Food and Chemical Toxicology

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1 Food and Chemical Toxicology 46 (2008) Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: The Inhalation toxicity of di- and triethanolamine upon repeated exposure A.O. Gamer, R. Rossbacher, W. Kaufmann, B. van Ravenzwaay * BASF SE, Product Safety, Z 470, D Ludwigshafen, Germany article info abstract Article history: Received 23 January 2007 Accepted 19 February 2008 Keywords: Diethanolamine Triethanolamine Neurotoxicity Respiratory tract Irritation Inhalation Systemic and respiratory tract (RT) toxicity of triethanolamine (TEA) was assessed in a 28-day nose-only inhalation study in Wistar rats (10 animals/sex, concentrations: 0, 20, 100, 500 mg/ ; 5 days/week, 6 h/ day). In two nose-only 90-day inhalation studies, with similar exposure design, Wistar rats were exposed to 0, 15, 150, 400 mg/ diethanolamine (DEA) (DEA Study 1:13 animals/sex, general subchronic study) and to 0, 1.5, 3, 8 mg/ (DEA Study 2:10 animals/sex) to specifically investigate respiratory tract toxicity. Only DEA induced systemic toxicity at or above 150 mg/ (body and organ weight changes, clinical- and histo-pathological changes indicative for mild blood, liver, kidney and testicular effects). Neurotoxicity was not observed for both substances. Exposure to both substances resulted in laryngeal epithelial changes starting fro mg/ for DEA (reversible metaplasia at the base of the epiglottis, inflammation at higher concentrations extending into the trachea) or from 20 mg/ for TEA (focal inflammation, starting in single male animals). TEA appears to be less potent with respect to systemic toxicity and RT irritancy than DEA. The 90-day no adverse effect concentration (NOAEC) for changes due to TEA exposure in the respiratory tract was 4.7 mg/ derived by extrapolation from the NOAEC of the 28 day study. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Diethanolamine (DEA, CAS No: ) and triethanolamine (TEA, CAS No: ) are alkanolamines (Fig. 1), are both used in a wide range of application, such the production of soaps and surfactants, in industrial uses for gas washing, grinding aid for concrete or cement, corrosion inhibitor, or metal working fluids, in textile processing, or a detergent and specialty cleaner formulations (Knaak et al., 1997), and therefore show a relevant potential for human exposure. Because of the low vapor pressure, dermal contact appears to be the most probable route of exposure but workers may also be exposed to DEA and TEA by inhalation of aerosols, e.g. during the use of lubricating or cleaning liquids. A comprehensive review of the toxicity of diethanolamine, triethanolamine, as well as monoethanolamine was published in 1997 (Knaak et al., 1997). BG-Chemie published an assessment of DEA and TEA in 1990 (BG-Chemie, 1990a,b). Both, DEA and TEA, show a low acute toxicity with LD 50 values ranging from 1.41 g/kg to 2.83 g/kg (DEA) and Abbreviations: DEA, diethanolamine; FOB, functional observational battery; GSD, geometric standard deviation; MEA, monoethanolamine; MMAD, mass median aerodynamic diameter; NOAEC, no observed adverse effect concentrations; NOEC, no observed effect concentration; NTP, national toxicology program; RT, respiratory tract; TEA, triethanolamine; URT, upper respiratory tract. * Corresponding author. Tel.: ; fax: address: bennard.ravenzwaay@basf.com (B. van Ravenzwaay) g/kg to g/kg (TEA), respectively (Anon., 1983). If not neutralized, they are irritant to skin and eyes and cause systemic toxicity mainly in liver, kidney, red blood cells and the nervous system following oral and/or dermal exposure in laboratory animals (BIBRA, 1990, 1993; Anon., 1983). Local effects reflect the irritant properties of these alkaline compounds (pk a of 8.88 for DEA, and of 7.76 for TEA, at 25 C) (Knaak et al., 1997). Two-week inhalation studies with TEA in rats and mice up to 2000 mg/ (Mosberg et al., 1985a,b) showed slight laryngeal epithelial inflammation. In addition increase in liver weights occurred at the high dose in male rats and kidney weights were increased in males starting with 500 mg/ and in females at 250 mg/ and above, which did not show any histopathological effects. Thus, kidney weight increase may indicate an adaptive response rather than a frank adverse effect. DEA and TEA are negative for skin sensitization in animal tests (RCC, 1990/NTP, 2005). Human experience indicates that in contrast to TEA (MAK, 2001b, 2007), DEA has a low skin sensitizing potential predominantly observed in workers frequently exposed to cutting fluids. Its stronger irritant potential and other factors responsible for impairment of the skin barrier appear to be important contributing factors (MAK, 2001a). To elucidate the toxicity of DEA and TEA following aerosol inhalation exposure in rats, several repeated exposure inhalation studies have been conducted (Table 1). In addition potential neurofunctional impairment after inhalation exposure was investigated. Moreover, upper respiratory tract (URT) irritation and its concentration response relationship were addressed in detail. We report /$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi: /j.fct

