U. S. Environmental Protection Agency Hazard Characterization Document September, 2014 SCREENING-LEVEL HAZARD CHARACTERIZATION Benzyltrimethylammonium Chloride (CASRN 56-93-9) The High Production Volume (HPV) Challenge Program1 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 Setl1'2) 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, AT SDR, 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 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 1 U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm. 2 U.S. EPA. HPV 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 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) 56-93-9 Chemical Abstract Index Name Benzenemethanaminium, N,N,N-trimethyl-, chloride Structural Formula SMILES: c 1 (C[N+] (C)(C)C)ccccc 1. [C1-] Summary Benzyltrimethylammonium chloride is a solid with negligible vapor pressure and high water solubility. It is expected to have moderate mobility in soil. Volatilization of benzyltrimethylammonium chloride is low. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is moderate; however, this is not likely to be an important environmental fate process since this substance is not likely to exist in the vapor phase in the atmosphere. Benzyltrimethylammonium chloride is not readily biodegradable. Benzyltrimethylammonium chloride is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). Acute oral toxicity of benzyltrimethylammonium chloride to rats is moderate. Following repeated oral gavage exposures of rats and mice to benzyltrimethylammonium chloride for 13 weeks, clinical signs of toxicity included cholinergic effects at non-lethal dose levels and deaths in some animals at 100 mg/kg-day; the NOAEL is 25 mg/kg-day for both mice and rats. No specific reproductive toxicity studies are available; however, in the 13-week repeated dose studies, no adverse effects on reproductive organs, sperm or estrous cycle were observed. Benzyltrimethylammonium chloride did not induce gene mutations in bacteria in vitro, but did induce chromosomal aberrations in mammalian cells in vitro and micronuclei in mouse peripheral blood cells in vivo. The acute 48-h EC so of benzyltrimethylammonium chloride is 11.94 mg/L for aquatic invertebrates. There were no data or inadequate data for acute toxicity to fish and toxicity to aquatic plants. Data gaps for the developmental toxicity, acute toxicity to fish and toxicity to aquatic plants endpoints were identified under the HPV Challenge program 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 The sponsor, Bayer Corporation, submitted a Test Plan and Robust Summaries to EPA for benzyltrimethylammonium chloride (CASRN 56-93-9; CA Index name: benzenemethanaminium, N,N,N-trimethyl-, chloride) on November 14, 2003. EPA posted the submission on the ChemRTK HPV Challenge website on December 17, 2003 (http://www.epa.gov/hpv/pubs/summaries/bnztricl/cl4845tc.htm). EPA comments on the original submission were posted to the website on April 27, 2004. Public comments were also received and posted to the website. 1. Chemical Identity 1.1 Identification and Purity The test plan states that benzyltrimethylammonium chloride (CASRN 56-93-9) is used as a solvent for cellulose, as a gelling inhibitor in polyester resins, as an intermediate, as a dye assistant for acrylics, and as a phase-transfer agent. The benzyltrimethylammonium chloride solutions evaluated in the submitted studies have a purity of 60% v/v or > 99%, unless otherwise indicated. 1.2 Physical-Chemical Properties The physical-chemical properties of benzyltrimethylammonium chloride are summarized in Table 1. Table 1. Physical-Chemical Properties of benzyltrimethylammonium chloride1 Property Value CASRN 56-93-9 Molecular Weight 185.70 Physical State Solid2 Melting Point 239°C (measured with decomposition)3'4 Boiling Point Decomposes3'4 Vapor Pressure 2.3xl0"8mm Hg at 25°C (estimated)5 Dissociation Constant (pKa) Not applicable Henry's Law Constant <1,0x 10"10 atm-m3/mole (estimated)5 Water Solubility 8.0><106 mg/L at 20°C (Millipore MSDS) Log Kow -2.17 (measured) 1 Bayer Chemicals LLC. 2003. Test Plan and Robust Summary for Benzyltrimethylammonium chloride. Available online at http://www.epa.gov/hpv/pubs/summaries/bnztricl/cl4845tc.htm as of April 2, 2012. 2The pure compound is a solid in the form of colorless or off-white crystals. However, the compound is marketed as a ca. 60% aqueous solution. 3SRC. 2012. The Physical Properties Database (PHYSPROP). Syracuse, NY: Syracuse Research Corporation. Available online at http://www.svrres.com/esc/phvsprop.htm as of April 2, 2012. 4A value of <-50°C was reported for the melting point and a value of >135°C was reported for the boiling point (with decomposition) in the test plan; however, this was for the commercial (60% aqueous solution) product, not the neat substance. 5U.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 April 2, 2012. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 2. General Information on Exposure 2.1 Production Volume and Use Pattern Benzyltrimethylammonium chloride 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 for the chemical were claimed confidential. 2.2 Environmental Exposure and Fate Benzyltrimethylammonium chloride is expected to have moderate mobility in soil. It achieved 1% of its theoretical biochemical oxygen demand (BOD) using an activated sludge and the modified MITI (OECD 301C) test over the course of a 4-week incubation period. Volatilization of benzyltrimethylammonium chloride is low since this substance is a quaternary ammonium compound. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is moderate; however, this is not likely to be an important environmental fate process since this substance is not likely to exist in the vapor phase in the atmosphere. Benzyltrimethylammonium chloride is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). The environmental fate properties of benzyltrimethylammonium chloride are summarized in Table 2. Table 2. Environmental Fate Properties of Benzyltrimethylammonium chloride1 Property Value CASRN 56-93-9 Photodegradation Half-life 7.5 hours (estimated)2 Hydrolysis Half-life Stable Biodegradation 1% after 28 days (not readily biodegradable)3 Bioaccumulation Factor BCF = <0.2 (measured in carp at 2 mg/L)3; BCF = <1.5 (measured in carp at 0.2 mg/L)3; BAF = 0.9 (estimated)2 Log Koc 2.5 (estimated)2 Fugacity (Level III Model)2 Air (%) <0.1 Water (%) 17.6 Soil (%) 82.7 Sediment (%) 0.2 Persistence4 P2 (moderate) Bi oaccumul ati on4 Bl (low) 1 Bayer Chemicals LLC. 2003. Test Plan and Robust Summary for Benzyltrimethylammonium chloride. Available online at http://www.epa.gov/hpv/pubs/summaries/bnztricl/cl4845tc.htm as of April 2, 2012. 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 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 March 16, 2012. 3National Institute of Technology and Evaluation. 2002. Biodegradation and Bioaccumulation of the Existing Chemical Substances under the Chemical Substances Control Law. Available online at http://www.safe.nite.go.ip/english/kizon/KIZON start hazkizon.html as of April 2, 2012. 4Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204. Conclusion: Benzyltrimethylammonium chloride is a solid with negligible vapor pressure and high water solubility. It is expected to have moderate mobility in soil. Volatilization of benzyltrimethylammonium chloride is low. The rate of hydrolysis is expected to be negligible. The rate of atmospheric photooxidation is moderate; however, this is not likely to be an important environmental fate process since this substance is not likely to exist in the vapor phase in the atmosphere. Benzyltrimethylammonium chloride is not readily biodegradable. Benzyltrimethylammonium chloride is expected to have moderate persistence (P2) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of the health effects data submitted for SIDS endpoints is provided in Table 3. Acute Oral Toxicity Fischer 344 rats (5 males/dose) were administered benzyltrimethylammonium chloride (purity not indicated) at 125, 175, 210 or 250 mg/kg by oral gavage. Each rat concurrently received a subcutaneous injection of either saline, neostigmine or atropine sulfate. Duration of the observation period and number of deaths per dose group were not specified in the robust summary. LDso =180 mg/kg Repeated-Dose Toxicity (1) Fischer 344 rats (10 rats/sex/dose) were administered benzyltrimethylammonium chloride (60% v/v purity) at 0, 12.5, 25, 50 or 100 mg/kg-day by oral gavage 5 days/week for 13 weeks. Two female rats at the high-dose level died as the result of pharmacological effects on the cardiovascular system. Little effect was noted on body weights; final mean weights in the exposed groups were within 8% of control animals. Cholinergic effects including chromodacyorrhea, lacrimation, salivation, papillary constriction, altered gait and mild tremors were observed at non-lethal doses (cholinergic effects observed at each non-lethal dose were not specified in the robust summary). No clinically significant effects on hematological or clinical chemistry parameters were observed. No effects on the absolute or relative organ weights or treatment-related gross or histopathological lesions were observed. LOAEL = 50 mg/kg-day (based on cholinergic effects) NOAEL = 25 mg/kg-day 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (2) B6C3F1 mice (10 mice/sex/dose) were administered benzyltrimethylammonium chloride (60% v/v purity) at 0, 12.5, 25, 50 or 100 mg/kg-day by oral gavage 5 days/week for 13 weeks. One mouse of each sex at the high-dose level died as the result of pharmacological effects on the cardiovascular system. Little effect was noted on body weights; final mean weights in the exposed groups were within 3% of control animals. Cholinergic effects including chromodacyorrhea, lacrimation, salivation, papillary constriction, altered gait and mild tremors were observed at non-lethal doses (cholinergic effects observed at each non-lethal dose were not specified