U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 SCREENING-LEVEL HAZARD CHARACTERIZATION SPONSORED CHEMICAL 2-Amino-2-hydroxymethyl-l,3-propanediol (TRIS AMINO) CASRN 77-86-1 SUPPORTING CHEMICALS 2-Amino-2-methyl-l,3-propanediol (AMPD) CASRN 115-69-5 2-Amino-2-methyl-l-Propanol (AMP) CASRN 124-68-5 The High Production Volume (HPV) Challenge Programi was conceived as a voluntary initiative aimed at developing and making publicly available screening-level health and environmental effects information on chemicals manufactured in or imported into the United States in quantities greater than one million pounds per year. In the Challenge Program, producers and importers of HPV chemicals voluntarily sponsored chemicals; sponsorship entailed the identification and initial assessment of the adequacy of existing toxicity data/information, conducting new testing if adequate data did not exist, and making both new and existing data and information available to the public. Each complete data submission contains data on 18 internationally agreed to "SIDS" (Screening Information Data Setll 2) endpoints that are screening-level indicators of potential hazards (toxicity) for humans or the environment. The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is evaluating the data submitted in the HPV Challenge Program on approximately 1400 sponsored chemicals by developing hazard characterizations (HCs). These HCs consist of an evaluation of the quality and completeness of the data set provided in the Challenge Program submissions. They are not intended to be definitive statements regarding the possibility of unreasonable risk of injury to health or the environment. The evaluation is performed according to established EPA guidance2-3 and is based primarily on hazard data provided by sponsors; however, in preparing the hazard characterization, EPA considered its own comments and public comments on the original submission as well as the sponsor's responses to comments and revisions made to the submission. In order to determine whether any new hazard information was developed since the time of the HPV submission, a search of the following databases was made from one year prior to the date of the HPV Challenge submission to the present: (ChemID to locate available data sources including Medline/PubMed, Toxline, HSDB, IRIS, NTP, ATSDR, IARC, EXTOXNET, EPA SRS, etc.), STN/CAS online databases (Registry file for locators, ChemAbs for toxicology data, RTECS, Merck, etc.), Science Direct and ECHA4. OPPT's focus on these specific sources is based on their being of high quality, highly relevant to hazard characterization, and publicly available. OPPT does not develop HCs for those HPV chemicals which have already been assessed internationally through the HPV program of the Organization for Economic Cooperation and Development (OECD) and for which Screening Initial Data Set (SIDS) Initial Assessment 1 U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm. 2 U.S. EPA. EtPV Challenge Program - Information Sources; http://www.epa.gov/chemrtk/pubs/general/guidocs.htm. 3 U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm. 4 European Chemicals Agency, http://echa.europa.eu. ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Reports (SIAR) and SIDS Initial Assessment Profiles (SIAP) are available. These documents are presented in an international forum that involves review and endorsement by governmental authorities around the world. OPPT is an active participant in these meetings and accepts these documents as reliable screening-level hazard assessments. These hazard characterizations are technical documents intended to inform subsequent decisions and actions by OPPT. Accordingly, the documents are not written with the goal of informing the general public. However, they do provide a vehicle for public access to a concise assessment of the raw technical data on HPV chemicals and provide information previously not readily available to the public. 