2 2174 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) HO here the results of these inhalation studies and compare the local and systemic effects of both alkanolamines. 2. Materials and methods 2.1. Test substance N H DEA was supplied by BASF (DEA Study 1), or the Dow Chemical Company (DEA Study 2), respectively, and had a purity of > 99%. DEA is a white crystalline solid at room temperature and a viscous liquid above 28 C. Its vapor pressure at 25 C is hpa (NLM, 2004). TEA was produced by BASF AG and had a purity of 98.9%. Above 20.5 C TEA is a colourless clear liquid. Its vapor pressure at 25 C is hpa (Pors and Fuhlendorff, 2002). The stability of DEA and TEA under the specific storage conditions was ensured for the study duration by covering with N Generation of the inhalation atmosphere for DEA and TEA OH C 4 H 11 NO 2 Diethanolamine CAS: HO N OH OH C 6 H 15 NO 3 Triethanolamine CAS: Fig. 1. Chemical formula and structure of diethanolamine and triethanolamine. For each concentration group, the liquid test substances were supplied to a twocomponent atomizer by means of a continuous infusion pump and were atomized with compressed air. Due to the relatively high freezing point of DEA (28 C) and TEA (20.5 C) (Knaak et al., 1997) the syringes, tubes and atomizers of the aerosol generation systems were heated. The test substances were sprayed into cyclone separators and, after dilution with conditioned air, the aerosol was supplied to the cylindrical past-flow head nose exposure systems, constructed from stainless steel (DEA Studies 1 and 2:90 L volume, TEA study: 55 L volume, BASF AG). For exposure the rats were transferred to glass exposure tubes and only the animals snouts protruded into the inhalation systems. The inhalation concentrations were adjusted by the infusion pump rates. The exposure systems were operated with an air exchange of above 50 times/h and a positive pressure relative to the laboratory air. Air flow rates as well as relative humidity and temperature of the inhalation atmospheres were monitored continuously. The measurements were within the range of the OECD guideline requirements for temperature (20 24 C) and humidity (30 70%) or as scheduled for the air flow rates (around 3 /h). Due to the lack of a more specific analytical method, gravimetric determination of the concentrations of TEA was performed (6 (hourly) samples per concentration and exposure), which was validated during the technical trials of the range finding studies by adding a dye to the test substance, which then was quantified photometrically. In addition the low vapor pressure allows the use of gravimetry. Measurement of DEA concentrations (two samples per concentration and exposure) was carried out by spectrophotometry of absorption samples in distilled water after derivatization with biuret agent, although the low vapor pressure might also have allowed for using gravimetry. Additionally, the stability of the concentrations during the daily exposure periods was measured with scattered light photometers (RAM, Mie, USA). Particle size analyses were performed, using cascade impactors (MARK III, Andersen, USA for TEA and DEA Study 1 and Marple 298, Andersen, USA for DEA Study 2). Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the measured mass distributions. Two measurements per concentration were performed for TEA, 3 measurements per concentration for DEA Study 1 and 6 8 measurements per concentration in DEA Study Animals and husbandry The studies were conducted according to the provisions of the German animal welfare regulations. Healthy male and female Wistar rats were purchased at an age of about 7 weeks from Dr. K. Thomae, Biberach, Germany (strain Chbb:THOM) (for DEA Study 1 and TEA study) or from Charles River, Sulzfeld, Germany (strain CrlGlxBrIHan:WI) (for DEA Study 2). During the exposure-free period animals were housed individually in Makrolon wire cages type MD III (Becker & Co, Castrop-Rauxel, Germany) and received KLIBA laboratory diet (Klingenthalmühle AG, Kaiseraugust, Switzerland) and tap water ad libitum. Animal rooms were maintained air-conditioned at C and 30 70% relative humidity. The day/night rhythm was 12 h light from 6:00 AM to 6:00 PM and 12 h dark from 6:00 PM to 6:00 AM. The animals were allocated to individual test groups based on weight and sex at random and identified uniquely by ear tattoo. For the studies with motor activity investigations, these were conducted in polycarbonate cages (Ehret, Emmendingen, Germany) with wire covers and bedding Study design A total of five studies were conducted (Table 1) with one short-term exposure study for each test substance serving as concentration range finder. All studies were conducted following the OECD (OECD, 1981a,b), EU (EEC, 1988) as well as EPA Guidelines (EPA, 1985, 1987, 1998) and according to Good Laboratory Practice (German Chemicals Act, OECD Principles). Head nose exposure of animals was used to minimize exposure to the test substances by dermal contact and oral ingestion. For adaptation to the exposure conditions the animals were exposed to clean air in the exposure system on 3 to 5 days before start of the exposure period under comparable conditions in each study. Table 1 Study design for diethanolamine (DEA) and triethanolamine (TEA) Substance DEA TEA Study identification DEA range finding DEA Study 1 DEA Study 2 TEA range finding TEA study study study Special Focus Neurotoxicity URT toxicity Neurotoxicity URT toxicity Number of exposures (days/6 h) Number of animals/sex/ group a Recovery groups 10 females treated with 0, 3, 8 mg/ Concentration (mg/ ) b 0, 100, 200, 400 0, 15, 150, 400 0, 1.5, 3, 8 0, 100, 200, 400 0, 20, 100, 500 URT: upper respiratory tract. a In the TEA study and DEA Study 1 animal numbers per group were higher than proposed by the OECD guideline in order to increase statistical power for neurofunctional testing and make use of animals for perfusion fixation. b Groups are also defined as control, low exposure, intermediate exposure and high exposure groups, respectively.

