U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 SCREENING-LEVEL HAZARD CHARACTERIZATION SPONSORED CHEMICAL Hexanedinitrile Hydrogenated, High-Boiling Fraction CASRN 68411-90-5 SUPPORTING CHEMICAL Bis(hexamethylene) triamine CASRN 143-23-7 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 Set1'2) 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.) and 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. 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 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 68411-90-5 Supporting Chemical 143-23-7 Chemical Abstract Index Name Sponsored Chemical Hexanedinitrile Hydrogenated, High-Boiling Fraction (BHMT) Supporting Chemical Bis(hexamethylene)triamine Structural3 Formula H Representative SMILES: NCCCCCCNCCCCCCN Summary Hexanedinitrile, hydrogenated, high-boiling fraction (crude BHMT) is a variable composition mixture that consists primarily of 1,6-hexanediamine, Nl-(6-aminohexyl)- (CASRN 143-23-7). It has low vapor pressure and high water solubility. It is expected to have moderate mobility in soil. Volatilization of BHMT is considered low since the primary constituent of this substance will exist as a cation under environmental conditions and cations do not volatilize. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is considered rapid. BHMT is expected to be readily biodegradable. BHMT is expected to have low persistence (PI) and low bioaccumulation potential (Bl). The acute oral toxicity of crude BHMT is moderate in rats; the acute dermal toxicity to rabbits is high. In a 13-week oral gavage repeated-dose toxicity test with crude BHMT, mortality and an increased incidence of interstitial pneumonia were observed in rats at 50 mg/kg-day; the NOAEL for systemic toxicity is 20 mg/kg-day. Respiratory tract lesions (hypertrophy, hyperplasia, squamous metaplasia and ulceration of the mucociliary epithelium) were observed in rats exposed to aerosols of crude BHMT at 0.01 mg/L during a 13-week repeated-dose inhalation study; the NOAEC for systemic toxicity is not established. A reproductive toxicity study is not available for crude BHMT; however, no macroscopic or microscopic effects on reproductive organs were observed in the repeated-dose studies described above. A prenatal oral gavage 5 1 Hexanedinitrile, hydrogenated, high-boiling fraction is a variable composition mixture that consists of approximately 50 - 70 % 1,6-hexanediamine, Nl-(6-aminohexyl)- (CASRN 143-23-7), 20 - 35 % oligomeric amines, 0-10 % C10 amines, 0-10 % hexamethylenediamine, 0-10 % caprolactam, 0 - 5 % adiponitrile and 0 - 5 % 6-aminocapronitrile and small amounts of related compounds. 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 developmental toxicity screening test with crude BHMT showed increased mortality and post implantation loss in dams at 50 mg/kg-day; the NOAELs for maternal and developmental toxicity are not established. Crude BHMT was mutagenic in bacterial cells in vitro but not mammalian cells in vivo. It did not induce chromosomal aberrations in vivo or unscheduled DNA synthesis in vitro. Crude BHMT is corrosive to rabbit skin and eyes. No adequate data were submitted for crude BHMT to evaluate acute toxicity to fish, aquatic invertebrates, and aquatic plants. Acute toxicity to fish, aquatic invertebrates, and aquatic plants were identified as data gaps under the HPV Challenge Program. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 The sponsor, E.I. du Pont de Nemours and Company, Inc., submitted a Test Plan and Robust Summaries to EPA for hexanedinitrile hydrogenated, high-boiling fraction (crude BHMT, CASRN 68411-90-5) on February, 13, 2003. EPA posted the submission on the ChemRTK HPV Challenge website on February, 25, 2003. (http://www.epa.gov/oppt/chemrtk/pubs/summaries/hexdihyd/cl4298tc.htm). EPA comments on the original submission were posted to the website on July, 9, 2003. Public comments were also received and posted to the website. In its original comments, EPA stated that the developmental toxicity study submitted for the sponsored chemical, hexanedinitrile hydrogenated, high-boiling fraction (crude BHMT; CASRN 68411-90-5) was inadequate; however, upon further