2 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Chemical Abstract Service Registry Number (CASRN) Sponsored Chemical 77-86-1 Supporting Chemicals 115-69-5 124-68-5 Chemical Abstract Index Name Sponsored Chemical 1,3-Propanediol, 2-amino-2-(hydroxymethyl)-(TRIS AMINO) Supporting Chemicals 1,3-Propanediol, 2-amino-2-methyl- (AMPD) 1-Propanol, 2-amino-2-methyl-(AMP) Structural Formulae Sponsored Chemical h2n HO^WoH HO SMILES: OCC(N)(CO)CO Supporting Chemicals OH H9N J ji, HO SMILES: OCC(N)(CO)C H,N qui 2 v 3 >CHS HO SMILES: OCC(N)(C)C Summary 2-Amino-2-(hydroxymethyl)-l,3-propanediol (TRIS AMINO) is a white, crystalline solid with low vapor pressure and high water solubility. It is expected to have high mobility in soil. Volatilization is low since this substance will primarily exist as a cation under environmental conditions and cations do not volatilize. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is moderate. TRIS AMINO is not readily biodegradable. TRIS AMINO is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). The acute oral toxicity of TRIS AMINO to mice and rats is low. In a 90-day oral gavage repeated-dose toxicity study with the supporting chemical, 2-amino-2-methyl-l-propanol (AMP), at pH 11 in rats, decreased survival was seen at the lowest dose of 500 mg/kg-day, and no 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 animals survived at 1700 mg/kg-bw/day; the NOAEL is not established. No mortalities occurred at pH 7; however, limited data were reported and so the NOAEL was not determined. In a dietary 8-week repeated-dose toxicity study in rats with the supporting chemical AMP, a dose- dependent increase in hepatocyte vacuolation was observed in rats starting at the lowest dose (244 in males and 436 in females); the NOAEL is not established. In a similar dietary 8-week repeated dose study in mice with the supporting chemical AMP, no treatment-related effects were observed up to the highest dose tested; the NOAEL is 14.4 and 27.7 mg/kg/day for males and females, respectively. In a 90-day dietary repeated dose toxicity study in dogs with the supporting chemical AMP, decreased prothrombin time in males, increased liver and/or livenbody weight ratios in both sexes, and minimal fatty changes in the liver were observed at 22 mg/kg-bw/day; the NOAEL is 0.9 mg/kg-bw/day. In an oral gavage combined reproductive/developmental toxicity screening test in rats with TRIS AMINO, significant point of entry effects on the stomach including irritation and histopathology changes occurred at 300 mg/kg-day; the NOAEL for local effects in parental animals is 100 mg/kg-day. In this study, no effects on reproductive or developmental parameters were observed at the doses tested; the NOAEL for reproductive and developmental toxicity is 1000 mg/kg-day, the highest dose tested. The supporting chemical, AMP, was not mutagenic to bacteria and mammalian cells in vitro, and did not induce micronuclei in mice in vivo. TRIS AMINO administered by injection is irritating to rat skin, but is not irritating to rabbit eyes. The supporting chemical AMP is irritating to rabbit skin and is not sensitizing to guinea pig skin. For the supporting chemical, AMP, the 96-h LCso values for fish and aquatic invertebrates are 184, and 179 mg/L, respectively. For TRIS AMINO, the 96-h ECso for aquatic plants is >100 mg/L. No data gaps were identified under the HPV Challenge Program. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 The sponsor, Dow Chemical Company, submitted a Test Plan and Robust Summaries to EPA for 2-amino-2-hydroxymethyl-l,3-propanediol (also known as tris(hydroxymethyl)aminomethane or TRIS AMINO; CASRN 77-86-1; CA Index name: 1,3-propanediol, 2-amino-2-(hydroxymethyl)- dated November 14, 2006. EPA posted the submission on the ChemRTK HPV Challenge website on December 22, 2006 (http://www.cpa.gov/oppt/chciTntk/pubs/suiTiiTiarics/2aiTiino2h/ ltm). EPA comments on the original submission were posted to the website on May 13, 2009. Public comments were also received and posted to the website. Revised test plans and summaries were received and posted to the ChemRTK HPV Challenge website on December 1, 2009 and July 2, 2012. Justification for Supporting Chemicals Two supporting chemicals, 2-amino-2-methyl-1,3-propanediol (AMPD, CASRN 115-69-5) and 2-amino-2-methyl-l-propanol (AMP, CASRN 124-68-5), were proposed based on similarity in structure, physical chemistry, environmental fate, and ecological and mammalian toxicity to TRIS AMINO. The test plan provides sufficient evidence for the use of the supporting chemicals for data for aquatic invertebrates. For human