3 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) Diethanolamine For the concentration range finding study, groups of 10 animals/sex were exposed for 6 h/day over a time period of 2 weeks (10 exposures) to target concentrations of 100, 200, or 400 mg DEA/. No substance related findings were obtained at the two lower concentrations. Exposure to 400 mg/ resulted in decreased body weight and body weight gain in males and slightly decreased serum cholesterol in both sexes. Additionally, the relative and absolute liver weights were increased in the female animals. No histopathological findings were observed in the RT (nasal cavity, trachea, lungs), however, the examination of the larynx was not included in the range finding study. Based on these results, target concentrations of 15, 150 and 400 mg/ were chosen for the main DEA Study 1. Groups of 13 animals of each sex were exposed for 6 h/day on 65 days over a time period of 99 days (90-day study). The animals were exposed on all workdays, except the days of the functional observational battery (FOB). For the DEA Study 2, 10 animals/sex and group were exposed to target concentrations of 1.5, 3, and 8 mg/ with an identical procedure, except the conduct of a functional observation battery (FOB). Study 2 also included recovery groups of 10 additional female animals exposed to 3 or 8 mg/, which were observed for a post-exposure period of three months. For each study, control groups with the respective number of animals/sex were exposed to clean air Triethanolamine For the concentration range finding study, groups of 5 animals/sex were exposed 6 h/day for five consecutive days to 100, 200, or 400 mg/ (target concentrations). Five control animals of each sex were exposed to clean air. Up to the highest exposure concentration, no adverse effects occurred during clinical, clinico-chemical, hematological, and pathological examinations. Concentration-dependent laryngeal inflammation and edema were noticed histopathologically, with a no observed adverse effect concentration (NOAEC) of 100 mg/. Based on these results, target concentrations of 20, 100, or 500 mg TEA/ were chosen for the main study. The high concentration was increased to 500 mg/kg in the expectation to produce at least some overt systemic effects. Groups of 10 animals of each sex were exposed for 6 h/day on 20 workdays over a time period of 28 days. 10 control animals/sex were exposed to clean air Clinical examinations The animals were observed for evident signs of toxicity or mortality twice (DEA Study 2) or 3 times (DEA Study 1, TEA study) on all exposure days and once during preflow and post-exposure examination. In DEA Study 1 ophthalmoscopy was performed before the preflow period (on all animals) and at the end of the study (control and high exposure group), using an ophthalmoscope (Heine Optotechnik, Herrsching, Germany). The body weight was determined at the beginning of the preflow period, before the first exposure (TEA study: day 1; DEA studies: day 0), and once a week during exposure period. Comprehensive neurofunctional tests were conducted on 10 animals/sex (DEA Study 1), or 7 animals/sex (TEA study), using a functional observational battery (FOB), which includes various parameters of sensory and motor functions. The FOB was performed in a blind fashion on randomized animals by an experienced technician. In the DEA Study 1, FOB was conducted before the exposure period and in monthly intervals thereafter (4 times). In the TEA study, FOB was performed in about weekly intervals. The animals were observed in a standard arena. The reflex and sensorimotoric tests covered responses to visual, auditory, olfactory, and touch stimuli (e.g. pupillary reflex, startle response, blinking reflex) as well as neuromotor alterations (e.g. pain perception, grip strength). Motor activity was measured in the DEA Study 1 on the same day as the FOB, using a Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. The animals were assigned to the cages in a randomized order. The number of beam interrupts was counted over 18 intervals, each lasting 5 minutes. The assessment period for each animal started with the first beam interrupt and the measurement ended exactly 1.5 h thereafter. The number of beam interrupts that correlated to the activity was counted Clinical chemistry, hematology, and urinalysis For hematology and clinical chemistry examinations, blood was taken from the retro orbital venous plexus of non-fasted, not anesthetized animals in the morning following the last exposure in a randomized order. For urinalysis (only conducted in DEA Study 1) the individual animals were transferred to metabolism cages and urine was collected overnight from all animals. The analyses were performed under internal laboratory quality control conditions in 10 animals per test group (DEA Study 1), or in 5 animals per test group (TEA study), respectively. The hematological parameters included leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and platelet count and were determined by means of a cell counter (H1E, Bayer Diagnostics, Munich, Germany). The differential blood count and reticulocytes (only TEA study) were evaluated visually according to Schermer (1967) and Zeile et al. (1980). For the clotting analysis, the thromboplastin time was determined by the Hepato Quicḱs test according to Fischer and Falkensammer (1974), using a ball coagulometer (KC 10 A model, Amelung, Lemgo, Germany). The clinico-chemical parameters were measured using an automatic analyzer (Hitachi 737, Boehringer, Mannheim, Germany) and included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, c-glutamyltransferase, sodium, potassium, magnesium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, and cholesterol. Urinalysis was conducted semi quantitatively, either by microscopical or visual evaluation or using test strips (Combur-9-test RL, Boehringer, Mannheim, Germany) and a reflection photometer (Urotron RL9 Model, Boehringer, Mannheim, Germany). The urine was examined for volume, colour, turbidity, nitrite, ph, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity (by a urine refractometer), and sediment. In DEA Study 2, no blood and urine examinations were conducted Pathology and neuropathology for DEA studies In the DEA Study 1, from every test group, three animals of each sex were anaesthetized and killed by perfusion fixation using Sorensen s phosphate buffer as a rinsing solution followed by Karnovsky s fixative. These animals served for comprehensive neuropathological examinations. The relevant organs of the control and high-concentration group were embedded either in epoxy resin (mid-thigh sciatic nerve and the tibial nerve at knee) or in paraplast (brain: 6 cross sections, spinal cord: 2 cross sections). The animals not scheduled for perfusion were necropsied and assessed by gross pathology. A comprehensive histopathology investigation according to the respective test guidelines including cross sections of the nasal cavity (4 levels) and the larynx (3 levels) was performed in these animals. In the DEA Study 2 all animals were necropsied and assessed by gross pathology, and histopathology examination was conducted from all gross lesions. According to the focus on the respiratory tract, histopathological investigations of nasal cavity, larynx and trachea with bifurcation were performed in all test groups. Lungs, and mediastinal lymph nodes of the control and high exposure group were examined in addition. Nasal cavity and larynx of those recovery groups, which could contribute data on the reversibility of effects were also examined Pathology and neuropathology for TEA study The animals were sacrificed the day after the last exposure. As with DEA Study 1, 3 animals of each sex were perfused for neuropathological examinations. The sciatic nerves were embedded in epoxy resin for cross and longitudinal sectioning. The brain, the cervical cord (C3 C6), the thoracic cord (one part), and the lumbar cord (L1 L4) were embedded in paraplast. From the animals not scheduled for perfusion, the major organs, including brain, spinal cord and sciatic nerve, were fixed in 4% formaldehyde solution. Examinations of the haematoxylin eosin stained organs comprised respiratory tract (including longitudinal sections of pharynx/larynx region), brain (3 cross sections) and sciatic nerve. Brain weight measurements, macroscopical and histopathological examinations of the brain and sciatic nerve in the regular sacrificed animals did not reveal any neuropathological changes. In addition, clinical examinations did not give indications for any kind of treatmentrelated neurofunctional disorders. Therefore no further neuropathological examinations were carried out on the epoxy resin embedded or preserved material of the perfusion fixed animals Statistics for DEA and TEA studies For clinical examinations (body weight, body weight change, grip strength fore and hind limbs and hot plate test) the data were analyzed statistically using two-sided ANOVA (Cochran, 1957) and Dunnett s test (1955, 1964). For clinical chemistry and hematology, a one-way analysis of variance was done via the F-test (ANOVA; Winer, 1971). If the resulting p-value was less than 0.05 a pair-wise comparison of each test group with the control was carried out using the Dunnett s test (1955, 1964) for the hypothesis of equal means. Statistical analyses of absolute and relative organ weights were performed using non-parametric one-way analysis of variance (Kruskal Wallis H-test, Miller, 1981; Siegel, 1956). If the resulting p-value was less than 0.05, a pair-wise comparison of each group was done with the Wilcoxon U-test (Nijenhuis and Wilf, 1978; Hettmansperger, 1984) using the hypothesis of equal medians. The concentration response relation of laryngeal irritation was assessed using a benchmark multistage model posted on the web site of the US EPA ( epa.gov/ncea). 3. Results As results from range finding studies are briefly summarized in Section 2. This section is focused to the 3 main studies, DEA Study 1, DEA Study 2, and TEA study.