consideration, this information was deemed sufficient for the purposes of human health hazard characterization. The corrosive nature of this substance and the excessive mortality observed in treated dams obviates the need for further testing. Justification for Supporting Chemical The sponsor provided data for the supporting chemical, bis(hexamethylene)triamine (purified BHMT; CASRN 143-23-7), the main component of the hexanedinitrile hydrogenated, high- boiling fraction mixture (crude BHMT). In its original comments, EPA stated that data for the octanol-water partition coefficient are adequate for the purposes of the HPV Challenge Program. The data submitted for purified BHMT for the ecological and biodegradation endpoints are inadequate because they may not adequately account for the complex variable composition of the sponsored substance. For the health effects endpoints, data for purified BHMT are considered adequate as this data served to supplement existing data provided for the sponsored substance. 1. Chemical Identity 1.1 Identification and Purity The following information was taken from the 2003 Test Plan: Crude BHMT (CASRN 68411-90-5) is the high-boiling fraction produced from the distillation of hexamethylene diamine. Because this substance is the residue of a distillation procedure in which processing times and conditions are adjusted to maximize yield, crude BHMT exists as a mixture of variable composition. It generally consists (on a dry weight basis) of approximately 50-70% (BHMT), 20-35% oligomeric amines, 0-10% Cio amines, 0-10% hexamethylenediamine, 0-10%) caprolactam, 0-5% adiponitrile, 0-5% 6-aminocapronitrile and small amounts of related compounds. The purity of BHMT, where indicated, is high (97.1%>); however, the relative abundance of this substance in crude BHMT may vary. 1.2 Physical-Chemical Properties The physical-chemical properties of hexanedinitrile, hydrogenated, high-boiling fraction (crude BHMT) are summarized in Table 1. This substance has low vapor pressure and high water solubility. 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 1. Physical-Chemical Properties of Hexanedinitrile, hydrogenated, high-boiling fraction 1 Property Value CASRN 68411-90-5 Molecular Weight 215.39 Physical State Brown, viscous semi-liquid or paste Melting Point 32 - 34 °C (measured); 33 - 36 °C (measured for CASRN 143-23-7) Boiling Point 249 °C at 100 mm Hg (measured); 220°C at 20 mm Hg (measured for CASRN 143-23-7); 320 °C at 760 mm Hg (estimated)2 Vapor Pressure 7 mm Hg at 180 °C (measured); 82.5 mm Hg at 250 °C (measured for CASRN 143-23-7); 0.00017 mm Hg at 25 °C (estimated)2 Dissociation Constant (pKa) 9.33 (estimated)3'4; 10.33 (estimated)3'4; 11.03 (estimated)3'4 Henry's Law Constant <1.0><10"10 atm-m3/mole (estimated)4'5 Water Solubility 10,000 mg/L at 25 °C (measured) Log Kow 1.80 (estimated)4'5 'E.I. du Pont de Nemours Inc. and Solutia Inc. February 2003. Test Plan and Robust Summary for Crude BHMT. Available online at: http://www.epa.gov/chemrtk/pubs/summaries/hexdihvd/cl4298tc.htm as of December 13,2011. 2 NOM05. 1987. Programs to Enhance PC-Gems Estimates of Physical Properties for Organic Compounds. The Mitre Corp. 3 SPARC. 2011. Online pKa/Property Calculator, w4.2.1405-s4.2.1408. University of Georgia, Athens, GA, USA. http://ibmlc2.chem.uga.edu/spare/ as of January 9,2012. 4 Estimation based on the chemical structure of 1,6-hexanediamine, Nl-(6-aminohexyl)- (CASRN 143-23-7) 5 U.S. EPA. 2011. 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 December 13, 2011. 2. General Information on Exposure 2.1 Production Volume and Exposure Hexanedinitrile, hydrogenated, high-boiling fraction (crude BHMT) had an aggregated production and/or import volume in the United States between 10 to 50 million pounds during calendar year 2005. Non-confidential information in the IUR indicated that the industrial processing and uses of the chemical include other basic organic chemical manufacturing as intermediates and corrosion inhibitors and anti-scaling agents. Non-confidential commercial and consumer uses of this chemical include paints and coatings; and "other." 