in the robust summary). No clinically significant effects on hematological or clinical chemistry parameters were observed. Kidney weights were increased in male mice at 50 and relative kidney weight was increased 100 mg/kg-bw/day. Relative heart weights were increased in males dosed at 25 mg/kg-day and higher. Since no treatment-related gross or histopathological lesions were observed in any animals, the changes in organ weight were not considered to be related to treatment. LOAEL = 50 mg/kg-day (based on cholinergic effects) NOAEL = 25 mg/kg-day (3) Crj:CD (SD) rats (5/sex/dose) were administered benzyltrimethylammonium chloride (60% v/v purity) at 0, 30, 60 or 120 mg/kg-bw/day by an unspecified oral route for 28 days. One high- dose female died during the fourth week of the dosing period. Histopathology of the deceased rat revealed hepatocellular swelling and eosinophilic bodies. No histopathological effects were noted in surviving of control animals. Observations at the mid-dose level males included increased salivation. Observations at the high- dose level included increased salivation, lacrimation and soiled fur in both sexes, piloerection in females and decreased body weight gain, decreased food consumption and alterations in hematological parameters (increased hemoglobin, mean cell volume and mean corpuscular hemoglobin levels) in males. LOAEL (female) = 120 mg/kg-bw/day (based on death, piloerection, increased salivation, lacrimation and soiled fur) NOAEL (female) = 60 mg/kg-bw/day LOAEL (male) = 60 mg/kg-bw/day (based on increased salivation, a muscarinic-type cholinergic symptom) NOAEL (male) = 30 mg/kg-bw/day Reproductive Toxicity No specific reproductive toxicity studies are available. (1) In the 13-week oral repeated-dose toxicity study in Fischer 344 rats described previously, samples were collected from animals treated at 0, 25, 50 or 100 mg/kg-day for sperm motility and vaginal cytology evaluations. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the female animals were moistened with saline, if necessary, and samples of fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelia cells and large squamous epithelial cells were determined and used to ascertain estrous cycle stage, length of estrous cycle and percentage of cycle spent in estrous. The left testis, left epididymis and left caudal epididymis of male animals were isolated, weighed and evaluated for spermatid heads per testis and per gram testis; spermatid counts; and epididymal spermatozoa motility and concentration. The reproductive organs of male and female animals in the control and high-dose 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 groups were also examined histologically. There were no exposure-related effects on sperm or reproductive tissues in males compared to controls. A minimal shortening of diestrous and prolongation of proestrous occurred females at 25 mg/kg-day, but not at higher dose levels. There was no alteration in the length of the estrous cycle or histopathological effects on female reproductive organs. (2) In the 13-week oral repeated dose toxicity study in B6C3F1 mice described previously, samples were collected from animals treated at 0, 25, 50 or 100 mg/kg-day for sperm motility and vaginal cytology evaluations. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the female animas were moistened with saline, if necessary, and samples of fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelia cells and large squamous epithelial cells were determined and used to ascertain estrous cycle stage, length of estrous cycle and percentage of cycle spent in estrous. The left testis, left epididymis and left caudal epididymis of male animals were isolated, weighed and evaluated for spermatid heads per testis and per gram testis; spermatid counts; and epididymal spermatozoa motility and concentration. The reproductive organs of male and female animals in the control and high-dose groups were also examined histologically. There were no exposure-related effects on sperm or reproductive tissues in males and estrous cycle or histopathological effects on reproductive organs in females compared to controls. Developmental Toxicity No Data Genetic Toxicity - Gene Mutations In vitro (1) In an Ames test, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2mrA were exposed to benzyltrimethylammonium chloride (> 99% purity) in distilled water at concentrations of 156, 313, 625, 1250, 2500 or 5000 |j,g/plate in the presence and absence of metabolic activation. Positive and solvent controls were tested concurrently, but results of control responses were not provided. Three plates per concentration were tested in each replicate and two replicates were conducted. No data were provided regarding cytotoxicity. No further details were provided. No evidence of mutagenicity was noted in any of the strains. Benzyltrimethylammonium chloride was not mutagenic in this assay. (2) In a reverse mutation assay, Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were exposed to benzyltrimethylammonium chloride (purity not indicated) at concentrations up to 10,000 |j,g/plate in the presence and absence of metabolic activation. No data were provided on the use or response of solvent and positive controls. No evidence of cytotoxicity was observed at any dose level. No further details were provided. No evidence of mutagenicity was noted in any of the strains. Benzyltrimethylammonium chloride was not mutagenic in this assay. 