responsible for liver tumor promotion in mice. 59 The localization of β-cartenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F1 mice exposed dermally to mg/kg bw DEA for 2 yrs were characterized, and genetic alterations in the Catnb and H-ras genes were evaluated. 60 A lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas led the researchers to suggest that the signal transduction pathway is not involved in the development of liver tumors following DEA administration. RISK ASSESSMENT The Occupational Safety and Health Administration time-weight average (TWA) for DEA is 3 ppm, and the American Conference of Industrial Hygienists threshold limit value-twa is 0.46 ppm or 2 mg/m According to a review of DEA by the International Agency for Research on Cancer (IARC) Working Group, there is inadequate evidence in humans, for the carcinogenicity of DEA. 61 There is limited evidence in experimental animals for the carcinogenicity of DEA. The overall evaluation of the IARC is that DEA is not classifiable as to its carcinogenicity to humans (Group 3). 17

58 TABLES Table 1. Definition and Structure Ingredient CAS No. Definition Synonyms Function in Cosmetics Diethanolamine ph adjuster The product of the ammonolysis of 2 stoichiometric equivalents of ethylene oxide N,N-diethanolamine 2,2 -dihydroxydiethylamine 2,2 -iminobisethanol Formula/Structure HO H N OH Table 2. Physical and Chemical Properties Property Value Reference Physical Form clear viscous liquid white crystalline solid at room temperature viscous liquid above 28 C Color colorless Odor ammoniacal Molecular Weight /Specific Gravity 20 C Vapor Pressure 0.01 mm Hg ( C not specified) Melting Point 28.0 C Boiling Point C Water Solubility soluble Other Solubility soluble in alcohol, ethanol, and benzene log K ow 25 C Disassociation Constant pka 25 C Table 3. Current and historical frequency and concentration of use according to duration and type of exposure # of Uses Conc. of Use (%) data year Totals Duration of Use Leave-On Rinse Off Exposure Type Eye Area NR NR NR NR Possible Ingestion NR NR NR NR Inhalation NR 3 NR NR Dermal Contact Deodorant (underarm) NR NR NR NR Hair - Non-Coloring Hair-Coloring NR Nail 1 NR NR NR Mucous Membrane NR Bath Products 4 NR NR NR Baby Products NR NR NR NR NR - no reported use 18

59 Table 4. Percutaneous penetration study details (Study 1) Shampoo A Shampoo B Hair Dye C Hair Dye D Body Lotion E Body Lotion F [ 14 C]DEA (µci) free DEA (%) Cocamide DEA (%) x x Lauramide DEA (%) x x Oleamide DEA (%) x TEA (%) product dose (mg/cm 2 ) DEA dose (µg)/cm Application Time 5 min 5 min 30 min 30 min 24 h 24 h Monitoring Time (h) 24 h 24 h 24 h 24 h 24 h; 72 h 24 h amount absorbed (in receptor fluid; % of applied dose) total penetration (% of applied dose) total found in skin (% of applied dose) found in epidermis and dermis (% of dose) number of samples x indicates that ingredient was part of the formulation Table 5. Percutaneous penetration study details (Study 2) Shampoos A/B Shampoo C/D Bubble Bath E Moisturizer F Semi-Permanent Oxidative Hairdye G Hairdye H DEA (%) Cocamide DEA (%) Lauramide DEA (%) TEA (%) Dose Applied 100 µl/cm µl/cm µl/cm 2 5 mg/cm mg/cm mg/cm 2 Dilution 1:10 aq. 1:10 aq. 1: :1 w/h 2 O 2 Application Time 10 min 10 min 30 min 48 h 30 min 30 min Monitoring Time (h) penetration (% of dose) found in skin (% of dose) 24 h (B) or 48 h (A) A: 0.019% B: 0.025% A: 0.063% B: 0.078% 24 h 24 h 48 h 24 h 24 h C: 0.034% D: 0.011% C 0.037% D: 0.04% 0.508% 1.923% found in dermis (% of dose) number of samples cocamide DEA contained 11.0% free DEA lauramide DEA contained 2.79% free DEA TEA contained 0.28% free DEA fresh skin: 0.605% frozen skin: 0.456% fresh skin: % frozen skin: % fresh skin: 0.877% frozen skin: 0.534% fresh = 13 frozen =

60 REFERENCES 1. Elder RE (ed). Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. J Am Coll Toxicol. 1983;2:(7). 2. Dow Chemical Company. The alkanolamines handbook Midland, MI: The Dow Chemical Company.Secondary reference in Knaak et al Dow Chemical Company. Sales Specification: Diethanolamine Secondary reference in IARC Lehman-McKeeman LD and Gamsky EA. Choline supplementation inhibits diethanolamine-induced morphological transfomation in Syrian hamster embryo cells: Evidence for a carcinogenic mechanism. Toxicol Sci. 2000;55: Gottschalck T.E. and Bailey, J. E. eds. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal Care Products Council, Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Washington, DC: FDA.Updated May Personal Care Products Council. Concentration of use data - submitted by industry in response to a Council survey Unpulblished data submitted by the Council. 8. Bailey J. DEA in consumer products is safe. FASEB J. 2007;21:(1): James AC, Stahlhofen W, Rudolf G, and et al. Annex D. Deposition of inhaled particles. Annals of the ICRP. 1994;24:(1-3): Oberdorster G, Oberdorster E, and Oberdorster J. An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 2005;113:(7): Bowers D. Unpublished information on hair spray particle sizes provided at the September 9, 1999 CIR Expert Panel meeting Johnson MA. The influence of particle size. Spray Technology and Marketing. 2004;Nov European Commission. Cosing Database. Annex II. List of substances which must not form part of the composition of cosmetic products. Secondary alkyl- and alkanolamines and their salts, including diethanolamine Heatlh Canada.Cosmetic Ingredient Hotlist Accessed Food and Drug Administration.Everything Added to Food in the United States (EAFUS) Accessed Sun JD, Beskitt JL, Tallant MJ, and Frantz SW. In vitro skin penetration of monoethanolamine and diethanolamine using excised skin from rats, mice, rabbits, and humans. J Toxicol -- Cut & Ocular Toxicol. 1996;15:(2): Mathews JM, Garner CE, and Matthews HB. Metabolism, bioaccumulation, and incorporation of diethanolamine into phospholipids. Chem Res Toxicol. 1995;8:(5): National Toxicology Program. Toxicology and carcinogenesis studies of diethanolamine (CAS No ) in F344/N rats and B6C3F 1 mice. (Dermal studies.) NTP TR Waechter JM, Bormett GA, and Stewart HS. Diethanolamine: pharmacokinetics in Sprague-Dawley rats following dermal or intravenous administration Secondary reference in Knaak et al Mathews JM, Garner CE, Black SL, and Matthews HB. Diethanolamine absorption, metabolism and disposition in rat and mouse following oral, intravenous and dermal administration. Xenobiotica. 1997;27:(7):

61 21. Craciunescu CN, Niculescu MD, Guo Z, Johnson AR, Fischer L, and Zeisel SH. Dose response effects of dermally applied diethanolamine on neurogenesis in fetal mouse hippocampus and potential exposure of humans. Toxicol Sci. 2009;107:(1): Mathews JM and Jeffcoat AR. Absorption and dispositoin of diethanolamine (DEA) in rats and mice after oral, dermal, and intravenous administration. RTI/3662/00-12P Secondary reference in Knakk et al Mendrala AL, Waechter JM, Bormett GA, Bartels MJ, and Stott WT. The pharmacokinetics of diethanolamine in Sprague- Dawley rats following intravenous administration. Food Chem Toxicol. 2001;39: Yourick JJ, Marks A, Lockhead RY, and Bronaugh RL. Diethanolamine (DEA) in vitro dermal absorption in Fuzzy rat skin. Unpublished manuscript. Secndary reference in OECD Kraeling MEK, Yourick JJ, and Bronaugh RL. In vitro human skin penetration of diethanolamine. Food Chem Toxicol. 2004;42: Brain KR, Walter KA, Green DM, Brain S, Loretz LJ, Sharma RK, and Dressler WE. Percutaneous penetration of diethanolamine through human skin in vitro: Application from cosmetic vehicles. Food Chem Toxicol. 2005;43: National Toxicology Program. Toxicity studies of diethanolamine (CAS NO ) administered topically and in drinking water to F344/N rats and B6C3F1 mice. Tech. Rep. Series No Seconary reference in OECD Mellert W, Kaufmann W, Rossbacher R, and van Ravenzwaay B. Investigations on cell proliferation in B6C3F 1 mouse liver by diethanolamine. Food Chem Toxicol. 2004;42: Melnick RL, Mahler J, Bucher JR, Hejtmancik M, Singer A, and Persing RL. Toxicity of diethanolamine. 2. Drinking water and topical application exposures in B6C3F 1 mice. J Appl Toxicol. 1994;14:(1): Melnick RL, Mahler J, Bucher JR, Thompson M, Hejtmancik M, Ryan MJ, and Mezza LE. Toxicity of diethanolamine. 1. Drinking waer and topical application exposures in F344 rats. J Appl Toxicol. 1994;14:(1): Environmental Health Research and Testing. Screening of priority chemicals for reproductive hazards. Monoethanolamin, diethanolamine, and triethanolamine, ETOX PB Secondary reference in OECD Gulf South Research Institiute. Subchronic test of diethanolamine (C55174) in B6C3F1 mice and Fischer 344 rats Secondary reference in OECD Eastman Kodak Company. Health and safety studies for diethanolamine, with cover letter. TSCATS, OTC , Doc ID Secondary reference in OECD Gamer AO, Rossbacher R, Kaufmann W, and van Ravenzwaay B. The inhalation toxicity of di- and triethanolamine upon repeated exposure. Food Chem Toxicol. 2008;46: Lee ID, Lee GS, Moon HJ, Seok JH, Yang JY, Chae SY, Lee YW, Kim SM, Kim SH, and Jeong SY. A study of the reproductive and developmental toxicity of diethanolamine. (Unofficial translation.). The Annual Report of KFDA : EHQ A; Submission by Dow Chemical Co. 36. Marty MS, Neeper-Bradely TL, Neptun DA, and Carney EW. Developmental toxicity of diethanolamine applied cutaneously to CD rats and New Zealand White rabbits. REg Toxicol Pharmacol. 1999;30: Gulati DK, Grimes LK, Heindel J, and Schwetz BA. Range finding studies: Developmental toxicity of diethanolamine when administered via gavage in CD Sprague-Dawley rats, NTP 89-RF/DT Secondary reference in Marty et al Center for Life Science and Technology. Final study report. Developmetnal toxicity screen for diethanolamine (CAS No ) administered by gavage to Sprgue-Dawley (CD) rats on gestational days 6 through 19: Evaluaton of dams and pups through postnatal day Prepared for the National Toxicology Program; NTP Study No. TER BASF AG. Range-finding study on the prenatial inhalation toxicity of diethanolamin in pregnant rats. BASF Project No 11R0233/ Secondary reference in OECD