health effects, even though the available information is not conclusive, AMP can be used as a worst-case supporting chemical for the repeated-dose toxicity endpoint due to its relatively higher lipid solubility. Comparison of reproductive and developmental toxicity studies for AMP and TRIS AMINO as well as an apparent lack of toxicity in humans by the known use of TRIS AMINO as a pharmaceutical buffering agent by the intravenous route both support this assumption. AMP has a published Hazard Characterization document (http://www.epa.gov/chemrtk/hpvis/hazchar/124685 AMP March 2012.pdf) and an ECHA dossier for REACH registration (http://www.echa.europa.eu/web/guest/information-on- chemical s/regi stered-sub stances). 1. Chemical Identity 1.1 Identification and Purity TRIS AMINO is a white crystalline solid used in a wide variety of applications as a buffer, solubilizer, and neutralizing agent. No information on the purity of the chemical is provided in the robust summaries. 1.2 Physical-Chemical Properties TRIS AMINO is a white, crystalline solid with low vapor pressure and high water solubility. The physical-chemical properties of TRIS AMINO are summarized in Table 1. 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 1. Physical-Chemical Properties of TRIS AMINO and Supporting Chemicals1 Property SPONSORED CHEMICAL TRIS AMINO SUPPORTING CHEMICAL AMPD SUPPORTING CHEMICAL AMP CASRN 77-86-1 115-69-5 124-68-5 Molecular Weight 121.14 105.14 89.14 Physical State White, crystalline solid White, crystalline solid White, crystalline solid Melting Point 171-172°C (measured) 110 °C (measured) 30.5 °C (measured) Boiling Point 219-220°C at 10 mm Hg (measured); >300°C (estimated)2 151 - 152 at 10 mm Hg (measured); 288 °C (estimated)2 165.5 mm Hg (measured) Vapor Pressure 2.7xl0"5 mm Hg at 25°C (estimated)2 6.5xl0"3 mm Hg at 20°C (measured) 0.34 mm Hg at 20°C (measured) Dissociation Constant pKb = 5.7 (measured) pKb = 5.2 (measured)3 pKb = 3.8 (measured)3 Henry's Law Constant <1.0><10"10 atm-m3/mole (estimated)4 <1.0xl0"10 atm-m3/mole (estimated)4 1,2x 10"7 atm-m3/mole (estimated)4 Water Solubility 8.00xl05 mg/L at 25°C (measured) 2.5xl06 mg/L at 25°C (measured) Miscible Log Kow -2.31 at 20 °C (measured) -3.8 (measured) -0.63 'The Dow Chemical Company. 2009. Test Plan and Robust Summary for 2-Amino-2-hydroxymethyl-l,3- propanediol. December 2009. Available online at http://www.epa.gov/chemrtk/pubs/summaries/2amino2h/cl6455tc.htm as of June 15, 2012. 2N0M05. 1987. Programs to Enhance PC-Gems Estimates of Physical Properties for Organic Compounds. The Mitre Corp. 3SRC. 2012. The Physical Properties Database (PHYSPROP). Syracuse, NY: Syracuse Research Corporation. Available online at http://www.svrres.com/esc/phvsprop.htm as of June 15, 2012. 4U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of June 15, 2012. 2. General Information on Exposure 2.1 Production Volume and Use Pattern CASRN 77-86-1 may be produced by reduction or catalytic hydrogenation of the corresponding nitro compound. CASRN 77-86-1 had an aggregated production and/or import volume in the United States between 1 and 10 million pounds during calendar year 2005. Industrial processing and uses, and commercial and consumer uses for the chemical were claimed confidential. The Test Plan states that CASRN 77-86-1 is used as an emulsifier for 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 cosmetics, mineral oil and wax emulsions, leather dressings, textiles, cleaners, pharmaceuticals, and as a chemical intermediate buffer. Table 2. Environmental Fate Properties of TRIS AMINO1 Property SPONSORED CHEMICAL TRIS AMINO SUPPORTING CHEMICAL AMPD SUPPORTING CHEMICAL AMP CASRN 77-86-1 115-69-5 124-68-5 Photodegradation Half-life 3.8 hours (estimated)2 4.3 hours (estimated)2 5.0 hours (estimated)2 Hydrolysis Half- life Stable Stable Stable Photodegradation Half-life 3.8 hours (estimated)2 4.3 hours (estimated)2 5.0 hours (estimated)2 Hydrolysis Half- life Stable Stable Stable Biodegradation 0% after 28 days (not readily biodegradable) 40% after 28 days (not readily biodegradable) 96.7% after 22 days (inherently biodegradable) Bioaccumulation Factor BAF = 0.9 (estimated)2 BAF = 0.9 (estimated)2 BAF = 0.9 (estimated)2 Log Koc 0.0 (estimated)2 0.0 (estimated)2 0.4 (estimated)2 Fugacity (Level III Model)2 Air (%) Water (%) Soil (%) Sediment (%) <0.1 37.7 62.2 <0.1 <0.1 37.9 62.0 <0.1 0.8 42.5 56.6 0.1 Persistence3 P2 (moderate) PI (low) PI (low) Bi oaccumul ati on3 Bl (low) Bl (low) Bl (low) 'The Dow Chemical Company. 