4 2176 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) Analytical test substance concentrations The measured mean concentrations of DEA and TEA were very close to the target concentrations with low daily variations (DEA Study ± 2.3, 153 ± 20 and 410 ± 45 mg/ ; DEA Study ± 0.3, 3.3 ± 0.8 and 8.2 ± 1.5 mg/ ; TEA Study 20 ± 0.8, 100 ± 6 and 500 ± 19 mg/ ). The DEA particle size distribution measured by cascade impactor analyses yielded mass median aerodynamic diameters (MMADs) between 0.6 and 1.9 lm for DEA Study 1 and between lm for DEA Study 2. The MMAD of the TEA aerosol particles were between 0.7 and 1.1 lm. No clearcut relation between particle size measurements and concentrations was present. Geometrical standard deviations were in the range of 2 to 3. Overall for both DEA and TEA a near to complete respirability of the particles was achieved, which ensured deposition in all compartments of the respiratory tract. Using a ventilation rate of 0.8 l/min and kg (Snipes, 1989) the test concentrations correspond to doses of about 5.8, 29 and 144 mg/kg/ day for TEA and 0.4, 0.9, 2.3, 4.3, 43 and 115 mg/kg/ day for DEA (c.f. Discussion section) Clinical examination DEA 1 study No animals died during the course of the study. The clinical examination revealed a clear substance related reduction in body weight development in male animals in the high exposure group when compared with the control (Table 2). No other differences from normal were noticed in any exposure group during prestudy and exposure period, including the FOB and ophthalmoscopic investigations. A few isolated significant differences from controls were observed in the motor activity measurements that were judged incidental because they were not time or concentration related DEA 2 study From the recovery groups, one female each of the control group and of the high exposure group died during exposure and recovery period, respectively. Both deaths were regarded as incidental. No substance-related clinical signs or effects on body weight and body weight change were observed TEA study No animals died during the course of the study. The low and intermediate concentration groups showed no clinical findings Table 3 Effects of TEA on body weight (g) and selected relative organ weights (mg/100 g) in male (m) and female (f) rats TEA Study (20 exposures) Control 20 mg/ 100 mg/ 500 mg/ Body weights Day-1 m 268 ± ± ± ± 9 Day ± ± ± ± 14 Day-1 f 186 ± ± ± ± 6 Day-27 f 225 ± ± ± ± 9 Organs Liver m 4044 ± ± ± ± 351 f 4217 ± ± ± ± 125 Kidneys m 705 ± ± ± ± 35 f 750 ± ± ± ± 23 Brain m 573 ± ± ± ± 39 f 805 ± ± ± ± 66 Lungs 56 ± ± ± ± 28 f 409 ± ± ± ± 23 Note: mean values and standard deviations are given (n = 10), statistical significance was not obtained. related to the treatment. In the animals of the high concentration group, bloody crusts on the nasal edges were noticed during the second half of the exposure period. The neurofunctional tests revealed isolated, statistically significant findings that were, however, not time and concentration related and thus assumed not to be caused by treatment (data not shown). Body weight data are provided in Table Clinical chemistry, hematology, and urinalysis DEA 1 study The following changes in clinical pathology parameters were present in the mid and high concentration groups, showing concentration dependence, if both concentrations were concerned: Exposure to 400 mg/ led to statistically significant decreases in red blood cells, hemoglobin, hematocrit and mean corpuscular volume in male and female animals (Table 4). Anisocytosis was only marginally increased at this concentration. In mid and highconcentration animals, enzyme examinations revealed slight, but significantly increased serum alkaline phosphatase in both sexes, and decreased serum alanine aminotransferase in males. In females of the mid and high-concentration groups, serum levels of calcium, total protein, albumin, and globulin were increased. An Table 2 Effects of DEA on body weight (g) and selected relative organ weights (mg/100 g) in male (m) and female (f) rats DEA Study 1 (65 exposures) DEA Study 2 (65 exposures) Control 15 mg/ 150 mg/ 400 mg/ Control 1.5 mg/ 3 mg/ 8 mg/ Body weights Day 0 m 280 ± ± ± ± ± ± ± ± 9 Terminal 1 m 433 ± ± ± ± 27 ** 327 ± ± ± ± 20 Day 0 f 200 ± ± ± ± ± ± ± ± 5 Terminal 1 f 266 ± ± ± ± ± ± ± ± 12 Organs Liver m 2834 ± ± ± ± 121 * 2532 ± ± ± ± 198 f 3171 ± ± ± 215 ** 3771 ± 204 ** 2624 ± ± ± ± 291 * Kidneys m 652 ± ± ± 45 * 734 ± 58 ** 629 ± ± ± ± 46 f 795 ± ± ± 39 ** 922 ± 72 ** 676 ± ± ± ± 41 Brain m 510 ± ± ± ± 40 * 630 ± ± ± ± 25 f 787 ± ± ± ± ± ± ± ± 43 Lungs 19 ± ± ± ± ± ± ± ± 12 f 451 ± ± ± ± ± ± ± ± 24 Note: mean values and standard deviations are given (DEA Study 1: n = 13 for body weight, n = 10 for organ weight; DEA Study 2: n = 10). * P < 0.05, ** P < 0.01, significantly different from control; DEA Study 1: Dunnet test; DEA Study 2: Kruskal Wallis H + Wilcoxon test (both two-sided). 1 DEA Study 1: Terminal=day 98; DEA Study 2: Terminal=day 103.