62 40. Gamer AO, Hellwig J, and Hildebrand B. Study of the prenatal toxicity of diethanolamin n rats after inhalation, Project No. 31R0233/90010; BASF Secondary reference in Marty et al BASF AG. Study of the prentatl inhalation toxicit of diethanolamine in rats after inhalation. BASF Project No. 31R0233/ Secondary reference in OECD Tennant RW, French JE, and Spalding JW. Identifying chemical carcinogens and assessing potential risk in short-term bioassays using transgenic mouse models. Environ Health Perspect. 1995;103: Spalding JW, French J, Stasiewicz S, Furedi-Machacek M, Conner F, Tice RR, and Tennant RW. Responses of transgenic mouse lines p53 +/- and Tg AC to agents tested in conventional carcinogenicity bioassays. Toxicol Sci. 2000;53: Kimber I, Basketter DA, Butler M, Gamer A, Garrigue JL, Gerberick GR, Newsome C, Steiling W, and Vohr HW. Classification of contact allergens to potency. Proposals, Food Chemical Toxciol : Secondary reference in OECD Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine. Data analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60: RCC. Contact hypersensitivity to Diethanolamin, rein in albino Guinea pigs, Maximization test, RCC Project Unpublished report Secondary reference in OECD CIT. Skin sensitization test in guinea pigs on FORAFAC (Maximization method of Magnusson and Kligman.) Laboratory study umber TSG EHQ Stott WT, Bartels MJ, Brzak KA, Mar M-H, Markham DA, Thorton CM, and Zeisel SH. Potential mechanisms of tumorigenic action of diethanolamine in mice. Toxicol Lett. 2000;114: Saghir SA, Brzak A, Markham DA, Bartels MJ, and Stott WT. Investigation of the formation of N-nitrosodiethanolamine in B6C3F1 mice following topical administration of triethanolamine. REg Toxicol Pharmacol. 2005;43: Munson AE, White KL, and McCay JA. Immunotoxicity of diethanolamine in female Fischer 344 rats. Report to the National Toxicology Program Secondary reference in OECD Lehman-McKeeman LD, Gamsky EA, Hicks SM, Vassalo JD, Mar M-H, and Zeisel SH. Diethanolamine induces hepatic choline deficiency in mice. Toxicol Sci. 2002;67: Lehman-McKeeman LD. Incorporating mechanistic data into risk assessment. Toxicology. 2002; : Kamendulis LM and Klaunig JE. Species differences in the induction of hepatocellular DNA synthesis by diethanolamine. Toxicol Sci. 2005;87:(2): Craciunescu CN, Wu R, and Zeisel SH. Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus. FASEB J. 2006;20: Niculescu MD, Wu R, Guo Z, da Costa KA, and Zeisel SH. Diethanolamine alters proliferation and choline metabolism in mouse neural precursor cells. Toxicol Sci. 2007;96:(2): Zeisel SH and Blusztajn JK. Choline and human nutrition. Annu Rev Nutr. 1994;14: Kamendulis LM, Smith DJ, and Klaunig JE. Species differences in the inhibition of gap junctional intercellular communication (GJIC) by diethanolamine. Toxicologist :(S-1): 58. Bachman AN, Kamednulis LM, and Goodman JI. Diethanolamine and phenobarbital produce an altered pattern of methylation in GC-rich regions of DNA in B6C3F1 mouse hepatocytes similar to that resulting from choline deficiency. Toxicol Sci. 2006;90:(2): Leung H-W, Kamendulis LM, and Stott WT. Review of carcinogenic activity of diethanolamine and evidence of choline deficiency as a plausible mode of action. REg Toxicol Pharmacol. 2005;43: Hayashi S-M, Ton VT, Hong H-HL, Irwin RD, Haseman JK, Devereux TR, and Sills RC. Genetic alterations in the Catnb gene but not the H-ras gene in hepatocellular neoplasms and hepatoblastomas of B6C3Fa mice following exposure to diethanolamine for 2 years. Chemico-Biological Interactions : Secondary reference in OECD

63 61. International Agency for Research on Cancer (IARC). Diethanolamine. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans. 2000;77: Knaak JB, Leung H-W, Stott WT, Busch J, and Bilsky J. Toxicology of mono-, di-, and triethanolamine. Review of Environmental Contamination and Toxicology. 1997;149:

64 Re-Review of Ethanolamine Introduction... 1 Chemistry... 1 Use... 1 Cosmetic... 1 Non-Cosmetic... 1 Toxicokinetics... 2 Absorption, Distribution, Metabolism and Excretion... 2 In-Vitro... 2 Dermal... 2 Non-Human... 2 Other... 3 Non-Human... 3 Toxicological Studies... 3 Acute (Single Dose) Toxicity... 3 Dermal... 3 Oral... 3 Inhalation... 3 Other... 3 Repeated Dose Toxicity... 3 Dermal... 3 Oral... 4 Inhalation... 4 Reproductive and developmental studies... 4 Dermal... 4 Oral... 4 Genotoxicity... 5 In Vitro... 5 Irritation and Sensitization... 5 Irritation... 5 Skin... 5 Mucosal... 5 Sensitization... 6 Provocative Testing... 6 Clinical Use... 6 Tables... 7 Table 1. Definition and Structure... 7 Table 2. Physical and Chemical Properties of MEA... 7 Table 3. Current and historical frequency and concentration of use of MEA according to duration and type of exposure.. 7 References... 8

65 INTRODUCTION Ethanolamine (MEA), an ingredient that functions in cosmetics as a ph adjuster, has previously been reviewed by the CIR Expert Panel. At that time, the INCI name for the ingredient was monoethanolamine. In 1983, the Expert Panel concluded that MEA is safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin, and MEA should only be used in rinse-off products. In that review, it was shown that MEA is a skin and eye irritant in animals, and clinical studies with formulations containing MEA indicated that it is a human skin irritant. Also, there was indication that the longer MEA was in contact with the skin, the greater the likelihood of irritation. This current document contains summaries of data obtained from a search of published literature, as well as summary information from the original safety assessment CHEMISTRY MEA is an amino alcohol. The structure and synonyms of MEA are given in Table 1, and chemical and physical properties are given in Table 2. MEA is produced commercially by aminating ethylene oxide with ammonia. The replacement of one hydrogen of ammonia with an ethanol group produces MEA. MEA typically contains a small amount of diethanolamine (DEA). MEA is reactive and bifunctional, combining the properties of alcohols and amines. At temperatures of C, MEA will react with fatty acids to form ethanolamides. Additionally, the reaction of ethanolamines and sulfuric acid produces sulfates, and, under anhydrous conditions, MEA may react with carbon dioxide to form carbamates. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 USE Cosmetic MEA functions in cosmetics as a ph adjuster. 2 In 1981, data provided through to the Food and Drug Administration (FDA) Voluntary Cosmetic Registration Program (VCRP) indicated that MEA was used in 51 formulations. 1 All but 2 of those products were rinse-off formulations, and 28 uses were in hair coloring products. Products contained MEA at concentrations 10%. VCRP data obtained in 2010 indicate that the use of MEA has increased significantly, as it is now reported to be used in 795 formulations. All of the uses, except 1, are rinse-off products, and 782 uses are in hair coloring formulations. 3 According to data submitted by industry in response to a recent survey conducted by the Personal Care Products Council (Council), MEA is used at concentrations of %. 4 All uses reported are in rinse-off products, with the exception of 3% in an aerosol hair color spray. The complete current and historical use data for MEA are shown in Table 3. According to data obtained from Health Canada, MEA is used at up to 10%. 5 Most of the uses are rinse-off, but some leave-on type products are reported. For example MEA is reported to be included at 3-10% in an eye make-up. MEA is listed by the European Commission in Annex III Part 1: the list of substances which cosmetic products must not contain, except subject to the restrictions and conditions laid down. 6 Monoalkylamines, monoalkanolamines, and their salts, including MEA, are allowed a maximum secondary amine content of 0.5% in finished product. Other limitations include: do not use with nitrosating agents; minimum purity of 99%; maximum secondary amine content of 0.5% (for raw materials); maximum nitrosamine content of 50 µg/kg; and must be kept in nitrite-free containers. Non-Cosmetic MEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes, mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate for resins, plasticizers, and rubber chemicals. It is also used as a lubricant in the textile industry, as a humectant and softening agent for hides, as an alkalizing agent and surfactant in pharmaceuticals, as an absorbent for acid gases, and in organic syntheses. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 1