2009. Test Plan and Robust Summary for 2-Amino-2-hydroxymethyl-l,3- propanediol. December 2009. Available online at: http://www.epa.gov/chemrtk/pubs/summaries/2amino2h/cl6455tc.htm as of June 15, 2012. 2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of June 15, 2012. 3Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204. 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 2.2 Environmental Exposure and Fate TRIS AMINO is expected to have high mobility in soil. TRIS AMINO achieved 0% biodegradation at initial concentrations of 8.7 and 13 mg/L over the course of a 28-day incubation period using an activated sludge inoculum and the closed bottle test (OECD 301D) test and was considered not readily biodegradable. In a second closed bottle test, the supporting chemical , AMPD, was 40% degraded over 28 days at an initial concentration of 2 mg/L. An inherent MITI (OECD 302C) test showed that supporting chemical AMP was 96.7% degraded after 22 days and was inherently biodegradable. Volatilization is low since TRIS AMINO will primarily exist as a cation under environmental conditions and cations do not volatilize. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is moderate. TRIS AMINO is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). The environmental fate properties of TRIS AMINO are provided in Table 2. Conclusion: TRIS AMINO is a white, crystalline solid with low vapor pressure and high water solubility. It is expected to have high mobility in soil. Volatilization is low since this substance will primarily exist as a cation under environmental conditions and cations do not volatilize. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is moderate. TRIS AMINO is not readily biodegradable. TRIS AMINO is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of health effects data for SIDS and other endpoints is provided in Table 3. The table also indicates where data for the supporting chemical AMP are read-across (RA) to the sponsored chemical. Acute Oral Toxicity TRIS AMINO (CASRN 77-86-1) (1) Mice (10/dose; sex and strain not specified) were administered TRIS AMINO via oral gavage at 2000, 3500, 5000, 7000 or 10,000 mg/kg. Additional information including mortality data was not provided. LDso = 5500 mg/kg (2) Wistar rats and Swiss mice (sex and number per dose not specified) were administered solutions of 20% and 5% respectively of TRIS AMINO via oral gavage at 1000, 2000 or 3000 mg/kg. No mortalities were observed. LD50 > 3000 mg/kg 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Repeated-Dose Toxicity AMP (CASRN124-68-5, supporting chemical) (1) In a 90-day oral gavage repeated-dose study, CD rats (male and female) were gavaged with 0, 500, 750, 1100 or 1700 mg/kg-day AMP for 5 days/week at both pH 7 and 11. At pH 11, decreased survival was seen at all doses, and no animals survived at 1700 mg/kg-day. Abdominal swelling was seen in pH 11 rats at 1100 and 1700 mg/kg-day; all these rats later died. No treatment-related mortality was seen in the pH 7 rats. Clinical signs of toxicity (e.g. hyperventilation, hyperirritability, hyperactivity, convulsions, and neuromuscular paralysis) were noted largely in the animals given pH 11 AMP, but to a lesser extent in pH 7 AMP as well. Skin exfoliation and some ulceration as well as hair loss were seen. Nasal and mouth bleeding was also noted. The lowest dose(s) at which these symptoms were observed were not stated. There were no significant differences in body weights. AMP-administered rats appeared to drink more frequently than controls. Neither the pH 7 nor the pH 11 females showed significant differences in hematocrit, hemoglobin, erythrocyte counts, or leukocytes. The males from the pH 11 group in the 1100 mg/kg-day dose group showed a significant decrease in hematocrit and hemoglobin, and red blood cell count due to a loss of blood; red blood cell counts were also decreased at all other pH 11 doses but statistical significance was not stated. pH 11 rats showed increased white blood cells without a clear dose response. In the pH 7 rats, decreased hematocrit and