5 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) Table 4 Effects of DEA on red blood cell parameters in male (m) and female (f) rats DEA study (65 exposures) Control 15 mg/ 150 mg/ 400 mg/ Erythrocytes (tera/l) m 8.6 ± ± ± ± 0.3 ** f 7.7 ± ± ± ± 0.2 ** Haemoglobin (mmol/l) m 9.7 ± ± ± ± 0.4 ** f 9.0 ± ± ± ± 0.2 ** Haematocrit (%) m 45.7 ± ± ± ± 2.0 ** Mean corpuscular volume (femtoliter) * P < ** P < f 409 ± ± ± ± 9 ** m 53.1 ± ± ± ± 1.9 * f 53 ± ± ± ± 1.0 * effect on total protein and albumin appeared in males only as a tendency towards higher concentrations. Urinalysis revealed a significantly increased blood content in animals of both sexes exposed to 400 mg/. Moreover, males of the two higher concentrations excreted significantly elevated amounts of renal tubular epithelial cells and sometimes granular casts TEA study No treatment related alterations in clinical chemistry and hematology were observed Pathology and neuropathology for DEA studies DEA 1 study Significant relative weight increases were observed in liver and kidneys of both sexes of the high exposure group (400 mg/ ), as well as in females (both organs) and in males (kidneys, only) at the intermediate exposure group (150 mg/, Table 2). The relative weight of the brain was significantly increased in high-concentration male animals. This is a typical finding for animals with reduced body weights relative to controls. Gross lesions on the epithelium of glandular stomach, i.e. erosions, were noticed in females of the intermediate and high exposure groups with concentration-dependent increase. No other macroscopic findings were attributed to DEA. The most apparent microscopic alterations were seen in the URT of both sexes (Table 5). All test animals of the high, mid and low exposure concentration (15 mg/ ) showed a focal squamous metaplasia of the ventral laryngeal epithelium at the base of the epiglottis (level 1). Additionally, animals of the mid and high exposure group revealed a concentration-dependent increase in laryngeal squamous hyperplasia, as well as in incidence and severity of local inflammation of larynx and trachea. In 2 of the high-concentration male animals inflammatory polyps were observed in the larynx (Figs. 2 6). Histopathological examination in kidneys revealed minimal or slight tubular hyperplasia in some females and intratubular lithiasis that was slightly more pronounced than in controls in some male animals of the mid and high-concentration groups. Neuropathology did not reveal morphological evidence of neurotoxicological effects of DEA. Table 5 Incidence and severity of changes of larynx following DEA exposure (blank fields imply no findings) DEA Study 2 DEA Study mg/ 3 mg/ 8 mg/ 15 mg/ 150 mg/ 400 mg/ Metaplasia, squamous (level 1: at the base of epiglottis) Present 3/0 9/9 10/10 10/10 10/10 Metaplasia, squamous (level 2: at the region of ventral pouch and arytenoid cartilages) Present 2/0 Hyperplasia, squamous Grade 1 3/1 0/1 Grade 2 3/6 2/7 Grade 3 4/1 Grade 4 2/0 Inflammatory cells Grade 1 1/0 0/1 Grade 2 2/2 1/3 Grade 3 1/0 Chronic inflammation Grade 2 1/4 2/1 Grade 3 9/6 5/8 Grade 4 3/1 n = 10 /sex. Note: The incidences are given for male (M) and female (F) animals (M/F). The severity of the findings is graded semiquantitatively in a grading scheme with 5 gradings as follows: Grade 1 minimal. Grade 2 slight. Grade 3 moderate. Grade 4 marked/severe. Grade 5 massive (not observed). For the focal squamous metaplasia a grading was not performed as this lesion was only noted as present, if this was the case. Fig. 2. Control (Study 1) transverse section through the larynx close to the base of the epiglottis. Arrow indicates ciliated laryngeal epithelium DEA 2 study Although not present in the low concentration group (15 mg/ ) of DEA Study 1, there was a significant increase of relative liver weight in high-concentration (8 mg/ ) females (Table 2). This shows the variability of different populations of animals used in independent studies. No significant weight changes were noticed in the recovery group after 3-month post-exposure (data not shown). At the high concentration, similar effects as in the DEA Study 1 were observed in the larynx (Table 5). Male and female animals revealed a focal squamous metaplasia of the laryngeal epithelium (level 1; Fig. 7 and 8) and submucosal inflammation in a few cases (Fig. 9). In 2 males, squamous metaplasia was also observed at level 2 of the larynx. Squamous metaplasia was noted in 3 of 10 males of the intermediate exposure group, which was not accompanied by epithelial inflammatory cell infiltration. In control animals, grade 1 (1 animal of each sex) and 2 (1 male) inflammatory cell infiltration was also observed occasionally. Examination of the URT after the recovery period of three months revealed no histological changes. No other parts of the RT showed substance-induced histomorphological changes at any concentration

6 2178 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) Fig. 3. DEA male-concentration group: 400 mg/ (Study 1) transverse section at a comparable level as in the control in Fig. 2 with the finding of a squamous metaplasia and a moderate hyperplasia of the normally ciliated laryngeal epithelium (arrows). Fig. 5. DEA male-concentration group: 400 mg/ (Study 1) transverse section at a comparable level as the level shown in the control of Fig. 4 with squamous metaplasia and a marked hyperplasia and focal ulceration (arrow) of the normally ciliated laryngeal epithelium. Fig. 4. Control (Study 1) transverse section through the larynx close to the base of the epiglottis. Arrow indicates ciliated laryngeal epithelium. in this study. No treatment related histopathological changes were observed in the larynx at 1.5 mg/ Pathology and neuropathology for TEA study No treatment related changes in organ weights were observed. Focal minimal to moderate inflammation in the submucosa of the larynx was observed histopathologically in a concentration related manner in male (all exposure groups) and in female (intermediate and high exposure groups) animals (Table 6). No substance-related effects were observed in other putative target organs, i.e. nasal cavity, trachea, liver, and kidneys. No indications of neurotoxicological effects of TEA were seen at pathological and histopathological examinations. 