66 MEA has uses as an indirect food additive; according to 21 CFR MEA is allowed for use as a component of adhexives. 7 MEA is also used as a rust inhibitor in water-based metalworking fluids. 8 TOXICOKINETICS MEA is the only naturally occurring ethanolamine in mammals, and it is excreted in the urine. MEA was converted to phosphatidylethanolamine (PE) in the liver, blood, and brain of female rats that were dosed intraperitoneally (i.p.) with radiolabeled MEA. The step-wise methylation of PE that converts it to phosphatidylcholine (PC) did not occur in the brain. Labeled respiratory carbon dioxide has been found in rats after i.p. administration of labeled MEA. A coenzyme-b 12 -dependent ethanolamine deaminase mediated the conversion of MEA to acetaldehyle and ammonia was demonstrated. Intraperitoneal administration of MEA to rats increased blood urea and blood glutamine, and the researchers suggested that ethanolamine was an ammonia source. Additional researchers, upon detection of labeled acetate in the urine of rats fed labeled MEA, suggested that MEA is phosphorylated by ATP in vivo, converted to acetaldehyde, ammonia, and inorganic phosphate, and the acetaldehyde is oxidized to acetate. The researchers further hypothesized that the removal of phosphorylated MEA by its conversion to acetate may exert a regulatory effect on PE biosynthesis. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Ethanolamine. 1 Absorption, Distribution, Metabolism and Excretion In-Vitro The dermal penetration of [1,2-14 C]ethanolamine HCl was evaluated in vitro using split-thickness skin from weanling Yorkshire pigs. 9 An ethanolic solution was prepared so that a 5 µl application to 0.8 cm 2 of skin gave a chemical dose of 4 µg/cm 2 and a radioactive dose of approximately 0.05 µci. After 50 h, 11% of the dose was lost to evaporation, 5% penetrated percutaneously, and 62% was recovered in the skin residue. Seven to nine times more radioactivity was recovered in the upper 100 µm layer, compared to recovery from the remaining dermis. Three full thickness skin preparations from CD rats, CD-1 mice, and New Zealand White rabbits, and 6 from female mammoplasty patients were used to compare the dermal penetration of MEA through skin of different species. 10 [ 14 C]MEA (98.8% purity; specific activity [sp. act.] 15.0 mci/mmol) was applied to the skin sample undiluted or as an aqueous (aq.) solution at a dose of 4 mg/cm 2. Dose volumes of 7 µl undiluted [ 14 C]MEA or 32 µl of the aq. solution (22% w/w) were applied to the exposed surface of the skin (1.77 cm 2 ). These volumes maintained an infinite dose during the 6 h exposure period. With undiluted MEA, the cumulative dose absorbed was 5.98, 16.92, 8.66, and 0.61% through rat, mouse, rabbit, and human skin, respectively. With aq. MEA, 1.32, 24.79, 1.87, and 1.11%, was the cumulative dose absorbed through rat, mouse, rabbit, and human skin, respectively. In vitro absorption of undiluted and aq. MEA was much greater through mouse skin than human skin, and human skin was the least permeable of all the skin samples. With human skin samples, the cumulative dose absorbed was greater with aq. MEA as compared to undiluted MEA. The researchers hypothesized that enhanced penetration of aq. MEA may be attributable to elevated skin hydration. Dermal Non-Human The distribution and metabolism of MEA was determined using groups of 5 male athymic nude mice with human skin grafts and ungrafted athymic nude mice. 9 [1,2-14 C]Ethanolamine HCl in ethanol was applied at a dose of 4.0 µg (3.6 µci) to a 1.45 cm 2 area of grafted or non-grafted skin. Penetration appeared similar for both groups. Radioactivity in expired carbon dioxide (CO 2 ) appeared 30 min after dosing, and the amount recovered at 30 min was % of the dose for grafted and ungrafted skin, respectively. After 24 h, % of the dose was in expired CO 2. Distribution was also similar for both groups. At 24 h after dosing, % of the dose was recovered in the liver and % in the kidneys. At the application site, 18.4% of the radioactivity was recovered in the grafted skin, and 12.1% was recovered in the ungrafted skin. The amount of radioactivity recovered in the urine was 4.6 and 5.2% for grafted and ungrafted nude mice, respectively. 2

67 Unchanged MEA comprised 10.2% of the urinary radioactivity. The major urinary metabolites were urea and glycine, comprising 39.9 and 20.4% of the urinary radioactivity. Minor urinary metabolites were serine, uric acid, choline, and unidentified ninhydrin-positive compounds. The radioactivity in proteins and amino acids isolated from the liver, human skin grafts, and mouse skin was determined as an evaluation of the metabolism of MEA. The researchers stated that the appearance of 14 C in skin and hepatic amino acids and proteins, and the incorporation of MEA into phospholipids, was evidence of extensive metabolism of the absorbed MEA. The liver was the most active site of MEA metabolism. Other Non-Human Five male athymic nude mice were dosed intraperitoneally (i.p.) with 4.0 µg (3.6 µci) of [1,2-14C]ethanolamine HCl in ethanol, and the distribution and metabolism were examined following dosing. 7 Radioactivity was detected in expired CO 2, increasing gradually over time. After 24 h, 18% of the radioactivity was detected in expired air. TOXICOLOGICAL STUDIES Acute (Single Dose) Toxicity Dermal The acute dermal toxicity of a mixture containing MEA (as well as hydroxylamine, diglycolamine, propylene glycol, catechol, and water; percentages not specified) was determined by applying 2g/kg of the mixture to the shaved back of 5 male and 5 female rabbits. 11 The dermal LD 50 of the mixture was >2 g/kg. In a similar study with a formulation containing MEA (and hydroxylamine, diglycolamine, gallic acid, and water; percentages not specified), the LD 50 of the mixture in rabbits was 1.24 g/kg bw. 12 Oral The acute oral toxicity of MEA, concentration not specified, ranged from g/kg for rats. Using rats, 20% aq. MEA had an LD 50 of g/kg, based on results of studies performed over a 10 yr time period. With groups of 10 rats, a hair preparation containing 5.9% MEA had an LD 50 of 14.1 g/kg when diluted and 12.9 ml/kg when undiluted. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 The acute oral toxicity of a mixture containing MEA (as well as hydroxylamine, diglycolamine, propylene glycol, catechol, glycolic acid and water; percentages not specified) was determined using groups of 5 male and 5 female rats. 13 The oral LD 50 of the mixture was 0.95 g/kg. In a similar study with a formulation containing MEA (and hydroxylamine, diglycolamine, gallic acid, and water; percent in formulation not specified), the oral LD 50 of the mixture was g/kg bw. Inhalation The acute inhalation toxicity of a mixture containing MEA (as well as hydroxylamine, 1-amino-propano-2-ol, catechol, and water; percent of each not specified) was determined using groups of 5 male and 5 female rats. The inhalation LC 50 of the mixture was estimated to be 2.48 mg/l. 13 Other The intraperitoneal (i.p.) LD 50 of MEA was 1.05 g/kg for mice. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Dermal Repeated Dose Toxicity Percutaneous application of 4 mg/kg/day MEA to rats resulted in nonspecific histological changes in the heart and lung, fatty degeneration of the liver parenchyma, and subsequent focal liver necrosis. The duration of dosing was not specified. 3

68 Oral From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Groups of 10 rats were fed g/kg/day MEA for 90 days. Heavy livers and kidneys were observed at 0.64 g/kg/day, and deaths and major pathology occurred with 1.25 g/kg/day. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Inhalation The dominant effects of continuous exposure of dogs, guinea pigs, and rats to 5-6 ppm MEA vapor were skin irritation and lethargy. No dogs or rodents exposed to ppm MEA vapor for 90 days died, but some dogs exposed to 102 MEA vapor for 2 days and some rodents exposed to ppm MEA vapor for days did die. Dogs and rodents exposed to ppm MEA vapor had behavioral changes, pulmonary and hepatic inflammation, hepatic and renal damage, and hematological changes. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 REPRODUCTIVE AND DEVELOPMENTAL STUDIES Dermal The developmental toxicity of dermally-applied aq. MEA was evaluated using groups of gravid Sprague- Dawley rats and 15 gravid New Zealand White rabbits. 14 MEA was applied at doses of 0, 10, 25, 75, and 225 mg/kg bw/day to the backs of rats on days 6-15 of gestation, with a dose volume of 1 ml/kg. Significant skin irritation, consisting of erythema followed by necrosis, scabs, and scar formation, occurred with 225 mg/kg MEA, but not at the other dose levels. No effects on kidney or liver weights were reported. Despite maternal effects in the 225 mg/kg dose group, no effects on reproductive parameters were observed at any dose, and there were no treatment-related increases in the visceral or skeletal malformations. The no-observed effect levels (NOELs) for maternal and embryonal/fetal toxicity in rats were 75 and 225 mg/kg/day, respectively. In rabbits, 0, 10, 25, and 75 mg/kg bw/day MEA was applied to the back (2 ml/kg). Severe skin irritation at the application site, consisting of necrosis, exfoliation, and crusting, was observed in the 75 mg/kg group, and crusting, transient erythema, and edema were observed in a few rabbits of the 25 mg/kg dose group. No effects on kidney or liver weights were reported. As with the rats, no effects on reproductive parameters were observed at any dose, and there were no treatmentrelated increases in the visceral or skeletal malformations. The NOEL for both maternal and embryonal/fetal toxicity in rabbits was 75 mg/kg/day. Oral No reproductive or developmental effects were observed in groups of gravid rats given a hair dye and base containing 22% MEA in the diet at concentrations 7800 ppm on days 6-15 of gestation. No differences in reproductive or developmental parameters were observed when male rats were fed treated diet for 8 wks prior to and during mating with undosed females or when female rats were fed treated feed for 8 wks prior to dosing until day 21 of lactation. These females were mated with undosed males. Additionally, no teratologic effects were induced by dosing gravid rabbits with 19.5 mg/kg/day of the hair dye and base by gavage on days 6-18 of gestation. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Ethanolamine. 1 Groups of 10 gravid Long-Evans rats were dosed by gavage with mg/kg aq. MEA on days 6-15 of gestation, and a negative control group of 34 gravid rats were dosed with water only. 15 The animals were killed and examined on day 20 of gestation. Significant maternal toxicity was not noted. Embryolethality was significantly increased in the 500 mg/kg group, with male pups affected more than female pups. Male pups were more severely affected than female pups at all dose levels in regards to intrauterine growth retardation and increased gross structural anomalies that were considered indicative of depressed fetal growth. However, pups of either sex who were contiguous to male siblings were more adversely affected that those contiguous to one or more female siblings. Male pups contiguous to male siblings (mmm) were, apparently, resorbed 4