hemoglobin was seen at 1700 mg/kg-day. Also at pH 7, alanine aminotransferase was increased at 1100 mg/kg-day (males) and 1700 mg/kg-day (both sexes). Urinalysis was conducted only for the pH 11 rats. Some proteinuria was seen, but doses were not noted. There were no treatment-related grossly-recognizable changes observed at necropsy. The doses at which other effects were observed at pH 7 are not provided, so the LOAEL and NOAEL could not be determined for pH 7. LOAEL (pH 11) = 500 mg/kg-day (based on mortality) NOAEL (pH 11) = Not established (2) Sprague-Dawley rats (10/sex/dose) were administered AMP in the diet at 0, 1000, 2000, 4000, 8000 or 16,000 ppm (~ 0, 244, 474, 912, 1901 and 4698 mg/kg-bw/day for males; ~ 0, 436, 806, 1628, 3424 and 7579 mg/kg-bw/day for females) for 8 weeks. Rats were observed daily for changes in general behavior and appearance. Individual body weights and sex group food consumption were recorded weekly. Animals were necropsied (all animals dosed at 8000 or 16,000 ppm, 2/sex/group elsewhere) and livers and any gross lesions were examined microscopically. Two female rats died at 16,000 ppm. Male and female rats at 16,000 ppm showed alopecia, focal skin erosions, microscopic skin lesions and ulcerations and gained slightly less weight (statistical significance not indicated) compared to control rats. Hepatocyte vacuolation was observed in rats from all treatment groups, with dose-dependent increases in severity. LOAEL (males) = 244 mg/kg-bw/day (based on hepatocyte vacuolization) LOAEL (females) = 436 mg/kg-bw/day (based on hepatocyte vacuolization) NOAEL = Not established (3) CD-I mice (10/sex/dose) were administered AMP in the diet at 0, 200, 400, 800, 1600 or 3200 ppm (~ 0, 1.2, 2.1, 3.4, 9.0 and 14.4 mg/kg-day in males; ~ 0, 1.6, 3.1, 6.1, 12.9 and 27.7 mg/kg-day in females) for 8 weeks. Mice were observed daily for changes in general behavior 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 and appearance. Individual body weights and sex group food consumption were recorded weekly. Animals were necropsied (all surviving animals dosed at 3200 ppm and 2/sex/group elsewhere) and livers and any gross lesions were examined microscopically. No treatment- related effects were observed in any parameter evaluated. NOAEL (males) = 3200 ppm (-14.4 mg/kg-bw/-day) (highest dose tested) NOAEL (females) = 3200 ppm (-27.7 mg/kg-bw/-day) (highest dose tested) (4) In a 90-day oral repeated-dose study, beagle dogs (4/sex/dose) were fed 25, 600 or 2500 ppm (approximately 0.9, 22, and 90 mg/kg-bw/day) AMP in the diet (Parekh, 1981). Body weight was decreased by 10% at the highest dose, although the change was not statistically significant. Decreased hemoglobin was seen in females at the highest dose (p < 0.05) with similar trend at lower doses and decreased prothrombin time was seen in males at the mid and high doses (p < 0.01). The authors noted clinical chemistry changes (higher bilirubin, lower cholesterol, and elevations of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase), increased liver weights, and histopathologic findings (vacuolization, periportal cirrhosis, and bile duct hyperplasia) at the 2500 ppm dose level in the diet (approximately 90 mg/kg-bw/day) that are indicative of the liver as a target organ. At the mid and high dose, increased relative and absolute kidney weights were seen but without accompanying changes in urinalysis. LOAEL = 600 ppm (22 mg/kg-bw/day; based on decreased prothrombin time in males, increased liver and/or liver: body weight ratios in both sexes, and minimal fatty changes in the liver) NOAEL = 25 ppm (0.9 mg/kg-bw/day) Reproductive Toxicity TRIS AMINO (CASRN 77-86-1) In a combined reproductive/developmental toxicity screening test, Sprague Dawley rats (10/sex/dose) were administered TRIS AMINO daily, by gavage, at doses of 0 (control), 100, 300, or 1000 mg/kg-day. Female rats were dosed once daily for approximately two weeks prior to breeding, through breeding (up to two weeks), gestation (three weeks), and lactation (four days) up to termination. Male rats were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 30). Effects on gonadal function, mating behavior, conception, development of the conceptus, and parturition were examined. In addition, a gross necropsy and histopathology