4. Discussion In the present studies, DEA and TEA showed toxicity to the upper respiratory tract (URT), in form of epithelial changes in the Fig. 6. DEA male-concentration group: 400 mg/ (Study 1) transverse section at a comparable level as the level shown in the control (Fig. 4) with development of an inflammatory polyp with a marked squamous hyperplasia and keratinization (arrow) of the normally ciliated laryngeal epithelium. larynx in a concentration-dependent manner (Tables 5 and 6; Fig. 10). In addition to these local portal of entry effects, DEA induced substance related systemic changes in hematology (regenerative, normochromic, microcytic anemia), clinical chemistry (changes in liver enzymes, calcium and blood protein levels) and urinalysis (blood, tubular cell and granular cast excretion) that were accompanied by changes in liver and kidney weights and histopathological alterations in the kidneys. In contrast, TEA did not induce systemic toxicity at the tested concentrations Local toxicity in the respiratory tract DEA exposed rats show increased incidences of squamous metaplasia at the base of the epiglottis and increased incidence and severity of inflammation and squamous hyperplasia of the laryngeal epithelia in both 90-day studies; (DEA Studies 1 and 2) at or above 3 mg/ (starting with squamous metaplasia in males,

7 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) Fig. 7. Control (Study 2) transverse section through the larynx close to the base of the epiglottis at the area of the arythenoid cartilage. Arrows indicate laryngeal epithelium below the ventral glands and ciliated epithelium (inlet). Fig. 8. DEA - male-concentration group: 8 mg/ (Study 2) - transverse section at a comparable level as in the control with a minimal focal squamous metaplasia of the normally ciliated laryngeal epithelium (arrows in overview and inlet). Those changes at this area belong to the first local signs of irritation of the laryngeal epithelium and would be according to recommendations of a recent ESTP expert workshop re-worded as focal epithelial alteration of the larynx. only). At 8 mg/, all lesions in the larynx were reversible within three months after the end of the exposure period. No examinations on reversibility have been conducted for the higher exposure concentrations in DEA Study 1. The observed squamous metaplasia at the base of the epiglottis, i.e. the replacement of laryngeal respiratory epithelium by squamous epithelium, appears to be a frequently observed reaction of the mucosal epithelium to the inhalation of irritant substances (Burger et al., 1989). Burger et al. state that the base of epiglottis is sensitive to metaplasia due to local dosimetry leading to a relatively high tissue dose in the larynx as compared e.g. to the nasal cavity, in which no epithelial changes were observed. Moreover, anatomically this region is the caudal extension of the epiglottis, which is covered with squamous epithelium. Burger et al. (1989) argue that the observed metaplasia should be considered as an adaptive instead of a toxic response. This is in agreement with results of a recent expert workshop on the finding larynx squamous metaplasia (ESTP expert workshop on larynx squamous metaplasia, final minutes, 2007). For an overview of larynx squamous metaplasia in rats also see Osimitz et al. (2007). After slide review of laryngeal epithelial alterations from inhalation studies with irritating compounds, the experts stated that minimal and focal laryngeal squamous metaplasia at the base of the epiglottis with just absence of cilia and flattening of the normally cuboidal, laryngeal epithelium does not fulfill the criteria of a complete laryngeal squamous metaplasia (Dungworth et al., 2001). The workshop introduced for such focal and minimal lesions at the base of the epiglottis the new term of a laryngeal epithelial alteration. This morphological correlate for only a slight irritation was regarded to be adaptive in character, as no significant dysfunction of the larynx is to be expected. Hence, the reversible squamous metaplasia at 3 mg/ is regarded to be of borderline toxicological significance and is assessed to represent the no observed adverse effect concentration (NOAEC). A 90-day No Observed Effect Concentration (NOEC) is shown at 1.5 mg/. The 28-day exposure to TEA also led to inflammation in the laryngeal mucosa, however, the extent and severity of which were less pronounced compared to the 90-day DEA studies (Fig. 10): 6-h exposures to all concentration resulted in concentrationdependent focal inflammation of larynx epithelium in part of the animals. Larynx inflammations were also observed after five exposures in the TEA range finding study at 200 and 400 mg/ (data not shown). An unpublished repeated concentration inhalation

8 2180 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) Fig. 9. DEA male-concentration group: 8 mg/ (Study 2) minimal focal squamous metaplasia of the normally ciliated laryngeal epithelium (arrows) with submucosal inflammatory cell infiltrates. Table 6 Incidence and severity of changes of larynx following TEA exposure (blank fields imply no findings) TEA study 20 mg/ 100 mg/ 500 mg/ Inflammation, focal Grade 1 2/0 1/1 0/2 Grade 2 1/0 1/1 3/2 Grade 3 1/0 n = 7 /sex. Note: The incidences are given for male animal/female animals. The severity of the findings is graded semiquantitatively as follows: Grade 1 minimal. Grade 2 slight. Grade 3 moderate. Grade 4 marked/severe (not observed). Grade 5 massive (not observed). mean score (grade 1:minimal...grade 3:moderate) Grading of laryngeal inflammation in the DEA 90-day studies and the TEA 28-day study for male and female rats mg/ DEA males females mg/ TEA Fig. 10. Grading of laryngeal inflammation in the DEA 90-day studies and the TEA 28-day study for male and female rats (n = 10 /sex (DEA) and