2.2 Environmental Exposure and Fate Hexanedinitrile, hydrogenated, high-boiling fraction (Crude BHMT) is expected to have moderate mobility in soil. Volatilization of hexanedinitrile, hydrogenated, high-boiling fraction is considered low since the primary constituent of this substance will exist as a cation under 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 environmental conditions and cations do not volatilize. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is considered rapid. Hexanedinitrile, hydrogenated, high- boiling fraction is expected to be readily biodegradable based on ready biodegradation data for BHMT-HP Polyamine, purity 92% by wt., which achieved 100% of its Theoretical Oxygen Demand (ThOD) in 28 days, using the Closed Bottle Test (OECD Guideline 301 D), indicating that it is readily biodegradable. Hexanedinitrile, hydrogenated, high-boiling fraction is expected to have low persistence (PI) and low bioaccumulation potential (Bl). The environmental fate properties for crude BHMT are provided in Table 2. Table 2. Environmental Fate Properties of Hexanedinitrile, hydrogenated, high-boiling fraction (Crude BHMT)1 Property Value Photodegradation Half-life 0.8 hours (estimated)2'3 Hydrolysis Half-life Stable Biodegradation 100%) after 28 days (readily biodegradable. OECD 301 D)4 Bioaccumulation Factor BAF = 7.3 (estimated)2'3 Log Koc 3.6 (estimated)2'3 Fugacity (Level III Model)2'3 Air (%) Water (%>) Soil (%) Sediment (%>) <0.1 15.6 82.4 2.0 Persistence5 PI (low) Bi oaccumul ati on5 Bl (low) 1 E.I. du Pont de Nemours Inc. and Solutia Inc. February 2003. Test Plan and Robust Summary for Crude BHMT. Available online at: http://www.epa.gov/chemrtk/pubs/summaries/hexdihvd/cl4298tc.htm as of December 13, 2011. 2U.S. EPA. 2011. 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 December 13, 2011. Estimation based on the chemical structure of 1,6-hexanediamine, Nl-(6-aminohexyl)- (CASRN 143-23-7) 4The Robust Summary for Crude BHMT does not provide biodegradation data for Crude BHMT, however it provides biodegradation data for BHMT-HP Polyamine, purity>92% by wt. (see Robust Summary - Appendix B online at http://www.epa.gov/chemrtk/pubs/summaries/hexdihvd/cl4298rs.pdf 5 Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204. Conclusion: Hexanedinitrile, hydrogenated, high-boiling fraction is a variable composition mixture that consists primarily of 1,6-hexanediamine, Nl-(6-aminohexyl)- (CASRN 143-23-7). It has low vapor pressure and high water solubility. It is expected to have moderate mobility in soil. Volatilization of hexanedinitrile, hydrogenated, high-boiling fraction is considered low since the primary constituent of this substance will exist as a cation under environmental conditions and 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 cations do not volatilize. The rate of hydrolysis is negligible. The rate of atmospheric photooxidation is considered rapid. Hexanedinitrile, hydrogenated, high-boiling fraction is expected to be readily biodegradable. Hexanedinitrile, hydrogenated, high-boiling fraction is expected to have low persistence (PI) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of health effects data submitted for SIDS endpoints is provided in Table 3. Acute Oral Toxicity Crude BHMT (CASRN 68411-90-5) Sprague-Dawley rats (2 or 3/sex/dose) were administered crude BHMT (composition not reported) via oral gavage at 316, 398, 501 or 631 mg/kg and observed for up to 7 days. Mortality occurred in males from the two highest dose groups (1/2 and 3/3 at 501 and 631 mg.kg, respectively) and in females from the three highest dose groups (1/2, 3/3 and 2/3 at 398, 501 and 631 mg/kg, respectively) within 1-2 hours of treatment. LDso = 450 mg/kg Acute Inhalation Toxicity Crude BHMT (CASRN 68411-90-5) Male albino rats (6/concentration) were exposed to crude BHMT (composition not reported) as a vapor at the "saturation level at ambient temperature" (approximately 0.75 ppm) for 6 hours and observed for up to 10 days. No mortalities occurred. No effects at vapor saturation. Acute Dermal Toxicity Crude BHMT (CASRN 68411-90-5) (1) One male New Zealand White rabbit was exposed to crude BHMT (50-60% relative abundance) at 200 mg/kg via topical application