69 (mrm). The number of malformed pups per dam was significantly increased in animals dosed with 300 mg/kg MEA. The number of malformed pups, when evaluated as a percent of the litter affects, was significantly increased for all 3 dose groups. The teratogenic potential of MEA was evaluated in a Chernoff-Kavlock postnatal mouse screening assay. 16 Gravid CD-1 mice were dosed orally with 850 mg/kg/day MEA on days 6-15 of gestation, resulting in 16% mortality of maternal mice and reduced numbers of viable litters. Litter size, percentage survival of pups, birth weight, and pup weight gains were not affected. A preliminary study was performed in which gravid Wistar rats were dosed orally with mg/kg by/day aq. MEA; details not provided. 17 Maternal effects were observed in the high dose animals, but no embryonal/fetal effects were observed at any dose. Based on these results, groups of 40 Wistar rats were dosed by gavage, with 0, 40, 120, or 450 mg/kg bw/day aq. MEA on days 6-15 of gestation. On day 20 of gestation, 25 dams/group were killed and necropsied, while the remaining 15 were allowed to litter, and then killed on day 21 of lactation. Despite evidence of maternal toxicity in the 450 mg/kg group, as indicated by statistically significant decreases in maternal body weights and feed consumption, no effects on reproductive parameters, incidences of visceral or skeletal malformations, or postnatal growth were observed at any dose The NOELs for maternal and developmental toxicity were 120 and 450 mg/kg/day, respectively. GENOTOXICITY In Vitro MEA, with and without metabolic activation using liver preparations from rats induced with a polychlorinated biphenyl mixture, was not mutagenic to Salmonella typhimurium TA100 or TA1535. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 MEA was negative in most Ames tests; 18 however weak mutagenic effects were reported in one study. 19,20 MEA, µg/ml, was negative in a cell transformation assay using hamster embryo cells. 21 MEA was clearly cytotoxic at 500 µg/ml. MEA did not produce chromosomal aberrations in rat liver cells, 18 but a weak positive response was reported in human lymphocytes. 19 IRRITATION AND SENSITIZATION Irritation Skin Non-Human MEA was corrosive to rabbit skin with single semi-occlusive patches of 30, 85 and 100% on intact and abraded skin. When applied to rabbit ears using non-occlusive applications and to shaved skin of the abdomen under semi-occlusive patches, 10% was corrosive, >1% was extremely irritating, and 1% was irritating.. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Human In a study in which a formulation containing 5.9% MEA was applied to the backs of 12 female subjects for 23 h/day for 21 days, the formulation was considered an experimental cumulative irritant. Results observed during the induction phase of a patch test of 165 subjects using a formulation containing 11.47% MEA were interpreted by the Expert Panel as irritation. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Mucosal Non-Human Using 6 rabbits, 30% aq. MEA was moderately irritating to rabbit eyes. Undiluted MEA, instilled as a volume of ml, produced severe injury to rabbit eyes. A hair preparation containing 5.96% MEA had a maximum avg. irritation score of 0.7/110 for rinsed and unrinsed eyes. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 5

70 Sensitization In Vitro A local lymph node assay was performed to evaluate the sensitization potential of MEA (as the hydrochloride salt). 8,22 Groups of 6 female CBA/Ca mice were treated on the ear with 10, 40, or 70% w/w aq. MEA HCl. MEA HCl did not have a skin sensitizing effect. Non-Human In a maximization study using 15 Dunkin-Hartley guinea pigs, intradermal and epicutaneous inductions with 0.6% and 10.3% aq. MEA, respectively, were performed after 10% sodium lauryl sulfate pre-treatment. 8 (MEA contained less than 0.1% DEA and TEA.) At challenge with 0.41, 2.05, or 4.1% MEA, 3, 2, and 3 animals, respectively, reacted positively after 3 days. Two of the 15 test animals reacted to the vehicle, but none of the control animals reacted to MEA or the vehicle. Possible cross-reactions to 5% TEA occurred in 3 animals, and to 7% DEA in 2 animals. In a second test using the same protocol, no animals reacted to 0.41% MEA, 2 reacted to 2.05% MEA, and 1 reacted to 4.1% MEA. Additionally, 1 animal reacted to 10% TEA and 2 reacted to 7% DEA. The control animals did not react to any of the ethanolamines. Human A formulation containing 5.9% MEA, tested undiluted, and one containing 11.47% MEA, tested at 5% in 25% alcohol, were not sensitizing in clinical studies. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Provocative Testing A patch test using 2% MEA in petrolatum (pet.) was performed on 370 patients with suspected dermatitis to MEAcontaining metal-working fluids and on 452 control subjects. 23 A 10-fold higher reaction was seen in the patient group, with 12.2% of the patients having positive reactions, as compared to 1.3% in the control group. Over a 3-yr period, 11/595 (1.9%) hairdresser clients and 7/401 (1.8%) female hairdressers reacted positively to 2% MEA pet. 8 In 22 patients with suspected intolerance to oxidative hair dye components, 4 had a positive reaction to 2% MEA pet. Over a 15-yr period, provocative patch testing using MEA was performed on 9602 subjects. There were 363 (3.8%) positive reactions to MEA, and most of the reactions (277; 2.9%) were weak positives. There were 55 (0.6%) irritant reactions reported. Cosensitization was reported; 38% of the patients that reacted to MEA also tested positive to DEA. Occupational sensitization was reported; of 5884 male patients, 2.9% that did not work in the metal industry had positive reactions as opposed to 7.0 and 15.2% of those working in the metal industry and those exposed to water-based metalworking fluids, respectively. CLINICAL USE MEA inhalation by humans has been reported to cause immediate allergic responses of dyspnea and asthma and clinical symptoms of acute liver damage and chronic hepatitis. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 A case of occupational asthma in an industrial worker exposed to a detergent containing 8% ethanolamine was reported. 24 Since the detergent was used in hot water, released\ of MEA vapor was greater than if the detergent temperature was lower.. Exposure to vapors from MEA in cleaning solutions can irritate the nose, throat, and lungs. 25 The Occupational Safety and Health Administration Permissible Exposure Level for MEA is 3 ppm, and the American Conference of Governmental Industrial Hygienists 15 min short term exposure limit is 6 ppm. 6

71 TABLES Table 1. Definition and Structure Ingredient CAS No. Ethanolamine Definition Synonyms Function(s) in Cosmetics Formula/Structure The product of the ammonolysis of one stoichiometric equivalent of ethylene oxide 2-aminoethanol 2-hydroxyethylamine monoethanolamine ph adjuster HO NH 2 Table 2. Physical and Chemical Properties of MEA Property Value Reference Physical Form clear viscous liquid Color colorless Odor ammoniacal Molecular Weight Specific Gravity (25 C) Viscosity (25 C) Vapor Pressure 0.48 mm Hg (20 C) Melting Point (20 C) Boiling Point 171 C Water Solubility miscible with water Other Solubility miscible with methanol and acetone Table 3. Current and historical frequency and concentration of use of MEA according to duration and type of exposure # of Uses Conc. of Use (%) data year Totals Duration of Use Leave-On (coloring hair spray) Rinse Off Exposure Type Eye Area 1 NR NR Possible Ingestion NR 1 NR NR Inhalation NR NR NR 3 Dermal Contact Deodorant (underarm) NR NR NR NR Hair - Non-Coloring Hair-Coloring Nail NR NR NR NR Mucous Membrane 3 NR Bath Products NR NR NR NR Baby Products NR NR NR NR NR - no reported use 7

72 REFERENCES 1. Elder RE (ed). Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. J Am Coll Toxicol. 1983;2:(7). 2. Gottschalck T.E. and Bailey, J. E. eds. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal Care Products Council, Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Washington, DC: FDA. 4. Personal Care Products Council. Concentration of use data - submitted by industry in response to a Council survey Heatlh Canada.Cosmetic Ingredient Hotlist Accessed European Commission.European Commission Enterprise and Industry. Cosmetics - Cosing. Annex III/Part 1, 61 - Monoalkylamines, nomoalkanolamines and their salts Accessed Food and Drug Administration.Everything Added to Food in the United States (EAFUS) Accessed Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine. Data analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60: Klain GC, Reifenrath WG, and Black KE. Distribution and metabolism of topically applied ethanolamine. Fund Appl Toxicol. 1985;5:S127-S Sun JD, Beskitt JL, Tallant MJ, and Frantz SW. In vitro skin penetration of monoethanolamine and diethanolamine using excised skin from rats, mice, rabbits, and humans. J Toxicol -- Cut & Ocular Toxicol. 1996;15:(2): Anonymous. Letter to the Environmental Protection Agency reporting an actue dermal toxcity study of a formulation containing DEA Anonymous. Letter to the Environmental Protection Agency reporting actue dermal and oral toxcity studies of a formulation containing DEA Anonymous. Letter to the Environmental Protection Agency reporting an actue inhalaton toxcity studies of a formulation containing DEA Liberacki AB, Neeper-Bradley TL, Breslin WJ, and Zielke GJ. Evaluation of developmental toxicity of dermally applied monoethanolamine in rats and rabbits. Fund Appl Toxicol. 1996;31: Mankes RF. Studies on the embryopathic effects of ethanolamine in Long-Evans rats: Preferential embryopathy in pups contiguous with male siblings in utero. Teratog.Carcinog Mutagen. 1986;6: Pereira M, Barnwell P, and Bailes W. Screening of priority chemicals for reproductive hazards, monoethanolamine, diethanolamine,, triethanolamine. NIOSH Contract No Environmental Health Research and Testing, Inc. REport No. ETOX Hellwig J and Liberacki AB. Short communication. Evaluation of the pre-, peri-, and postnatal toxicity of monothanolamine in rats following repeated oral adminstration during organogenesis. Fund Appl Toxicol. 1997;40: Dean BJ, Brooks TM, Hodson-Walker G, and Hutson DH. Genetic toxicology testing of 41 industrial chemicals. Mutat Res. 1985;153: Arutyunyan RM, Zalinyan RM, Mugnetsyan EG, and Gukasyan LA. Mutagenic action of latex polumerization stabilizers in different test systems. Tsitol Genet : Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, and Zeiger E. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environ Mutagen. 1986;8:(Suppl 7):

73 21. Inoue K, Sunakawa T, Okamoto K, and Tanaka Y. Mutagenicity tests and in vitro transformation assays on triethanolamine. Mutat Res. 1982;101: BASF. Ethanolamine hydrochloride Murine Local Lymph Node Assay (LLNA) Geier J, Lessmann H, Schnuch A, and Uter W. Diagnostic quality of patch test preparation monoethanolamine 2% pet. Contact Derm. 2005;52: Savonius B, Keskinen H, Tupperainen M, and Kanerva L. Occupational asthma caused by ethanolamines. Alergy. 1994;49: Bello A, Quinn MM, Perry MJ, and Milton DK. Characterization of occupational exposures to cleaning products used for common cleaning tasks - A pilot study of hospital cleaners. Envron Health. 2009;8:11. 9

74 COSMETIC Ethanolamine, Personal Care Products Council Committed to Safety, Quality & Innovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDffiNT REVWW (CIR) John Bailey, Ph.D. Industry Liaison to the CW Expert Panel DATE: October 13, 2010 SUBJECT: Concentration of Use - Diethanolamine and Triethanolamine th Street, N.W., Suite 300 Washington, D.C (fax)