of the adults were conducted with an emphasis on organs of the reproductive system. Localized point of contact irritation and histopathologic changes (hyperplasia, inflammation) occurred in the stomach of animals given 300 and 1000 mg/kg-day. There were no effects on any parameter of reproductive performance or offspring survival at any dose tested. LOAEL (parental) = 300 mg/kg-day (based on local irritation of the stomach) NOAEL (parental) = 100 mg/kg-day NOAEL (reproduction) = 1000 mg/kg-day (Based on no adverse effects on reproductive parameters; highest dose tested) 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Developmental Toxicity TRIS AMINO (CASRN 77-86-1) In the combined reproductive/developmental toxicity screening test described above, females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. In addition, pup clinical observations were recorded on each litter on PND 0 through 4. Early postnatal growth and survival were also evaluated. There were no effects on offspring growth, survival, gross pathology, litter size, or sex ratio at any dose tested. LOAEL (maternal toxicity) = 300 mg/kg-day (Based on local irritation of the stomach) NOAEL (maternal toxicity) = 100 mg/kg-day NOAEL (developmental toxicity) = 1000 mg/kg-day (Based on no adverse effects on developmental parameters; highest dose tested) Genetic Toxicity — Gene Mutation In vitro AMP (CASRN 124-68-5, supporting chemical) (1) In a bacterial reverse mutation assay using the plate incorporation method, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli (WP2 uvrA) were treated with up to 5000 [j,g/plate AMP (Wagner, 1996). Initial and confirmatory assays were used to evaluate the mutagenic potential of the test material. In triplicate tests, a minimum of 5 doses of the test article, with vehicle (water) and positive controls, were plated with tester strains in the presence and absence of metabolic (rat liver S9) activation. Neither precipitate nor appreciable toxicity was observed in the initial or confirmatory assays. In neither the initial nor confirmatory assay was there a positive response with any of the tester strains at any dose level, in the presence or absence of S9 activation. The mean of each positive control exhibited at least a 3-fold increase in the number of revertants over the mean value of the respective vehicle control, indicating a valid test. AMP was not mutagenic in this assay. (2) In a bacterial reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae (D-4) at test concentrations of 0.01 to 5.0 [j,L/plate in both the presence and absence of metabolic activation, AMP was negative for gene mutations. A negative (solvent control) was also run. Positive controls were used and showed appropriate responses. AMP was not mutagenic in this assay. (3) Mouse lymphoma cells (L5178Y/TK+-) were exposed to AMP at concentrations ranging from 1000 to 5000 [j,g/mL without metabolic activation or from 500 to 5000 [ig/mL with metabolic activation. Positive and negative controls were included. Positive controls responded 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 appropriately. No increase in mutant frequency was observed in the presence or absence of metabolic activation. AMP was not mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations In vivo AMP (CASRN124-68-5, supporting chemical) In a mouse micronucleus assay, ICR mice (5/sex/dose) received a single IP injection of 16, 30 or 60 mg/kg AMP; the positive control (cyclophosphamide) was administered in the same manner concurrently. At the scheduled sacrifice times (24 and 48 hours), marrow from the femurs was extracted and slides were prepared of the bone marrow cells. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes. There was no treatment-related mortality at any dose level. Clinical signs noted on the day of dosing were limited to lethargy in males and females at 60 mg/kg (highest dose). The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice. AMP did not induce micronuclei in mice in this assay. Additional Information Skin Irritation TRIS AMINO (CASRN 77-86-1) (1) Five rats (sex and strain not specified) were administered TRIS AMINO via an unspecified injection route at 500 or 1000 mg/kg and observed for an unspecified duration. The 500 mg/kg dose caused irritation at the injection site and the 1000 mg/kg-bw dose caused formation of lesions. TRIS AMINO was irritating to rat skin in this study. (2) Five mice (sex and strain not specified) were administered TRIS AMINO via an unspecified injection route at 500 or 1000 mg/kg and observed for an unspecified duration. The 500 mg/kg dose caused irritation at the injection site and the 1000 mg/kg dose caused formation of lesions. TRIS AMINO was irritating to mouse skin in this study. AMP (CASRN 124-68-5, supporting chemical) In an acute dermal toxicity study, observed signs of skin irritation in rabbits included severe eschar and necrosis. AMP was irritating to rabbit skin in this study. 12 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Eye Irritation TRIS AMINO (CASRN 77-86-1) Rabbits (six animals; sex and strain not specified) received an ocular administration of TRIS AMINO in water at 40% concentration. No additional data were provided. TRIS AMINO was not irritating to rabbit eyes in this study. Sensitization AMP (CASRN 124-68-5, supporting chemical) (1) Guinea pigs were treated topically with 0.5 mL of a 10% AMP solution; after 24 hours, sites were cleaned and scored for erythema and edema according to the method of Draize. At 48 hours, AMP application was repeated, and continued 2-3 times per week until 10 applications were made; after 2 applications the concentration was lowered to 5% due to mild irritation observed at the 10% applications. Animals were allowed a 2 week recovery period, and then challenged at a virgin site with 2.5% and 5% solutions of AMP (contact for 24 hours). Sites were scored again at 3 and 48 hours. Negative and positive controls were also used. None of the animals induced with AMP or the vehicle control exhibited a positive response when challenged with AMP. AMP was not a dermal sensitizer in guinea pigs. (2) Guinea pigs were administered intradermal injections during induction and challenge (0.05 mL of a 1, 0.5 and 0.1% AMP solution for inductions and O.lmL of 0.05% and 0.01% for challenge). During the induction phase, the first injection at 1% and second injection at 0.5% AMP induced necrotic lesions, so the remaining 8 injections (administered 2-3 times/week) were made with 0.1% solutions. The challenge injections occurred after a 2-week recovery period. Negative and positive controls were also used. During the challenge period, one animal in the test group showed mild reactions with 0.05%. In a repeat challenge, none of the animals in the test group showed any reactions with AMP, so the material was considered to be non-sensitizing following intradermal application. AMP was not an intradermal sensitizer in guinea pigs. Conclusion: The acute oral toxicity of TRIS AMINO to mice and rats is low. In a 90-day oral gavage repeated-dose toxicity study with the supporting chemical, 2-amino-2-methyl-l-propanol (AMP), at pH 11 in rats, decreased survival was seen at the lowest dose of 500 mg/kg-day, and no animals survived at 1700 mg/kg-bw/day; the NOAEL is not established. No mortalities occurred at pH 7; however, limited data were reported and so the NOAEL was not determined. In a dietary 8-week repeated-dose toxicity study in rats with the supporting chemical AMP, a dose-dependent increase in hepatocyte vacuolation was observed in rats starting at the lowest dose (244 in males and 436 in females); the NOAEL is not established. In a similar dietary 8- week repeated dose study in mice with the supporting chemical AMP, no treatment-related effects were observed up to the highest dose tested; the NOAEL is 14.4 and 27.7 mg/kg/day for males and females, respectively. In a 90-day dietary repeated dose toxicity study in dogs with the supporting chemical AMP, decreased prothrombin time in males, increased liver and/or liver:body weight ratios in both sexes, and minimal fatty changes in the liver were observed at 22 mg/kg-bw/day; the NOAEL is 0.9 mg/kg-bw/day. In an oral gavage combined reproductive/developmental toxicity screening test in rats with TRIS AMINO, significant point 13 ------- U.S. Environmental Protection Agency September, 2014 Hazard Characterization Document of entry effects on the stomach including irritation and histopathology changes occurred at 300 mg/kg-day; the NOAEL for local effects in parental animals is 100 mg/kg-day. In this