to shaved, intact skin under occlusive conditions for 24 hours. Excess test material was subsequently washed from the skin and the animal was observed for 14 days. No mortality occurred. Additional animals were not treated due to the corrosive nature of the test substance. AT J) > 200 mg/kg (2) New Zealand White rabbits (1/sex/dose) were exposed to crude BHMT (composition not reported) via topical application to clipped, intact skin at 31.6, 50.1, 79.4, 126, 200, 316, 1000 or 2000 mg/kg under occlusive conditions for 24 hours. Animals were observed for up to 14 days. Treatment at >200 mg/kg was lethal. LD50 = 126 - 200 mg/kg 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Repeated-Dose Toxicity Crude BHMT (CASRN 68411-90-5) (1) Sprague-Dawley rats (15/sex/dose) were administered an aqueous solution of crude BHMT (65% relative abundance) via gavage administration at 0, 20, 50 or 120 mg/kg-day for 13 weeks. Clinical signs of toxicity (daily), body weight, food consumption (weekly), organ weights (brain, kidneys, adrenals, heart, spleen, liver, ovaries and testes), urinalysis, hematology, and clinical chemistry parameters (at terminal sacrifice) were evaluated. Rats sacrificed as moribund and those surviving to terminal sacrifice were examined for gross lesions at necropsy. All tissue masses and gross lesions were retained for microscopic examination. In addition, reproductive organs (ovaries, testes with epididymides and mammary glands) and other selected tissues (not specified) from the high-dose and control groups were prepared for histological evaluation. Mortality occurred in the high-(one male and one female) and mid-dose groups (one male). Clinical signs of toxicity included respiratory rales in high-dose males and females, as well as mid-dose females. Significantly decreased body weight (p < 0.05) was observed in high-dose females; however, this treatment effect was not apparent at the end of the study. High-dose males exhibited reduced food consumption and decreased absolute adrenal weights; mid-dose females showed increased relative liver weight. No histopathology was observed in any organ or tissue. An increased incidence of interstitial pneumonia was observed in all treatment groups (level of significance not specified); increased segmented neutrophil counts were noted in high- dose males and females (level of significance not specified). No other treatment-related effects were reported. LOAEL = 50 mg/kg-day (based on mortality and increased incidence of interstitial pneumonia) NOAEL = 20 mg/kg-day (2) Sprague-Dawley rats (12/sex/group) were exposed to crude BHMT (63.3% relative abundance) via aerosol inhalation at 0, 10, 30 or 60 mg/m3 (0, 0.01, 0.03 or 0.06 mg/L) 6 hours/day, 5 days/week for 13 weeks. Mean analytical values over the entire course of the study for the three exposure levels were 10, 31 and 62 mg/m3 (0.01, 0.031 and 0.062 mg/L). Clinical signs of toxicity (daily), body weight, food consumption (weekly), opthalmic examinations (pre- exposure and at 12 weeks), organ weights (brain, kidneys, adrenals, heart, spleen, liver, ovaries and testes with epididymides), urinalysis, hematology and clinical chemistry parameters (at terminal sacrifice) were evaluated. Rats sacrificed as moribund and those surviving to terminal sacrifice were examined for gross lesions at necropsy. All tissue masses and gross lesions were retained for microscopic examination. In addition, reproductive (ovaries, testes with epididymides, mammary gland and uterus) and other selected tissues (not specified) from control and 0.06 mg/L treatment groups were prepared for histological evaluation. Selected tissues from the respiratory tract (nasal passages, trachea and lungs) were evaluated in animals treated at 0.01 or 0.03 mg/L. No mortality occurred. Clinical signs of toxicity included respiratory wheezing in animals treated at the highest concentration and discoloration of fur in females. Animals treated at the highest concentration exhibited significantly decreased body weight (level of significance not specified), organ weight changes (not specified), elevated red blood cell counts with associated increases in hemoglobin and hematocrit