75 Concentration of Use by FDA Product Category Ethanolamine, Diethanolamine and Triethanolamine Ingredient Product Category Concentration of Use Ethanolamine Hair conditioners 4% Ethanolamine Hair straighteners 3% Ethanolamine Permanent waves 4-6% Ethanolamine Shampoos (noncolonng) 0.05% Ethanolamine Hair dyes and colors (all types requiring caution 3-13% statement and patch testing) Ethanolamine Hair color sprays (aerosol) 3% Ethanolamine Hair bleaches 4-18% Ethanolamine Bath soaps and detergents % Ethanolamine Shaving cream (aerosol, brushless and lather) % Ethanolamine Skin cleansing (cold creams, cleansing lotions, liquids % and_pads) Diethanolamine Shampoos (noncoloring) % Diethanolamine Tonics, dressings and other hair grooming aids 0.008% Diethanolamine Bath soaps and detergents 0.009% Diethanolamine Shaving cream (aerosol, brushless and lather) 0.06% Diethanolamine Moisturizing creams, lotions and powders 0.06% Triethanolamine Baby lotions, oils, powders and creams 0.2-2% Tnethanolamine Bubble baths % Triethanolamine Other bath preparations 1-2% Triethanolamine Eyebrow pencil 1-3% Triethanolamine Eyeliner 0.3-3% Tnethanolamine Eye shadow 0.4-3% Triethanolamine Eye lotion 0.2-3% Page 1 of 3

76 Triethanolamine Eye makeup remover % Triethanolamine Mascara 2-4% Triethanolamine Other eye makeup preparations 0.8-3% Triethanolamine Colognes and toilet waters % Tnethanolamine Perfumes % Triethanolamine Hair conditioners % Triethanolamine Hair sprays (aerosol fixatives) % Triethanolamine Hair straighteners 3% Triethanolamine Permanent waves 0.01% Triethanolamine Shampoos (noncoloring) % Triethanolamine Tonics, dressings and other hair grooming aids % Triethanolamine Other hair preparations (noncoloring) 0.5-3% Triethanolamine Hair dyes and colors (all types requiring caution 2-13% statement and patch testing) Triethanolamine Hair tints 1% Triethanolamine Hair rinses (coloring) 5% Triethanolamine Other hair coloring preparations 3% Triethanolamine Blushers (all types) % Triethanolamine Face powders 0.06% Triethanolamine Foundations 1-2% Triethanolamine Leg and body paints 6% Tnethanolamine Lipstick 0.2-1% Triethanolamine Makeup bases 1-3% Triethanolamine Other makeup preparations 0.7-2% Triethanolamine Basecoats and undercoats (manicuring preparations) 3% Tnethanolamine Cuticle softeners 0.5-2% Tnethanolamine Nail creams and lotions 0.2-1% Page 2 of 3

77 Triethanolamine Other manicuring preparations 0.5-2% Triethanolamine Bath soaps and detergents % Triethanolamine Deodorants (underarm) % Triethanolamine Feminine hygiene deodorants 0.1% Triethanolamine Other personal cleanliness products 0.2-3% Triethanolamine Aftershave lotions 0.5-2% Triethanolamine Beard softeners 0.7% Triethanolamine Preshave lotions (all types) 0.3% Triethanolamine Shaving cream (aerosol, brushless and lather) 3-10% Triethanolamine Other shaving preparations 0.3-6% Triethanolamine Skin cleansing (cold creams, cleansing lotions, liquids % and pads) Triethanolamine Depilatories 0.8% Triethanolamine Face and neck creams, lotions and powders 0.4-6% Triethanolamine Face and neck sprays % Triethanolamine Body and hand creams, lotions and powders 0.3-3% Triethanolamine Body and hand sprays % Tnethanolamine Foot powders and sprays 0.4-1% Triethanolamine Moisturizing creams, lotions and powders 0.2-2% Triethanolamine Night creams, lotions and powders 0.3-5% Triethanolamine Paste masks (mud packs) 1-3% Triethanolamine Skin fresheners 2% Triethanolamine Other skin care preparations 0.4-2%, 7% Triethanolamine Suntan gels, creams and liquids 2% Triethanolamine Indoor tanning preparations 0.3-2% 7% in a rinse-off skin care preparation Information collected in 2010 Table prepared October 13, 2010 Page 3 of 3

78 COSMETIC Personal Care Products Council Committed to Safety, Quality & Innovation Memorandum TO: FROM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (Cifi) John Bailey, Ph.D. Industry Liaison to the CJR Expert Panel DATE: November 8, 2010 SUBJECT: Updated Concentration of Use Triethanolamine th Street, N.W., Suite 30O Washington, D.C (fax)

79 Concentration of Use by FDA Product Category Ethanolamine, Diethanolamine and Triethanolamine Ingredient Product Category Concentration of Use Ethanolamine Hair conditioners 4% Ethanolamine Hair straighteners 3% Ethanolamine Permanent waves 4-6% Ethanolamine Shampoos (noncoloring) 0.05% Ethanolamine Hair dyes and colors (all types requiring caution statement and 3-13% patch testing) Ethanolamine Hair color sprays (aerosol) 3% Ethanolamine Hair bleaches 4-18% Ethanolamine Bath soaps and detergents % Ethanolamine Shaving cream (aerosol, brushless and lather) % Ethanolamine Skin cleansine (cold creams, cleansine lotions, liquids and cads) % Diethanolamine Shampoos (noncoloring) % Diethanolamine Tonics, dressings and other hair grooming aids 0.008% Diethanolamine Bath soaps and detergents 0.009% Diethanolamine Shaving cream (aerosol, brushless and lather) 0.06% Diethanolamine Moisturizine creams, lotions and nowders 0.06% Triethanolamine Baby lotions, oils, powders and creams 0.2-2% Triethanolamine Bubble baths % Triethanolamine Other bath preparations 1-2% Triethanolamine Eyebrow pencil 1-3% Triethanolamine Eyeliner 0.3-3% Triethanolamine Eye shadow 0.4-3% Triethanolamine Eye lotion 0.2-3% Triethanolamine Eye makeup remover % Triethanolamine Mascara 0.7-4% Triethanolamine Other eye makeup preparations 0.8-3% Triethanolamine Colognes and toilet waters % Page 1 of 3

80 Triethanolamine Perfumes % Triethanolamine Hair conditioners % Triethanolamine Hair sprays (aerosol fixatives) % Triethanolamine Hair straighteners 3% Triethanolamine Permanent waves 0.0 1% Triethanolamine Shampoos (noncoloring) % Triethanolamine Tonics, dressings and other hair grooming aids % Triethanolamine Other hair preparations (noncoloring) 0.5-3% Triethanolamine Hair dyes and colors (all types requiring caution statement and 2-13% patch testing) Triethanolamine Hair tints 1% Triethanolamine Hair rinses (coloring) 5% Triethanolamine Other hair coloring preparations 3% Triethanolamine Blushers (all types) % Triethanolamine Face powders. 0.06% Triethanolamine Foundations 1-2% Triethanolamine Leg and body paints 6% Triethanolamine Lipstick 0.2-1% Triethanolamine Makeup bases 1-3% Triethanolamine Other makeup preparations 0.7-2% Triethanolamine Basecoats and undercoats (manicuring preparations) 3% Triethanolamine Cuticle softeners 0.5-2% Triethanolamine Nail creams and lotions 0.2-1% Triethanolamine Other manicuring preparations 0.5-2% Triethanolamine Bath soaps and detergents % Triethanolamine Deodorants (underarm) % Triethanolamine Feminine hygiene deodorants 0.1% Triethanolamine Other personal cleanliness products 0.2-3% Triethanolamine Aftershave lotions 0.5-2% Triethanolamine Beard softeners 0.7% Page 2 of 3

81 Triethanolamine Preshave lotions (all types) 0.3% Triethanolamine Shaving cream (aerosol, brushless and lather) 3-10% Triethanolamine Other shaving preparations 0.3-6% Triethanolamine Skin cleansing (cold creams, cleansing lotions, liquids and pads) % Triethanolamine Depilatories 0.8% Triethanolamine Face and neck creams, lotions and powders 0.4-6% Triethanolamine Face and neck sprays % Triethanolamine Body and hand creams, lotions and powders 0.3-3% Triethanolamine Body and hand sprays % Triethanolamine Foot powders and sprays 0.4-1% Triethanolamine Moisturizing creams, lotions and powders 0.2-3% Triethanolamine Night creams, lotions and powders 0.3-5% Triethanolamine Paste masks (mud packs) 1-3% Triethanolamine Skin fresheners - 2% Triethanolamine Other skin care preparations 0.4-2%, 7% Triethanolamine Suntan gels, creams and liquids 2% Triethanolamine Indoor tanning preparations 0.3-2% 17% in a rinse-off skin care preparation Information collected in 2010 Table prepared October 13, 2010 Updated November 8, 2010 (Triethanolamine: lowered minimum for Mascara to 0.7; increased maximum Moisturizing to 3%) Page 3 of 3

82 TITLE PAGETLE PAGE PAGE STUDY TITLE Report Ethanolamine hydrochloride Murine Local Lymph Node Assay (LLNA) DATA REQUIREMENT OECD /73/EC, B.42. US EPA OPPTS AUTHOR(S) Dr. Armin O. Gamer (Study Director) Dr. Robert Landsiedel STUDY COMPLETED ON 11 Jan 2007 PERFORMING LABORATORY Experimental Toxicology and Ecology BASF Aktiengesellschaft Ludwigshafen, Germany LABORATORY PROJECT IDENTIFICATION 58H0372/ SPONSOR Alkanolamines CHEMSTAR Panel American Chemistry Council Arlington, VA This document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be used only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, publication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory agencies acting within the limits of their administrative authority. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 1 of 44

83 Report; Project No.: 58H0372/ BLANK PAGE THIS PAGE IS INTENTIONALLY LEFT BLANK This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 2 of 44

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85 Report; Project No.: 58H0372/ The following statement is for US EPA submissions only: FLAGGING CRITERIA The flagging criteria of 40 CFR are not applicable for this study type This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 4 of 44

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87 Report; Project No.: 58H0372/ CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL Head of Experimental Toxicology and Ecology: Dr. rer. nat. B. v. Ravenzwaay Management: Dr. rer. nat. R. Landsiedel Study director: Dr. med. vet. A. O. Gamer Statistics: Dipl. Statistician M. Dammann Analytical Chemistry: Dr. rer. nat. E. Fabian Quality Assurance Unit (QAU): J. Hajok Acute Toxicology: M. Remmele This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 6 of 44

