U.S. Environmental Protection Agency Hazard Characterization Document December 2012 SCREENING-LEVEL HAZARD CHARACTERIZATION TEST RULE CHEMICAL NAME Methane, isocyanato- (CASRN 624-83-9) SUPPORTING CHEMICAL Urea, N,N'-dimethyl- (CASRN 96-31-1) 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. In the HPV Challenge Program, companies have sponsored more than 2200 HPV chemicals, with approximately 1400 chemicals sponsored directly through the HPV Challenge Program and over 860 chemicals sponsored indirectly through international efforts. Other chemicals, however, remain unsponsored in the voluntary program.3 Basic hazard data for unsponsored chemicals are being obtained through regulatory efforts such as TSCA Section 4 Test Rules and TSCA Section 8(a)/8(d) Rules. EPA is also initiating actions, such as significant new use rules (SNUR), to manage risks from HPV unsponsored chemicals. The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is evaluating the data available for HPV chemicals by developing hazard characterizations (HCs). These HCs consist of an evaluation of the quality and completeness of the data set available. They are not intended to be definitive statements regarding the possibility of unreasonable risk of injury to health or the environment. 2 4 The evaluation is performed according to established EPA guidance ' and is based on hazard data provided by submitters in response to EPA's regulatory actions, as well as other available data; however, in preparing the hazard characterization, EPA considered its own comments and public comments on available data as well as the submitter's responses to comments. 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. Regulatory Actions for Unsponsored Chemicals: http://www.epa.gov/hpv/pubs/general/regactions.htm. 4 U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm. 1 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 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 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 December 2012 Chemical Abstract Service Registry Number (CASRN) Test Rule Chemical 624-83-9 Supporting Chemical 96-31-1 Chemical Abstract Index Name Test Rule Chemical Methane, isocyanato- SuDDorting Chemical Urea, N,N'-dimethyl- Structural Formula See Appendix Summary Methane, isocyanato- is a liquid with high vapor pressure that reacts rapidly with water to first form methylcarbamic acid, which immediately decomposes to form methylamine and carbon dioxide. The methylamine then reacts with remaining methane, isocyanato- to yield urea, N,N'- dimethyl- (CASRN 96-31-1) as the final hydrolysis product. Mobility in soil, volatilization and biodegradation are not important environmental fate properties for methane, isocyanato- due to the rapid rate of hydrolysis. The rate of atmospheric photooxidation is negligible; however, this substance will likely react with moisture in the atmosphere. Methane, isocyanato- is expected to have low persistence (PI) and low bioaccumulation potential (Bl). Human Health Hazard The acute inhalation toxicity of CASRN 624-83-9 is high in rats, mice and guinea pigs. The LC50 values for inhalation are 0.014, 0.028 and < 0.012 mg/L for rats, mice and guinea pigs, respectively. Although the available repeated-dose and reproductive/developmental toxicity studies do not meet the minimum exposure duration of standard test guidelines, these endpoints are considered to be satisfied due to: 1) the high toxicity that is observed in relatively short exposures, 2) the highly irritating nature of the substance which may preclude longer term testing, 3) the reactive nature of the substance (rapid rate of hydrolysis) and 4) the likelihood that the substance is only used as a chemical intermediate. In an 8-day repeated-exposure inhalation toxicity study, rats 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 exposed to CASRN 624-83-9 as a vapor at 0.0072 mg/L/day showed body weight loss, decreased food consumption, and hematological and respiratory tract effects. The NOAEC is 0.0014 mg/L/day. In a 4-day repeated-exposure inhalation toxicity study, rats exposed to CASRN 624-83-9 as a vapor at 0.0070 mg/L/day in males and 0.0065 mg/L/day in females showed mortality, decreased body weight and respiratory tract, spleen, thymus, heart and liver effects. The NOAEC is 0.0026 mg/L/day (males and females). In a 4-day repeated- exposure inhalation toxicity study in mice, respiratory tract lesions were observed at 0.0070 mg/L/day in males and 0.0065 mg/L/day in females. The NOAEC is 0.0026 mg/L/day (males and females). In a reproductive/developmental toxicity study in which male and female rats received a single 30-minute inhalation exposure to CASRN 624-83-9 as a vapor before mating, reproductive and developmental effects included reductions in numbers of implantations and live births, reduced fetal size, increased resorptions, and increased incidences of gross, visceral and skeletal abnormalities at 0.00050 mg/L; decreased maternal body weight was also observed at 0.00050 mg/L. The NOAECs for maternal and reproductive/developmental toxicity are not established. In