in males (9%) and females (12%), as well as elevated alanine aminotransferase, aspartate aminotransferase and phosphorus levels (males); no histopathological correlate was observed. Hypertrophy, hyperplasia, squamous metaplasia and ulceration with secondary inflammation of the mucociliary epithelium were observed in the respiratory tract (i.e., nasal passages, trachea and lungs) at all levels of treatment. Necropsy 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 findings revealed concentration-related increases in the incidence and severity of pulmonary emphysema. LOAEC ~ 0.01 mg/L (based on respiratory tract lesions) NOAEC = Not established Reproductive Toxicity Crude BHMT (CASRN 68411-90-5) No specific reproductive toxicity studies are available. (1) In the 13-week oral repeated-dose toxicity study described above, no macroscopic or microscopic effects on reproductive organs (ovaries, testes with epididymides and mammary glands) were observed in rats following gavage exposure to crude BHMT. (2) In the 13-week inhalation repeated-dose toxicity study described above, no macroscopic or microscopic effects on reproductive organs (ovaries, testes with epididymides and mammary glands) were observed in rats following inhalation exposure to aerosols of crude BHMT. Developmental Toxicity Crude BHMT (CASRN 68411-90-5) In a prenatal developmental toxicity study, pregnant Sprague-Dawley rats (25/dose) were exposed to crude BHMT (aqueous) via gavage administration at 0, 50, 100, or 250 mg/kg-day on gestation days (GD) 6-15. Animals were observed twice daily for mortality or clinical signs of toxicity. At terminal sacrifice (GD 20), all surviving rats were subjected to a gross postmortem examination in which the numbers of live and dead fetuses, early and late resorptions, implantation sites and corpora lutea were recorded. In addition, fetuses were weighed, sexed and examined for gross external malformations. Half of these animals were sectioned and examined for soft tissue abnormalities; the rest were processed and stained for skeletal examination. Mortality was observed at all levels of treatment (i.e., 1, 6, and 5 dams from the low, mid and high dose groups, respectively, died or were sacrificed as moribund). The specific cause of death was not determined; however, all treated rats showed signs of respiratory distress. Necropsy findings showed no treatment-related macroscopic lesions in surviving dams, or offspring (i.e., no abnormalities in fetal ossification, soft tissue or skeletal development); however, a decrease in the number of viable offspring (in association with decreased numbers of corpora lutea and total implantation sites, as well as increased post implantation loss) was observed at 50 and 100 mg/kg-day (level of significance not specified). Although the study authors stated that these effects are not likely to be treatment related (due to the absence of a dose-related trend), they are biologically relevant. The excessive mortality observed in treated dams at > 100 mg/kg-day effectively limits the ability to assess a dose response. LOAEL (maternal) = 50 mg/kg-day (based on mortality) NOAEL (maternal) = Not established LOAEL (developmental) = 50 mg/kg-day (based on increased post implantation loss) NOAEL (developmental) = Not established 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Genetic Toxicity - Gene Mutation In vitro Crude BHMT (CASRN 68411-90-5) In a reverse mutation assay, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA were exposed to crude BHMT (46% relative abundance) in ethanol at 0, 25, 75, 200, 600, 1800 or 5000 |j,g/plate with and without metabolic activation. Assay concentrations were selected based on results obtained in a preliminary range finding assay. All positive controls, except for those used with strain TA100 (not specified), responded appropriately in the presence of metabolic activation. Strain TA1537 induced a non- dose responsive increase (2.4-fold) in mutation frequency in the presence of metabolic activation. For clarification, strains TA100 and TA1537 were retested in a second assay at 0, 25, 75, 200, 600, 1800, 2500 or 5000 |j,g/plate in the presence of metabolic activation. Strain