study, no effects on reproductive or developmental parameters were observed at the doses tested; the NOAEL for reproductive and developmental toxicity is 1000 mg/kg-day, the highest dose tested. The supporting chemical, AMP, was not mutagenic to bacteria and mammalian cells in vitro, and did not induce micronuclei in mice in vivo. TRIS AMINO administered by injection is irritating to rat skin, but is not irritating to rabbit eyes. The supporting chemical AMP is irritating to rabbit skin and is not sensitizing to guinea pig skin. Table 3. Summary Table of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoints SPONSORED CHEMICAL TRIS AMINO (77-86-1) SUPPORTING CHEMICAL AMP (124-68-5) Acute Oral Toxicity LDso (mg/kg) 5500 - Acute Dermal Toxicity LDso (mg/kg) - - Repeated-Dose Toxicity NOAEL/LOAEL Oral (mg/kg-bw/day) No Data NOAEL = 0.9 LOAEL = 22 (RA) (rat; 8-wk) NOAEL = not established LOAEL = 244 (males); 436 (females) (dog; 90-d) NOAEL = 0.9 LOAEL = 22 Reproductive Toxicity NOAEL/LOAEL Oral (mg/kg/day) Reproductive Toxicity NOAEL = 1000 (highest dose tested) - Developmental Toxicity NOAEL/LOAEL Oral (mg/kg/day) Maternal Toxicity NOAEL = 100 LOAEL = 300 - Developmental Toxicity NOAEL = 1000 (highest dose tested) Genetic Toxicity - Gene Mutation In vitro No data Negative (RA) Negative Genetic Toxicity - Chromosomal Aberrations In vivo No data Negative (RA) Negative 14 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 3. Summary Table of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoints SPONSORED CHEMICAL TRIS AMINO (77-86-1) SUPPORTING CHEMICAL AMP (124-68-5) Additional Information Skin Irritation Eye Irritation Sensitization Irritating Not irritating Irritating Not sensitizing Bold = experimental data (i.e. derived from testing); (RA) = read across; - Indicates endpoint not assessed 4. Hazard to the Environment A summary of aquatic toxicity data submitted for SIDS endpoints is provided in Table 4. The table also indicates where data for the supporting chemicals are read-across (RA) to the sponsored chemical. Acute Toxicity to Fish 2-Amino-2-hydroxymethyl-1,3-propanediol (TRIS AMINO, CASRN 77-86-1) Golden orfes (Leuciscus idus) were exposed to TRIS AMINO at a measured concentration of 10,000 mg/L under unspecified conditions for 96 hours. 96-h LCso > 10,000 mg/L 2-A mino-2-methyl-1 -Propanol (AMP, CASRN 124-68-5, supporting chemical) European plaice (Pleuronectesplatessa) were exposed to AMP at 100, 320, 560 or 1000 mg/L under semi-static conditions for 96 hours. Test-water properties (dissolved oxygen, pH range, temperature, salinity) were monitored throughout the study. 96-h LCso = 184 mg/L 2-Amino-2-methyl-l,3-propanediol (AMPD CASRN 115-69-5, supporting chemical) Zebrafish (Brachydanio rerio) were exposed to AMPD at a measured concentration of 10,000 mg/L under static conditions for 96 hours. 96-h LCso > 10,000 mg/L Acute Toxicity to Aquatic Invertebrates AMP (CASRN 124-68-5, supporting chemical) (1) Brown shrimp (Crangon crangon) were exposed to AMP at unspecified concentrations for 96 hours. 96-h LCso = 179 mg/L 15 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (2) Water fleas (Daphnia magna) were exposed to AMP at unspecified concentrations for 48 hours. 48-h LCso = 193 mg/L Toxicity to Aquatic Plants Green algae (Selenastrum capricornutum) were exposed to TRIS AMINO for 96 hours at nominal concentration of at least 100 mg/L. Analytical monitoring was not conducted. Reported study details include: temperature of 22°C and constant illumination. ECOSAR version 1.1 predicted an EC so value of 749.5 mg/L as supporting the measured value. 96-h ECso >100 mg/L Conclusion: For the supporting chemical, AMP, the 96-h LC50 values for fish and aquatic invertebrates are 184, and 179 mg/L, respectively. For TRIS AMINO, the 96-h EC50 for aquatic plants is >100 mg/L. Table 4. Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Aquatic Toxicity Data Endpoints SPONSORED CHEMICAL TRIS AMINO (77-86-1) SUPPORTING CHEMICAL AMPD (115-69-5) SUPPORTING CHEMICAL AMP (124-68-5) Fish 96-h LCso (mg/L) No Adequate Data 184 (RA) >10,000 184 Aquatic Invertebrates 96-h LCso (mg/L) No Data 179 (RA) - 179 Aquatic Plants 96-h ECso (mg/L) > 100 - - Bold = experimental data (i.e. derived from testing); (RA) = read across; - no data submitted 16 ------- |