another single-exposure reproductive/developmental toxicity study in which female rats exposed to CASRN 624-83-9 for a 30-minute period were mated with untreated males, increased resorptions and decreased maternal body weight were observed at 0.00082 mg/L and increased incidences of gross and skeletal abnormalities were observed at 0.00050 mg/L. The NOAEC for reproductive and maternal toxicity is 0.00062 mg/L, while the NOAEC for developmental toxicity is not established. In a prenatal inhalation developmental toxicity study in which mice were exposed to CASRN 624-83-9 as a vapor on gestation days 14 through 17, a significant increase in fetal deaths was observed at 0.0023 mg/L but no signs of maternal toxicity were evident. The NOAEC for developmental toxicity is not established, while the NOAEC for maternal toxicity is 0.0070 mg/L, the highest concentration tested. In an inhalation reproductive/developmental toxicity study in which mice were exposed for 3 hours to CASRN 624-83-9 as a vapor on gestation day 8, increased fetal resorptions and decreased fetal weights were observed at 0.0047 mg/L, while decreased maternal body weight was observed in mice at 0.021 mg/L. The NOAEC for maternal toxicity is 0.014 mg/L and the NOAEC for reproductive/developmental toxicity is not established. CASRN 624-83-9 induced genetic mutations, sister chromatid exchange and chromosomal aberrations in mammalian cells in vitro, but did not induce genetic mutations in bacteria in vitro. CASRN 624-83-9 induced chromosomal aberrations, sister chromatid exchange and micronuclei in mice in vivo. CASRN 624-83-9 did not induce dominant lethal mutations in mice in vivo. CASRN 624-83-9 is irritating to the eyes of rabbits and skin of rabbits and guinea pigs. CASRN 624-83-9 is a dermal sensitizer in guinea pigs. Hazard to the Environment 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 The 96-h LC50 of supporting chemical CASRN 96-31-1 for fish is -10,000 mg/L. The 48-h EC50 of CASRN 96-31-1 for aquatic invertebrates is > 500 mg/L. The 72-h EC50 values of CASRN 96-31-1 for aquatic plants are 560 and > 500 mg/L for biomass and growth rate, respectively. No data gaps were identified under the HPV Program. Introduction Methane, isocyanato- (CASRN 624-83-9) was identified as a candidate chemical under the EPA Challenge program for high production volume chemicals. As it was not sponsored in the voluntary phase of the HPV Challenge Program, it was deemed as subject to testing requirements under a TSCA Section 4 Test Rule (Testing of Certain High Production Volume Chemicals, Final Rule, 71 FR 13708, March 16, 2006; Document ID EPA-HQ-OPPT-2005-0033-0197; available at http://www.regulations.gov/#!documentDetail;D=EPA-HO-OPPT-2005-0033-0197). The test rule required the following toxicological tests for CASRN 624-83-9: CI (consisting of acute toxicity to fish, acute toxicity to Daphnia, and toxicity to algae if log Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae if log Kow > 4.2; because CASRN 624-83-9 undergoes rapid hydrolysis to form carbon dioxide and urea, N,N'-dimethyl-, acute aquatic toxicity testing was considered appropriate). Testing for other SIDS human health and ecological endpoints was not required by the test rule because adequate data were available from the open literature to characterize those endpoints. In response to the test rule, Bayer CropScience submitted the following studies, which were conducted on the proposed analog test substance urea, N,N'-dimethyl- (CASRN 96-31-1), to satisfy the toxicological testing requirements: BASF AG. (1989) Department of Toxicology. Project 10F0173/895165. December 29, 1989 (unpublished study). Summary available at http://www.regulations.gov/#!documentDetail;D=EPA-HO-OPPT-2005-0033-0249 as of December 4, 2012. BASF AG. (1988a) Department of Ecology. Project 0383/88. April 20, 1988 (unpublished study). Summary available at http://www.regulations.gov/#!documentDetail;D=EPA-HQ- OPPT-2005-0033-0249 as of December 4, 2012. BASF AG. (1988b) Department of Ecology. Project 0383/88. August 12, 1988 (unpublished study). Summary available at http://www.regulations.gov/#!documentDetail;D=EPA-HQ- OPPT-2005-0033-0249 as of December 4, 2012. The submitted data, as well as other available data, are summarized in this hazard characterization. Justification for Supporting Chemicals 5 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Data provided for supporting chemical urea, N,N' -dimethyl- (CASRN 96-31-1) were used to assess the ecotoxicity of methane, isocyanato- (CASRN 624-83-9). EPA accepts this substance as a supporting chemical due to the rapid hydrolysis (with a half-life on the order of minutes) of CASRN 624-83-9 to CASRN 96-31-1 and carbon dioxide. 1. Physical-Chemical Properties 1.1 Identification and Purity Toxicological studies reviewed in this hazard characterization tested a substance with 90 - 99.9% purity. The structure of the compound is provided in the Appendix. 