TA1537 induced a non-dose responsive increase (2.9-fold) in mutation frequency in the second assay. No precipitate was observed; however, cytotoxicity occurred at > 1800 |j,g/plate. Strain TA1537 was retested (at the same concentrations) in two additional assays with reduced metabolic activation (5% and 15%). No positive mutagenic response was observed in the latter two assays; however, cytotoxicity occurred at > 3333 |j,g/plate. No precipitate was observed. Crude BHMT was mutagenic in this assay. Purified BHMT (CASRN 143-23-7, supporting chemical) In a reverse mutation assay, Salmonella typhimurium strains TA97a, TA98, TA100 and TA1535 and Escherichia coli strain WP2 uvrA (pKMlOl) were exposed to refined BHMT (97.1% purity) at 0, 10, 50, 100, 500, 1000, 2500 or 5000 |ig/plate with and without metabolic activation, three replicates were completed. Positive controls were tested concurrently, but these responses were not provided. No precipitate was observed. Cytotoxicity occurred at > 500 |ig/plate in the absence of metabolic activation and at > 1000 |ig/plate in the presence of metabolic activation. Purified BHMT was not mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations In vivo Crude BHMT (CASRN 68411-90-5) In a micronucleus assay, Sprague-Dawley rats (15/sex/dose) were administered crude BHMT (65%) relative abundance) in corn oil via gavage administration at 0 or 500 mg/kg. Test concentrations were selected based on results obtained in a preliminary range finding study. Bone marrow from each femur was removed and processed for cytogenetic analysis. Positive controls (5/sex; treated with cyclophosphamide) were sacrificed after 24 hours; treated animals (5/sex) were sacrificed at 6, 24, and 48 hours. Whenever possible, 50 metaphase cells were examined from each animal. Mean mitotic indices, mean chromosome numbers, percent aberrant cells, and the mean number of aberrations per cell for were determined for each treatment group. One male and one female died following treatment with crude BHMT. Clinical signs of toxicity included depressed motor activity, labored breathing, wheezing, soft feces and red stains around the nose and eyes. Decreased body weight was observed at 24 and 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 48 hours post exposure. Positive controls exhibited significant increases in the average number of chromosomal aberrations per cell and the percent of cells with aberrations, a significantly decreased mitotic index, and chromosome number, thereby confirming the sensitivity of the assay to known mutagens. A comparison of the negative controls and crude BMHT treatment groups revealed no differences in the frequency of aberrant cells. Crude BHMT did not induce chromosomal aberrations in this assay. Genetic Toxicity — Other In vitro Crude BHMT (CASRN 68411-90-5) In an unscheduled DNA synthesis (UDS) assay, primary hepatocytes from Fischer 344 rats were exposed to crude BHMT (65% relative abundance) at 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000 or 5000 |j,g/L in a preliminary assay and at 0, 10, 50, 100, 500 or 750 |j,g/mL in triple replicate assays. Cytotoxicity was observed at 1000 and 5000 |j,g/mL in the preliminary assay. Positive controls were tested concurrently and produced an appropriate response. Crude BHMT did not induce unscheduled DNA synthesis in this assay. Additional Information Skin Irritation Crude BHMT (CASRN 68411-90-5) One male New Zealand White rabbit was exposed to crude BHMT (50-60% relative abundance) via topical application to shaved, intact skin at 200 mg/kg under occlusive conditions for 24 hours. Excess test material was washed away and the animal was observed for clinical signs of toxicity at 3 hours post exposure, then once daily thereafter for 14 days (1 weekend excluded). Necrosis, mild to severe erythema, and mild to severe edema were observed throughout the study. Green discoloration, cratering and sloughing of the epidermis was observed at the test site. Additional rabbits were not treated due to the corrosiveness of the test substance. Crude BHMT was corrosive to rabbit skin in this study. Eye Irritation Crude BHMT (CASRN 68411-90-5) Two groups of