n = 7 /sex (TEA)). study sponsored by the National Toxicology Program (NTP) with F 344 rats (12 exposure days, 6 h/d, 5 animals/sex/group, 0, 125, 250, 500, 1000, 2000 mg/ TEA) showed mild acute inflammation of the laryngeal submucosa at 2000 mg/ (3/5 males; 2/5 females). The same lesion of minimal severity was observed in 1/5 males and 1/5 females at 1000 mg/ and in 1/5 males and 2/5 females from the 125 mg/ concentration group. The lesions were not observed in either sex of the 500 or 250 mg/ group or the control group. Laryngeal inflammation was characterized by an infiltration of few lymphocytes into the mucosa and around the mucosal glands. It often appeared only in one section out of several from the same larynx (Mosberg et al., 1985a). An identically designed study with B6C3F 1 mice showed similar effects as in rats with no laryngeal effects at 125 mg/ or in controls (Mosberg et al., 1985b). In consequence, these earlier studies with a second strain of rats and mice suggests that only slight effects have to be expected after subacute exposure to 125 mg/ or above (Mosberg et al., 1985a,b). In the present study, however, in single animals minimal signs of inflammatory reactions were also noted at a concentration of 20 mg/, while other animals showed no adverse effects at concentrations more than 20-fold higher. The higher heterogeneity of individual susceptibility against TEA than DEA has also been observed in other studies (Mosberg et al., 1985a,b). At the present state of the investigations no definitive explanation for this effect can be given. Comparing the local irritant effects of TEA and DEA it can be concluded that DEA has a higher irritant effect on the respiratory tract. A possible but somewhat speculative explanation for the higher local toxicity of DEA compared to TEA could be related to its higher pk a -value (8.88 for DEA vs for TEA at 25 C, respectively). In contrast to the DEA studies, exposure to TEA resulted only in submucosal inflammation but not in squamous metaplasia or squamous hyperplasia of the ciliated laryngeal epithelium. It should be pointed out, however, that the sensitivity to detect these changes was less than in the DEA study. This is related to the fact, that in the TEA study the larynx was examined in longitudinal sections, whereas in the DEA studies cross-sectional levels were used according to Sagartz et al. (1992), the latter method being more sensitive with respect to the detection of small focal changes. The fact that even at 500 mg/ no signs of metaplasia were observed makes it less likely that this particular change would play a significant role in TEA mediated laryngeal irritation. Extrapolation of laryngeal irritation data, conducted by calculation of benchmark concentrations for a 5% incidence of mucosal inflammation without consideration of severity using a multistage model with 95% confidence level, leads to point estimates (with lower confidence values in brackets) for DEA exposed rats of 7.6 (1.94) mg/ or 5.0 (2.0) mg/ in male and female animals, respectively. The respective benchmark concentrations for TEA were 36.8 (14.8) mg/ or 26.3 (14.1) mg/. The lower confidence values calculated for DEA are just in between the NOEC of 1.5 mg/ and NOAEC of 3 mg/ corroborating these values. This puts confidence into the use of the respective values in deriving the NOAEC for the TEA study. To assess longer term toxicity from a study with shorter duration usually time extrapolation factors are employed. For local effects in the respiratory tract in inhalation studies, a factor of 3 is proposed in the literature to extrapolate from sub-acute to subchronic exposure (Kalberlah et al., 1999). For TEA this means that based on a sub-acute benchmark lower confidence value of 14 mg/ a sub-chronic NOAEC of 4.7 mg/ can be calculated. This concentration is slightly higher than the NOAEC for DEA, which fits well to the somewhat lower alkalinity of TEA leading to a slightly lower irritation potential. These considerations could be used for the derivation of occupational exposure levels for the two ethanolamines Systemic toxicity Following inhalation exposure of DEA moderate effects on morphology and physiology of liver and kidneys were observed down

9 A.O. Gamer et al. / Food and Chemical Toxicology 46 (2008) to a concentration of 150 mg/ (internal dose: approx. 40 mg/kg and day, calculated using a default respiratory volume and assuming 100% absorption). An internal dose of 40 mg/ kg d as an effect dose after inhalation compares well to similar effects after oral and dermal exposure at or above approximately 50 mg/kg/day reported by Knaak et al. (1997). Somewhat lower systemic effect concentrations have been reported in an abstract by Hartung et al. (1970): Liver and kidney effects were found after continuous exposure of rats to 63 mg/ for 216 h. Exposure to 91 mg/, workday schedule, for 13 weeks resulted in a depression of growth rate, increased lung and kidney weight, and some death among male animals. Such effects have not been observed in the experiments reported here at comparable and higher concentrations (Table 2). As no details were provided (only abstract published) these results cannot be further assessed. Following inhalation exposure of TEA to concentrations up to 500 mg/ no signs of systemic toxicity were observed. These results are in agreement with the unpublished NTP inhalation study by Mosberg et al. (1985a), where TEA concentrations at or above 500 mg/ for 12-day period (6 h/d; 5 d/w) resulted in increased kidney weight in male and female F344 rats. At this concentration, no histological alterations were observed in the kidneys. For B6C3F 1 -mice, less consistent kidney effects were seen at 500 mg TEA/ or higher (Mosberg et al., 1985b). In the current study with 20 exposures only an insignificant increase of kidney weights occurred at 500 mg/ (Table 3). Using the default calculation indicated above, 500 mg/ would correspond to an internal dose of 140 mg/kg and day. In an oral study with rats, organ weight increases of liver and kidneys have been noticed