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89 Report; Project No.: 58H0372/ GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY) This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 8 of 44

90 Report; Project No.: 58H0372/ CONTENTS TITLE PAGE 1 BLANK PAGE 2 GLP COMPLIANCE STATEMENT 3 FLAGGING CRITERIA 4 SIGNATURE PAGE 5 CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL 6 STATEMENT OF THE QUALITY ASSURANCE UNIT 7 GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY) 8 CONTENTS 9 LIST OF ABBREVIATIONS SUMMARY INTRODUCTION OBJECTIVES SELECTION OF DOSES/CONCENTRATIONS TEST GUIDELINES TIME SCHEDULE RETENTION OF RECORDS MATERIALS AND METHODS TEST SUBSTANCE TEST ANIMALS HOUSING CONDITIONS TEST GROUPS AND DOSES/CONCENTRATIONS TEST-SUBSTANCE PREPARATION ANALYSES EXPERIMENTAL PROCEDURE Conduct of the study ³H-thymidine injection Terminal procedures Data evaluation Calculations and statistical analyses Historical control data 22 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 9 of 44

91 Report; Project No.: 58H0372/ Positive control EVALUATION OF RESULTS RESULTS ANALYSES CLINICAL OBSERVATIONS AND ASSESSMENT OF FINDINGS Cell counts, 3 H-thymidine incorporation and lymph node weights Ear weights Body weights Other findings DISCUSSION AND CONCLUSION 27 APPENDIX 28 CELL COUNT, 3 H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND STIMULATION INDICES 29 EAR WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND STIMULATION INDICES 29 CELL COUNT, 3 H-THYMIDINE INCORPORATION, LYMPH NODE WEIGHT AND EAR WEIGHT: INDIVIDUAL VALUES 30 BODY WEIGHT DATA 31 HISTORICAL CONTROL DATA 32 POSITIVE CONTROL 34 α-hexylcinnamaldehyde, techn. 85%, vehicle: acetone 34 α-hexylcinnamaldehyde, techn. 85%, vehicle 1% aqueous Pluronic L92 36 FLOW CHART FOR EVALUATION OF THE LLNA 37 ANALYSES 38 Analytical characterization of the test substance (Certificate of Analysis) 39 Concentration Control Analysis of Ethanolamine hydrochloride in 1 % Pluronic L 92 in doubly distilled water 40 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 10 of 44

92 Report; Project No.: 58H0372/ LIST OF ABBREVIATIONS a.m. approx. ca. cm d DPM EC e.g. g h L LLNA LSC = ante meridiem (before noon) = approximately = circa = centimeter = day = disintegrations per minute = estimated concentration that leads to the respective stimulation index = for example = gram = hour = liter = Local Lymph Node Assay = liquid scintillation counter µci = micro Curie µl = microliter µm = micrometer mg = milligram ml = milliliter mm = millimeter No./Nos. p PBS p.m. S.D. SPF SI TCA v v/v w w/w = number/s = significance level = phosphate buffered saline = post meridiem (after noon) = standard deviation = specific pathogen free = stimulation index = trichloro-acetic acid = volume = volume per volume = weight = weight per weight This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 11 of 44

93 Report; Project No.: 58H0372/ Symbols: C = degree celsius ~ = circa = = equivalent % = per cent - = not observed, not determined This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 12 of 44

94 Report; Project No.: 58H0372/ SUMMARY The skin sensitizing potential of Ethanolamine hydrochloride was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears. Groups of 6 female CBA/Ca mice each were treated with 10%, 30% and 70% w/w preparations of the test substance in 1% aqueous Pluronic (1% Pluronic L 92 Surfactant in doubly distilled water) or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was applied with 2 x 12.5 µl per ear of the respective test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 2 x 12.5 µl per ear of the vehicle alone. The total volume of 25 µl per ear and application was split into two portions in order to prevent run off of the test-substance preparations due to high volume. The second daily application was performed immediately after the first daily application was finished in all animals of the study collective. Three days after the last application the mice were injected intravenously with 20 µci of 3 H- thymidine in 250 µl of sterile saline into a tail vein. About 5 hours after the 3 H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content and 3 H-thymidine incorporation (indicator of cell proliferation) as well as the weight of each animal s pooled lymph nodes. Moreover a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. The mean stimulation indices (fold of change as compared to the vehicle control) for cell count, 3 H-thymidine incorporation, lymph node weight and ear weight are summarized for each test group in the table below. Test group Treatment Cell Count Stimulation Index ³H-thymidine incorporation Stimulation Index Stimulation Index 1 1% aqueous Pluronic % in 1% aqueous Pluronic # 3 30% in 1% aqueous Pluronic # % in 1% aqueous Pluronic 1.26 # 2.03 ## The statistical evaluations were performed using the WILCOXON-test ( # for p 0.05, ## for p 0.01 ) Lymph Node Weight Ear Weight Stimulation Index No signs of systemic toxicity were noticed. The statistically significant increases in cellularity, caused by the 70% concentration and in 3 H-thymidine incorporation into the lymph node cells, caused by the 70% and 30% preparations, failed to reach the cut off stimulation index of 1.5 or 3, respectively, and thus lie below the threshold of immunologic relevance. Lymph node weights were not statistically significantly increased. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 13 of 44

95 Report; Project No.: 58H0372/ Though not concentration related and statistically significant in mice treated with the 10% test substance preparation, only, the slight increases in ear weight stimulation indices indicate minimal ear skin irritation at all concentrations. Thus it is concluded that Ethanolamine hydrochloride does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen. 2. INTRODUCTION 2.1. OBJECTIVES The Murine Local Lymph Node Assay (LLNA) is recommended by international test guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans. The study was performed based on the method of Kimber I. et al. (1994) 1. The study procedure was extended according to the publications described in section Test Guidelines, which evaluate the skin sensitizing potential of test substances by determination of lymph node response using lymph node weight and lymph node cell count as parameters which are set into perspective to ear weight or thickness as an indicator of skin irritation SELECTION OF DOSES/CONCENTRATIONS The selection of concentrations took into account available information on the chemical/physical properties and the composition of the test substance. In addition the results of a pretest were considered, which showed slightly increased ear weights after application of a 70% test substance preparation in 1% aqueous Pluronic as indication of some ear irritation. No increase in lymph node weights was observed (details are archived with the raw data). The following dose levels were selected: 10%, 30% and 70% in 1% aqueous Pluronic The control and test group animals were treated according to the following treatment scheme: Induction Control group 1 Vehicle: 1% aqueous Pluronic Test group 2 Test substance 10% in 1% aqueous Pluronic Test group 3 Test substance 30% in 1% aqueous Pluronic Test group 4 Test substance 70% in 1% aqueous Pluronic 1 Kimber I., Dearman R.J., Scholes E.W., Basketter D.A. (1994). The Local Lymph Node Assay: Developments and Applications. Toxicology, 93, This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 14 of 44

96 Report; Project No.: 58H0372/ TEST GUIDELINES The study was conducted according to the following test guidelines and publications: - OECD Guideline for Testing of Chemicals No. 429, April 24, 2002 ( Skin Sensitization: Local Lymph Node Assay ) - EC directive 2004/73, No. L 216, B.42., June 16, 2004 ( Skin Sensitization Local Lymph Node Assay ) - U. S. EPA Health Effects Test Guidelines OPPTS , March 2003 ( Skin Sensitization ) - Vohr H.-W., Blümel J., Blotz A., Homey B. and Ahr H.J., An intralaboratory validation of the integrated Model for the differentiation of skin reaction (IMDS): discrimination between (Photo)allergic and (photo)irritant skin reactions in mice. Arch. Toxicol. 73: , Ulrich, P., Streich, J. and Suter, W., Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals. Arch. Toxicol. 74: , Ehling G., Hecht M., Heusener A., Huesler J., Gamer A.O., van Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R. and Vohr H.-W., An European interlaboratory validation of alternative endpoints of the murine local lymph node assay: First round, Toxicology 212, 60-68, Ehling G. Hecht M., Heusener A., Huesler J., Gamer A.O., van Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R. and Vohr H.-W., An European interlaboratory validation of alternative endpoints of the murine local lymph node assay 2nd round, Toxicology 212, 69-79, This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 15 of 44

97 Report; Project No.: 58H0372/ TIME SCHEDULE Test dates: Study initiation date (date of signature of the study protocol) Arrival of the animals and start of acclimatization period (at least 5 days before application) Experimental starting date (selection of animals (randomization) for the first treatment within a study) 05 Jul Jun Jul st induction 12 Jul nd induction 13 Jul rd induction 14 Jul H-thymidine injection. Sacrifice of the animals. 17 Jul 2006 Determination ear weight, lymph node weight and cell count. Cell washing and precipitation. Measurement of 3 H-thymidine incorporation 18 Jul 2006 Experimental completion date (documentation of check of raw data) 09 Nov RETENTION OF RECORDS GLP relevant records and materials are stored at BASF Aktiengesellschaft for at least the period of time specified in the GLP principles. Details concerning responsibilities or locations of archiving can be seen from the respective SOPs and from the raw data. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 16 of 44

98 Report; Project No.: 58H0372/ MATERIALS AND METHODS 3.1. TEST SUBSTANCE The analyses of the test substance were carried out at SIGMA-ALDRICH. Name of test substance: Ethanolamine hydrochloride Test-substance No.: 05/ Batch identification: 092K5318 CAS No.: Purity: Homogeneity: Storage stability: 100% (for details see Certificate of Analysis in the Appendix) The test substance was homogeneous by visual inspection. The stability under storage conditions over the study period was guaranteed (for details see Certificate of Analysis) TEST ANIMALS Test species / strain / quality: Reasons for the selection of the test species: Age at day 0: Sex: Supplier: Arrival in the testing facility: Identification: Body weight at day 0: Mouse / CBA/CaOlaHsd The female animals were nulliparous and non-pregnant. The test method was developed in mice weeks The somewhat younger age of the animals as compared to the range given in the test guideline does not influence the test according to our experience. Female Harlan Winkelmann GmbH, Gartenstr. 27, Borchen, Germany Acclimatization period (22 days before the first test-substance application) The single housed animals were identified by cage cards 18.3 g 24.2 g This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 17 of 44