male and female albino rabbits (two males and one female per group) received a single O.lmL instillation of (undiluted) crude BHMT (percent composition not reported) in the conjunctival sac of the right eye; the left (untreated) eye served as control. Eyes were washed with saline either one minute or 24 hours after treatment and evaluated at 10 minutes, 1 hour, and 3, 5, 7, and 14 days post exposure. A severe irritation response was evident within one hour or 10 minutes of test article application in the 1-minute and 24-hour treatment groups, respectively (determinations were made using the Draize method). Reported treatment effects included corneal cloudiness and copious discharge which progressively worsened, producing opacity, dullness, severe erythema and moderate edema within five days of treatment. Crude BHMT was corrosive to rabbit eyes in this study. 12 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Conclusion: The acute oral toxicity of crude BHMT is moderate in rats; the acute dermal toxicity to rabbits is high. In a 13-week oral gavage repeated-dose toxicity test with crude BHMT, mortality and an increased incidence of interstitial pneumonia were observed in rats at 50 mg/kg-day; the NOAEL for systemic toxicity is 20 mg/kg-day. Respiratory tract lesions (hypertrophy, hyperplasia, squamous metaplasia and ulceration of the mucociliary epithelium) were observed in rats exposed to aerosols of crude BHMT at 0.01 mg/L during a 13-week repeated-dose inhalation study; the NOAEC for systemic toxicity is not established. A reproductive toxicity study is not available for crude BHMT; however, no macroscopic or microscopic effects on reproductive organs were observed in the repeated-dose studies described above. A prenatal oral gavage developmental toxicity screening test with crude BHMT showed increased mortality and post implantation loss in dams at 50 mg/kg-day; the NOAELs for maternal and developmental toxicity are not established. Crude BHMT was mutagenic in bacterial cells in vitro but not mammalian cells in vivo. It did not induce chromosomal aberrations in vivo or unscheduled DNA synthesis in vitro. Crude BHMT is corrosive to rabbit skin and eyes. Table 3. Summary of Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoint SPONSORED CHEMICAL Hexanedinitrile hydrogenated, high-boiling fraction (crude BHMT) (68411-90-5) SUPPORTING CHEMICAL bis(Hexamethylene) triamine (purified BHMT) (143-23-7) Acute Oral Toxicity LDso(mg/kg) 450 - Acute Inhalation Toxicity LCso (mg/L) No effects at vapor saturation - Acute Dermal Toxicity LDso (mg/kg) 126-200 - Repeated-Dose Toxicity NOAEL/LOAEL Oral (mg/kg-day) NOAEL = 20 LOAEL = 50 - Repeated-Dose Toxicity NOAEC/LOAEC Inhalation (mg/L) NOAEC = Not established LOAEC ~ 0.01 - Reproductive Toxicity NOAEL/LOAEL Oral (mg/kg-day) No effects were seen following evaluation of reproductive organs in a 13-wk repeated-dose oral toxicity study in rats. - Reproductive Toxicity NOAEC/LOAEC Inhalation (mg/L) No effects were seen following evaluation of reproductive organs in a 13-wk repeated-dose inhalation study in rats. - 13 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 3. Summary of Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoint SPONSORED CHEMICAL Hexanedinitrile hydrogenated, high-boiling fraction (crude BHMT) (68411-90-5) SUPPORTING CHEMICAL bis(Hexamethylene) triamine (purified BHMT) (143-23-7) Developmental Toxicity NOAEL/LOAEL Oral (mg/kg-day) Maternal Developmental NOAEL = Not established LOAEL = 50 NOAEL = Not established LOAEL = 50 - Genetic Toxicity - Gene Mutation In vitro Positive Negative Genetic Toxicity - Chromosome Aberrations In vivo Negative - Genetic Toxicity- Other Unscheduled DNA Synthesis In vitro Negative - Additional Information Skin Irritation Eye Irritation Corrosive Corrosive - Measured data in bold; - indicates endpoint was not assessed 4. Hazard to the Environment Acute Toxicity to Fish No adequate data. Acute Toxicity to Aquatic Invertebrates No adequate data. Toxicity to Aquatic Plants No adequate data. Conclusion: No adequate data were submitted for crude BHMT to evaluate acute toxicity to fish, aquatic invertebrates, and aquatic plants. 14 ------- |