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (3) In an Ames test, Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were exposed to benzyltrimethylammonium chloride (60% v/v purity) at concentrations up to 10,000 |j,g/plate in the presence and absence of metabolic activation. Positive and solvent controls were tested concurrently, but results of control responses were not provided. Three plates per concentration were tested in each concentration and for each control. No evidence of cytotoxicity was observed at any dose level. No further details were provided. No evidence of mutagenicity was noted in any of the strains. Benzyltrimethylammonium chloride was not mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations In vitro Chinese hamster lung cells were exposed to benzyltrimethylammonium chloride (60% v/v purity) in saline at concentrations of 475, 950 or 1900 |j,g/mL in the presence and absence of metabolic activation and 1000, 1300, 1600 or 1900 |j,g/mL in the presence of metabolic activation in a confirmatory test. No data were provided on the use or response of solvent and positive controls. "Slightly increased incidences" of structural chromosomal aberrations with metabolic activation were observed in the initial assay. Clear reproducibility was obtained in the confirmatory study. No further details were provided. Benzyltrimethylammonium chloride induced chromosomal aberrations in this assay. In vivo At the end of the 13-week repeated dose oral study in B6C3F1 mice described previously, peripheral blood samples were obtained from animals of both sexes and evaluated for the presence of micronuclei. Analyses yielded positive trends in both male and female data. The highest dose tested (100 mg/kg-day) produced an increase in micronuclei that was significantly different from the controls. Benzyltrimethylammonium chloride induced micronuclei in peripheral blood in this assay. Conclusion: Acute oral toxicity of benzyltrimethylammonium chloride to rats is moderate. Following repeated oral gavage exposures of rats and mice to benzyltrimethylammonium chloride for 13 weeks, clinical signs of toxicity included cholinergic effects at non-lethal dose levels and deaths in some animals at 100 mg/kg-day; the NOAEL is 25 mg/kg-day for both mice and rats. No specific reproductive toxicity studies are available; however, in the 13-week repeated dose studies, no adverse effects on reproductive organs, sperm or estrous cycle were observed. Benzyltrimethylammonium chloride did not induce gene mutations in bacteria in vitro, but did induce chromosomal aberrations in mammalian cells in vitro and micronuclei in mouse peripheral blood cells in vivo. 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 3. Summary Table of the Screening Information Data Set under the U.S. HPV Challenge Program - Human Health Data Endpoints Benzyltrimethylammonium Chloride ( 56-93-9) Acute Oral Toxicity LDso (mg/kg) 180 Repeated-Dose Toxicity NOAEL/LOAEL (oral, mg/kg-day) (rat/mouse; 13-wk) NOAEL = 25 LOAEL = 50 Reproductive Toxicity NOAEL/LOAEL (mg/kg-day) In the 13-week repeated dose studies, no adverse effects on reproductive organs, sperm or estrous cycle were observed Developmental Toxicity NOAEL/LOAEL No Data Genetic Toxicity - Gene Mutation In vitro Negative Genetic Toxicity - Chromosomal Aberrations In vitro In vivo Positive Positive Measured data in bold 4. Hazard to the Environment A summary of aquatic toxicity data submitted for SIDS endpoints is provided in Table 4. Acute Toxicity to Fish No data were available for the fish endpoint. Acute Toxicity to Aquatic Invertebrates Water fleas (Daphniapulex) were exposed to the test substance for 48 hours under unspecified test conditions. No details on concentrations tested, number of animals per concentration or mortality/toxic signs at each dose level were provided. 48-h ECso = 11.94 mg/L Toxicity to Aquatic Plants No adequate data were available for the aquatic plants endpoint. 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Conclusion: The acute 48-h EC50 of benzyltrimethylammonium chloride is 11.94 mg/L for aquatic invertebrates. There were no data or inadequate data for acute toxicity to fish and toxicity to aquatic plants. Table 4. Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Aquatic Toxicity Data Endpoints Benzyltrimethylammonium chloride (56-93-9) Fish 96-h LCso (mg/L) No Data Aquatic Invertebrates 48-h ECso (mg/L) 11.94 Aquatic Plants 72-h ECso (mg/L) No Adequate Data Bold = measured data (i.e., derived from testing) 11 ------- |