99 Report; Project No.: 58H0372/ HOUSING CONDITIONS Room temperature / relative humidity: Day / night rhythm: Type of cage: The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of C for temperature and of 30 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study. 12 h / 12 h (6.00 a.m p.m. / 6.00 p.m a.m.) Makrolon cage, type I No. of animals per cage: 1 Feeding: Drinking water: Bedding: Kliba-Labordiät (Maus / Ratte Haltung GLP ), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum Tap water ad libitum Granulat Typ ¾ (staubfrei); SSNIFF 3.4. TEST GROUPS AND DOSES/CONCENTRATIONS Groups Number of animals Animal No. Control group Test group Test group Test group Control group 1 was used to rule out any influence of the vehicle, used for production of the test-substance preparations, on the parameters measured in the test. The concentrations used for induction are given in section 2.2 Selection of doses/concentrations. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 18 of 44

100 Report; Project No.: 58H0372/ TEST-SUBSTANCE PREPARATION Test-substance preparation: The test-substance preparation was produced on a weight per weight basis shortly before each application. After stirring with a magnetic stirrer the test substance is soluble in the vehicle. Vehicle: Reason for the vehicle: Form of application: 1% Pluronic L 92 Surfactant in doubly distilled water The test substance is highly soluble in the aqueous vehicle. By means of the hydrophobic surface-active agent, Pluronic L 92, run-off of the test-substance preparations was prevented. Pluronic L 92 was recommended by Ryan C.A. et al. as suitable to produce aqueous preparations for use in the Murine Local Lymph Node Assay (LLNA). 2 Solution 3.6. ANALYSES Conduct of analyses: Stability of the testsubstance preparation: Homogeneity of the testsubstance preparation: Concentration analysis of the test-substance preparation: The analyses were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft. The stability of the test substance in the vehicle was determined indirectly by the concentration analysis. For this purpose, the samples taken were stored at room temperature over the maximum duration of the administration period and were subsequently deep-frozen. Afterwards, these samples were analyzed. The homogeneity of the test-substance preparation in the vehicle was not determined by analysis. The verification of the concentration of the test-substance preparations (10%, 30%, 70%) was investigated once by analysis during the study. 2 Ryan C.A. et al., Examination of a vehicle for use with water soluble materials in the murine local lymph node assay, Food Chem. Toxicol 40, , 2002 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 19 of 44

101 Report; Project No.: 58H0372/ Analysis of feed: Analysis of drinking water: Analysis of bedding: The feed used in the study was assayed for chemical and microbiological contaminants. The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for the presence of microbes by a contract laboratory. The bedding is regularly assayed for contaminants (chlorinated hydrocarbons, heavy metals). Records on analyses of feed, drinking water and bedding are stored at Experimental Toxicology and Ecology of BASF Aktiengesellschaft for at least the period of time specified in the GLP principles EXPERIMENTAL PROCEDURE Conduct of the study The study comprised three treatment groups and a vehicle control group. Each group consisted of 6 mice. Randomization: Body weight determination: Signs and symptoms: Form of application: Application volume: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals. No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data. Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions. 2 x 12.5 μl per ear The total volume of 25 µl per ear and application was split into two portions in order to prevent run off of the test-substance preparations due to high volume. The second daily application was performed immediately after the first daily application was finished in all animals of the study. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 20 of 44

102 Report; Project No.: 58H0372/ Site of application: Frequency of application: Mortality: Dorsal part of both ears 3 consecutive applications (day 0 day 2) to the same application site Twice on Mondays through Fridays (beginning and end) and once on Saturdays, Sundays and on public holidays. The animals of test groups 1-4 were treated with vehicle or test-substance preparation as given in section ³H-thymidine injection On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 µci of 3 H-thymidine in 250 µl of sterile saline into a tail vein Terminal procedures The animals were sacrificed on study day 5 about 5 hours after 3 H-thymidine injection by cervical dislocation. Determination of the ear weight: Removal and weight determination of the lymph nodes: Preparation of cell suspension and determination of cell count: Measurement of 3 H- thymidine incorporation of the lymph node cells: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal. After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 ml of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy ton in a ratio 1:500. The cell count was determined using a Casy -Counter. The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3 H-thymidine into the cells was measured in a ß-scintillation counter. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 21 of 44

103 Report; Project No.: 58H0372/ Data evaluation Table(s) and/or figure(s) of measured parameters presented in the report were produced using PC based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. As a consequence, calculation of mean values using the individual data presented in the report will, in some instances, yield minor variations in value Calculations and statistical analyses Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of cell count, 3 H-thymidine incorporation, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Further statistical analyses were performed according to the table below. Parameter Cell count, 3 H-thymidine incorporation, lymph node weight as well as ear weight Statistical test WILCOXON - Test Historical control data Vehicle related historical control data, gathered over an appropriate time period, are presented in the Appendix. These data may be used as described in section 3.8 for the evaluation of the results in addition to the concurrent control values. The comparison of the present data to the historical controls was not necessary for evaluation of this study Positive control A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance α-hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen. The results of the most recent studies are included in the Appendix. 3 Siegel, S. (1956): Non-parametric statistics for behavioural sciences. McGraw-Hill New York This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 22 of 44

104 Report; Project No.: 58H0372/ EVALUATION OF RESULTS In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, 3 H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance. The increase stimulation index (SI) of cell count by a factor of >1.5 and/or of 3 H-thymidine incorporation by a factor of 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance. If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI. In addition the evaluation uses the following considerations: If a test substance does not show a statistically significant and/or biologically relevant increase in cell count, 3 H-thymidine incorporation and/or lymph node weight as compared to the vehicle control in the presence of statistically significant and/or biologically relevant increased ear weights as indication of skin irritation, it is considered not to be a sensitizer. If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in cell count, 3 H-thymidine incorporation and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test substance is considered to be a sensitizer. If statistically significant and/or biologically relevant increases in ear weights are running in parallel to the increase in cell count, 3 H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Then, for identification of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control values (cf. section Historical control data ) may be performed. If one or a combination of the measured parameters change statistical significance, evaluation on basis of the criteria described above may be possible. If the statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitization response. If a test substance does not elicit a biological relevant increase in cell count, 3 H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered. A flow chart on the evaluation procedure is given in the Appendix. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 23 of 44

105 Report; Project No.: 58H0372/ RESULTS Tables on the results of the clinical examinations are presented in the Appendix ANALYSES Stability of the testsubstance preparation: Homogeneity of the testsubstance preparation: Concentration analysis of the test-substance preparation: Analysis of feed: Analysis of drinking water: Analysis of bedding: The stability of the test substance in the vehicle for the maximum application period was confirmed indirectly by the concentration analysis. The test-substance preparation was visually homogeneous (solution). The verification of the concentration of the test-substance preparations (70%, 30% and 10%) for the first application was confirmed by analysis (details are available in the Appendix ). In view of the aim and duration of the study the contaminants occurring in commercial feed should not influence the results. In view of the aim and duration of the study there are no special requirements exceeding the specification of drinking water. In view of the aim and duration of the study there are no special requirements exceeding the specification of a commercial grade monitored by the manufacturer. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 24 of 44

106 Report; Project No.: 58H0372/ CLINICAL OBSERVATIONS AND ASSESSMENT OF FINDINGS Cell counts, 3 H-thymidine incorporation and lymph node weights The statistically significant increases in cellularity, caused by the 70% concentration and in 3 H-thymidine incorporation into the lymph node cells, caused by the 70% and 30% preparations, failed to reach the cut off stimulation index of 1.5 and 3, respectively, and thus lie below the threshold of immunologic relevance. Lymph node weights were not statistically significantly increased. The group mean values are presented in the figures below. The corresponding data, the cell count, 3 H-thymidine incorporation and lymph node weight stimulation indices as well as the individual values are given in the Appendix. Cell Count 70% in 1% aqueous Pluronic # 30% in 1% aqueous Pluronic 10% in 1% aqueous Pluronic 1% aqueous Pluronic 0 5,000,000 10,000,000 15,000,000 Cell Counts [Counts / lymph node pair] ³H-thymidine incorporation 70% in 1% aqueous Pluronic ## 30% in 1% aqueous Pluronic # 10% in 1% aqueous Pluronic 1% aqueous Pluronic ,000 1,500 ³H-thymidine [DPM / lymph node pair] The statistical evaluations were performed using the WILCOXON-test ( # for p 0.05, ## for p 0.01 ) This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 25 of 44

107 Report; Project No.: 58H0372/ Lymph Node Weight 70% in 1% aqueous Pluronic 30% in 1% aqueous Pluronic 10% in 1% aqueous Pluronic 1% aqueous Pluronic Weight [mg / lymph node pair] Ear weights The ear weight stimulation indices indicate minimal ear skin irritation at all concentrations though not concentration related. A slight but statistically significant increase in ear weights was observed in mice treated with the 10% test substance preparation, only. The group mean values are presented in the figures below. The corresponding data, the ear weight stimulation index as well as the individual values are given in the Appendix. Ear Weight 70% in 1% aqueous Pluronic 30% in 1% aqueous Pluronic 10% in 1% aqueous Pluronic # 1% aqueous Pluronic Weight [mg / animal] The statistical evaluations were performed using the WILCOXON-test ( # for p 0.05, ## for p 0.01 ) This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 26 of 44

108 Report; Project No.: 58H0372/ Body weights The expected body weight gain was generally observed in the course of the study. The individual body weights are shown in the Appendix Other findings No abnormalities were observed during general observation. 5. DISCUSSION AND CONCLUSION No signs of systemic toxicity were noticed. The statistically significant increases in cellularity, caused by the 70% concentration and in 3 H-thymidine incorporation into the lymph node cells, caused by the 70% and 30% preparations, failed to reach the stimulation index criterion of 1.5 or 3, respectively, and thus lie below the threshold of immunologic relevance. Lymph node weights were not statistically significantly increased. Though not concentration related and statistically significant in mice treated with the 10% test substance preparation, only, the slight increases in ear weight stimulation indices indicate minimal ear skin irritation at all concentrations. Thus it is concluded that Ethanolamine hydrochloride does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 27 of 44