not below 170 mg/kg/day for a 90-day administration (Anon., 1983). For DEA, clinical symptoms indicating a possible neurotoxic effect such as tremors were observed in a well conducted 13-week drinking water study in Fischer F 344 rats at dose levels corresponding to 202 mg/kg bw in males and 124 mg/kg bw in females [Melnick et al., 1994; NTP, 1992]. These doses caused other marked systemic toxicity and even mortality. Histopathology revealed demyelination of the medulla and spinal chord in both sexes at the above mentioned dose levels with a NOAEL in males of 97 mg/kg bw and females of 57 mg/kg bw. These histopathological changes were noted to a milder degree after dermal exposure of the same rat strain for 13 weeks at a dose level of 500 mg/kg bw in both gender and 250 mg/kg bw in females only. Thus a NOAEL was established at 125 mg/kg bw in females and 250 mg/kg bw in males. It is noteworthy that there were no clinical symptoms indicating a neurotoxic effect and demyelination was restricted to the medulla. No clinical symptoms as described in the 90-day drinking water study were noted and even a detailed FOB observation did not indicate any neurotoxic effects in our studies. In addition, detailed histopathology of the central nervous system (6 brain levels and 3 transverse sections of the spinal cord) after an in vivo fixation procedure (perfusion fixation) did not reveal demyelination nor any other treatment-related, neurostructural effects. This might be explained in that such doses were not achieved in our inhalation study (high concentration tested corresponds to a dose of 115 mg/ kg/day). No clinical symptoms indicative of neurotoxicity or any of the above described histopathological changes were noted in Fischer F344 rats treated dermally in a 13-week study with Triethanolamine at doses up to 2000 mg/kg bw (NTP, 1999b) and no systemic effects were noted in the present 28-day inhalation study Metabolism and mechanistic considerations Both, DEA and TEA appear to be well absorbed via all exposure routes (Knaak et al., 1997; Kohri et al., 1982). Dermal penetration was 80% of the applied dose of TEA at 72 h after application to the skin of mice (NTP, 1999b). The nearly complete disappearance of DEA and TEA from the gastrointestinal tract occurred within minutes or few hours (Mathews et al., 1997; Kohri et al., 1982; NTP, 1992). Following absorption of oral administered radiolabelled DEA, a high portion of approx. 70% remained in the liver and kidney tissue, whereas the residual substance was excreted mainly via urine, and faeces to a small extent (Mendrala et al., 2001). In urine, unchanged DEA and several metabolites with significant amounts of N-methyl-DEA and dimethyl-dea were found. In contrast to DEA, approximately 73% of TEA was excreted unchanged within 24 h (53% from the urine and 20% from the faeces) following administration of a single oral dose or repeated administration (Kohri et al., 1982; NTP, 1999b). In contrast to DEA and TEA, MEA is of natural occurrence as a structural component of phospholipids, which are the most important constituents in membrane bilayers. DEA and TEA, due to their structural similarity to MEA, might interact with the phospholipid metabolism and alter the membrane structure (NTP, 1992, 1999a). The observed differences in the toxicity of DEA and TEA appear to correlate with their different metabolism and the way they interact with membranes. MEA phospholipids are very abundant in the membrane bilayers. Diethanolamine seems to be incorporated into phospholipids, instead of monoethanolamine and choline (Barbee and Hartung, 1979a; Blum et al., 1972). The resulting diethylamino-phospholipides possess a longer half-life and therefore can accumulate in the body. DEA accumulation, especially in liver and kidneys but also in brain and spleen, was observed by Mathews et al. (1995, 1997). As a consequence, adverse effects may be induced by modification of cell membrane function or changes in the membrane enzyme activities. Alterations in hepatic and renal phospholipid metabolism were observed in rats treated with DEA (Barbee and Hartung, 1979a; Lundberg, 1992). By interference of DEA with phospholipid metabolism, a loss of mitochondrial integrity developed, that could influence physiological processes (Barbee and Hartung, 1979b). These changes might also be related to the observed anemia, as an accumulation of radiolabelled DEA in erythrocytes was observed by Mendrala et al. (2001). In contrast to the mechanism reported for DEA, TEA is not incorporated into hepatic phospholipids (Stott et al., 2000). However, the substance inhibited the incorporation of labeled phosphate into phospholipids (Anon., 1983). This different pattern might be responsible for the lower systemic toxicity of TEA. 5. Conclusions The results of the present study and a comparison with the data available in the literature indicate that TEA has a somewhat lower URT irritation potential than DEA after aerosol inhalation exposure, the target organ being the larynx. The NOAEC for local irritation of DEA was determined to be 3 mg/, following a 90-day exposure. For TEA a 90-day NOAEC of 4.7 mg/ was calculated based on a 28-day No Adverse Effect Concentration of about 14 mg/ derived as the lower confidence value of a benchmark calculation. Using the benchmark value is plausible as laryngeal irritation was only present in some male animals exposed to 20 mg/. With respect to systemic toxicity, TEA induced no adverse effects up to a concentration of 500 mg/, whereas for DEA a NOEC was observed at 15 mg/, based on some systemic effects at 150 mg/. Kinetic studies have shown that TEA, in contrast to DEA, demonstrates no strong potential for bioaccumulation. Therefore it seems implausible, that systemic effects following exposure to TEA would change in nature (i.e. type of lesion or severity of a lesion) nor in incidence (quantity of a particular lesion) when extending the exposure period from 28 days to 90- days. This can also be inferred from studies using oral administration of DEA and TEA with comparable duration.