109 Report; Project No.: 58H0372/ APPENDIX This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 28 of 44

110 Report; Project No.: 58H0372/ CELL COUNT, 3 H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND STIMULATION INDICES Cell Counts [Counts/Lymph Node Pair] Test Group Treatment Mean S.D. Stimulation Index 1 1 1% aqueous Pluronic 8,460, ,321, % in 1% aqueous Pluronic 9,549, , % in 1% aqueous Pluronic 8,760, ,238, % in 1% aqueous Pluronic 10,639, ,767, # ³H-thymidine incorporation [DPM/Lymph Node Pair] Test Group Treatment Mean S.D. Stimulation Index 1 1 1% aqueous Pluronic % in 1% aqueous Pluronic % in 1% aqueous Pluronic # 4 70% in 1% aqueous Pluronic ## Lymph Node Weight [mg/lymph Node Pair] Test Group Treatment Mean S.D. Stimulation Index 1 1 1% aqueous Pluronic % in 1% aqueous Pluronic % in 1% aqueous Pluronic % in 1% aqueous Pluronic EAR WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND STIMULATION INDICES Ear Weight [mg/animal] Test Group Treatment Mean S.D. Stimulation Index 1 1 1% aqueous Pluronic % in 1% aqueous Pluronic # 3 30% in 1% aqueous Pluronic % in 1% aqueous Pluronic test group x / test group 1 (vehicle control) # = statistically significant for the value p 0.05 ## = statistically significant for the value p 0.01 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 29 of 44

111 Report; Project No.: 58H0372/ CELL COUNT, 3 H-THYMIDINE INCORPORATION, LYMPH NODE WEIGHT AND EAR WEIGHT: INDIVIDUAL VALUES Group Treatment Animal No. Cell Counts/ Lymph Node Pair ³H-thymidine incorporation Lymph Node Weight Ear Weight [DPM/Lymph Node Pair] [mg//lymph Node Pair] [mg/animal] 1 9,456, ,806, % aqueous Pluronic 3 8,148, ,792, * 5,646, ,098, ,910, ** 2 10% in 1% aqueous Pluronic 9 8,952, ,870, ,050, ,966, ,846, ,992, % in 1% aqueous Pluronic 15 9,240, ,212, ,918, ,352, ,460, ,960, % in 1% aqueous Pluronic 21 7,848, ,742, ,876, ,948, * Values of this animal were not used for calculation of the mean values and evaluation. The body weight of the animal decreased during the study period and unusually small lymph nodes were dissected. These effects were not considered to be test-substance related. ** Animal died due to irregularities during i.v. application. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 30 of 44

112 Report; Project No.: 58H0372/ BODY WEIGHT DATA Group Body Weight d0 [g] Mean S.D. Body Weight d5 [g] Mean S.D. Body Weight Change d5-d0 [g] Mean S.D. Animal No * ** * Values of this animal were not used for calculation of the mean values and evaluation. The body weight of the animal decreased during the study period and unusually small lymph nodes were dissected. These effects were not considered to be test-substance related. ** Animal died due to irregularities during i.v. application. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 31 of 44

113 Report; Project No.: 58H0372/ HISTORICAL CONTROL DATA The historical control data gathered in the laboratory with the vehicle used (1% aqueous Pluronic ) are presented in the following table: Project-No. Animal No. Cell Counts/ ³H-thymidine incorporation Lymph Node Weight Ear Weight Ear Thickness Date Lymph Node Pair [DPM] [mg] [mg] [µm] 1 8,942, ,216, H0056/ ,268, H0450/ ,061, Apr ,518, ,440, ,219, ,955, H0113/ ,094, H0375/ ,996, Jun ,246, ,677, ,406, ,296, H0359/ ,884, H0511/ ,788, Oct ,524, ,252, ,330, H0502/ ,278, Oct ,176, ,684, ,760, ,968, ,656, H0100/ ,250, Oct ,930, ,442, ,497, ,494, ,370, H0666/ ,316, Feb ,430, ,174, ,404, ,054, ,960, H0666/ ,500, Feb ,358, ,924, ,360, ,600, ,762, ,492, H0651/ ,222, Sep ,082, ,892, ,130, This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 32 of 44

114 Report; Project No.: 58H0372/ HISTORICAL CONTROL DATA (continued) 25 10,086, ,378, H0041/ ,628, Mar ,906, ,636, ,702, ,706, ,498, H0727/ ,378, H0728/ ,378, Mar ,320, ,016, ,232, ,534, H0835/ ,910, Mar ,290, ,584, ,400, Historical control data Median 10,379, Mean 10,103, S.D. 2,520, Present study 70% in 1% aqueous Pluronic Median 10,710, Mean 10,639, S.D. 1,767, Positive control study 10% in acetone 58H0288/ Median 22,287, Mean 20,823, S.D. 5,074, Positive control study 10% in 1% aqueous Pluronic 45H0288/ Median 26,536, Mean 25,013, S.D. 6,849, This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 33 of 44

115 Report; Project No.: 58H0372/ POSITIVE CONTROL α-hexylcinnamaldehyde, techn. 85%, vehicle: acetone As recommended by the respective testing guidelines, the following study was performed to evaluate the sensitivity of experimental animals (mice, CBA/CaOlaHsd, Harlan Winkelmann GmbH, Borchen, Germany) and the ability of procedures to detect a known mild to moderate skin sensitizer. Name of test substance: Alpha-Hexylcinnamaldehyde, techn. 85% CAS number: Substance number: 98/288-1 Project No.: 58H0288/ Experimental starting date: 14 Feb 2006 Experimental completion date: 03 Mar 2006 The mean stimulation indices (fold of change as compared to the vehicle control) for cell count, 3 H-thymidine incorporation, lymph node weight, ear weight and ear thickness are summarized for each test group in the table below. Test group Treatment ³H-thymidine incorporation Stimulation Lymph Node Weight Stimulation Index Index 1 vehicle acetone % in acetone 1.75 # 4.56 ## 1.47 # % in acetone 2.36 ## 6.63 ## 1.98 ## # 4 30% in acetone 2.98 ## 9.86 ## 2.46 ## 1.22 ## 1.15 ## The statistical evaluations were performed using the WILCOXON-test ( # for p 0.05, ## for p 0.01 ) Discussion and Conclusion Cell Count Stimulation Index Ear Weight Stimulation Index Ear Thickness Stimulation Index No signs of systemic toxicity were noticed. One animal of test group 2 died after 3 H-thymidine injection. The test substance induced a statistically significant and biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) 1.5) in the auricular lymph node cell counts when applied as 3%, 10% or 30% preparation in acetone. Concomitantly, the increase of 3 H-thymidine incorporation into the cells was statistically significant and biologically relevant (increase above the cut off stimulation index of 3) at these concentrations, which additionally caused statistically significantly increased lymph node weights. The statistically significant increase in ear weights at 30%, which was accompanied by an increase in ear thickness, indicates some irritation of the ear skin. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 34 of 44

116 Report; Project No.: 58H0372/ A small but statistically significant increase in ear thickness was caused by the 10% preparation. This mild irritation, however, does not explain the lymph node response observed at this concentration. Thus it is concluded that the Murine Local Lymph Node Assay is able to show the skin sensitizing effect of α-hexylcinnamaldehyde, techn. 85% under the test conditions chosen. The threshold concentration for sensitization induction was < 3% under the test conditions chosen. The EC 1.5 for cell count and the EC 3 for 3 H-thymidine incorporation was calculated by semi-logarithmical regression from the results of all concentrations to be 1.9% and 1.7%, respectively. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 35 of 44

117 Report; Project No.: 58H0372/ α-hexylcinnamaldehyde, techn. 85%, vehicle 1% aqueous Pluronic L92 Evaluation of the sensitivity of mice, CBA/CaOlaHsd, Harlan Winkelmann GmbH, Borchen, Germany) to known sensitizers as recommended by the guideline. Name of test substance: α-hexylcinnamaldehyde, techn. 85% CAS number: Substance number: 98/288-1 Project No.: 45H0288/ Experimental starting date: April 13, 2004 Experimental completion date: May 19, 2004 The indices for lymph node weight, cell count and ear weight in relation to the vehicle control group are summarized in the table below. Test group Treatment Lymph node Weight Index Cell Count Index Ear Weight Index 1 vehicle 1% Pluronic L 92 in doubly distilled water % in 1% Pluronic L 92 in doubly distilled water 1.28 # 1.43 # 1.10 ## 3 10% in 1% Pluronic L 92 in doubly distilled water 1.81 ## 2.28 ## 1.41 ## 4 30% in 1% Pluronic L 92 in doubly distilled water 2.39 ## 2.92 ## 1.94 ## The statistical evaluations were performed using the WILCOXON-test ( # for p 0.05, ## for p 0.01 ) Discussion and Conclusion: No signs of systemic toxicity were noticed. The test substance induced a statistically significant response in the auricular lymph nodes, when applied as 3%, 10% or 30% preparations in 1% Pluronic L 92 in doubly distilled water. The statistically significant increases in ear weights indicate irritation of the ear skin at all concentrations. The high ear weights in test groups 3 and 4 were accompanied by scaling and incrustations, which were observed on the day of lymph node removal. The increase in cell count index induced by the 3% test substance preparation was at the border of biological significance. The marked increase at 10% and 30% cannot be explained by skin irritation alone. Based on the results of this study, it is concluded that the test conditions and animal strain chosen are able to reveal the known skin sensitizing effect of α-hexylcinnamaldehyde, techn. 85% in the Murine Local Lymph Node Assay. The threshold of skin sensitization induction of the compound lies just below 3% under the test conditions used. This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 36 of 44

118 Report; Project No.: 58H0372/ FLOW CHART FOR EVALUATION OF THE LLNA The evaluation procedure described in section 3.8. of the report is presented here in graphical form. Statistically significant and/or biologically relevant increase of ³H-Thymidine incorporation, cell count and/or lymph node weight No Statistically significant and/or biologically relevant increase of ear punch weight and/or ear thickness (Ear skin irritation) Yes Yes No Yes Statistically significant and/or biologically relevant increase of ear punch weight and/or ear thickness at concentrations without lymph node response Sufficiently high concentration tested? Yes No No Conclusive evaluation of lymph node responseear response-relation possible by using historical control data Perform test with higher concentrations Yes No Further investigations (e.g. challenge experiment) sensitizing not sensitizing This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 37 of 44

119 Report; Project No.: 58H0372/ ANALYSES Analytical characterization of the test substance (Certificate of Analysis) Concentration Control Analysis of Ethanolamine hydrochloride in 1 % Pluronic L 92 in doubly distilled water This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 38 of 44

120 Report; Project No.: 58H0372/ This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 39 of 44

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123 Report; Project No.: 58H0372/ This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 42 of 44

124 Report; Project No.: 58H0372/ This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 43 of 44

125 Report; Project No.: 58H0372/ This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors Page 44 of 44

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