1.2 Physical-Chemical Properties The physical-chemical properties of methane, isocyanato- are summarized in Table 1. Methane, isocyanato- is a liquid with high vapor pressure that reacts rapidly with water to first form methylcarbamic acid, which immediately decomposes to form methylamine and carbon dioxide. The methylamine then reacts with remaining methane, isocyanato- to yield urea, N,N'-dimethyl - as the final hydrolysis product (CASRN 96-31-1). Its primary use is as an intermediate in the production of carbamate pesticides. Table 1. Physical-Chemical Properties of Methane, isocyanato-1 Property Value CASRN 624-83-9 Molecular Weight 57.05 Physical State Liquid Melting Point -45°C (measured) Boiling Point 39.1 - 40.1°C (measured) Vapor Pressure 348 mm Hg at 20°C (measured) Dissociation Constant (pKa) Not applicable Henry's Law Constant Not applicable Water Solubility Not applicable2 Log Kow Not applicable 1 Bayer CropScience Inc. (2007) Methyl isocyanate test plan, CAS Number 624-83-9. EPA-HQ-OPPT- 2005-0033-0249. Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HO- QPPT-2005-0033-0249 as of August 6, 2012. 2 Methane, isocyanato- hydrolyzes rapidly in water to yield the methylamine, which immediately decomposes to form carbon dioxide and urea, N,N'-dimethyl- (CASRN 96-31-1). 2. General Information on Exposure 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 2.1 Production Volume and Use Pattern CASRN 624-83-9 had an aggregated production and/or import volume in the United States between 10 to 50 million pounds during calendar year 2005. Industrial processing and uses for the chemical were claimed confidential. No commercial and consumer uses were reported for the chemical. 2.2 Environmental Exposure and Fate The environmental fate characteristics of methane, isocyanato- are summarized in Table 2. Volatilization, mobility in soil, and bioaccumulation will not be important environmental fate processes for methane, isocyanato- due to the rapid rate of hydrolysis. Its only stable hydrolysis byproduct, urea, N,N'-dimethyl-, was degraded 90-100% as measured by dissolved organic carbon removal (DOC) using an activated sludge and the modified AFNOR (OECD 301 A) test. The rate of atmospheric photooxidation is negligible; however, this substance will likely react with moisture in the atmosphere. Methane, isocyanato- is expected to have low persistence (PI) and low bioaccumulation potential (Bl). Table 2. Environmental Fate Characteristics of Methane, isocyanato-1 Property Value CASRN 624-83-9 Photodegradation Half-life 79 days (estimated)2 Hydrolysis Half-life 20 minutes at 15°C (measured); 9 minutes at 25°C (measured) Biodegradation 90-100% after 21 days (readily biodegradable, OECD 301 A)1'3 Bioaccumulation Factor Not applicable due to hydrolysis Log Koc Not applicable due to hydrolysis Fugacity (Level III Model)2'4 Air (%) Water (%) Soil (%) Sediment (%) 99.7 0.1 0.1 <0.1 Persistence5 PI (low) Bioaccumulation5 Bl (low) 1 Bayer CropScience Inc. (2007) Methyl isocyanate test plan, CAS Number 624-83-9. EPA-HQ-OPPT- 2005-0033-0249. Available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ- QPPT-2005-0033-0249 as of August 6, 2012). 2 U.S. EPA. (2012) Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. Washington, DC: U.S. Environmental Protection Agency. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of August 1, 2012. 3 Data pertain to urea, N,N'-dimethyl- (CASRN 96-31-1). 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Table 2. Environmental Fate Characteristics of Methane, isocyanato-1 Property Value 4 Half-lives of 0.1 hours were used for the water, soil, and sediment compartments while a half-life of 1,872 hours was used for the atmosphere compartment. 5 Federal Register. (1999) Category for persistent, bioaccumulative, and toxic new chemical substances. U.S. Environmental Protection Agency. Federal Register 64(213):60194-60204. Conclusion: Methane, isocyanato- is a liquid with high vapor pressure that reacts rapidly with water to first form methylcarbamic acid, which immediately decomposes to form methylamine and carbon dioxide. The methylamine then reacts with remaining methane, isocyanato- to yield urea, N,N'-dimethyl- (CASRN 96-31-1) as the final hydrolysis product. Mobility in soil, volatilization and biodegradation are not important environmental fate properties for methane, isocyanato- due to the rapid rate of hydrolysis. The rate of atmospheric photooxidation is negligible; however, this substance will likely react with moisture in the atmosphere. Methane, isocyanato- is expected to have low persistence (PI) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of health effects data for SIDS endpoints is provided in Table 3. Acute Inhalation Toxicity Methane, isocyanato- (CASRN 624-83-9) (1) Fischer 344 rats (6/sex/group) were exposed whole-body to CASRN 624-83-9 (99.9% purity) as a vapor at concentrations of 0, 1, 2.4, 5.4, 10.5 or 20.4 ppm (0, 0.00233, 0.0056, 0.0126, 0.0245 and 0.0476 mg/L, respectively) for 6 hours and observed for 14 days following exposure. No mortalities were observed in animals exposed to 1.0 or 2.4 ppm. Percent mortality of males and females combined was ~ 83%, 50% and 100% for the 5.4, 10.5 and 20.4 ppm groups, respectively. (Dodd et al., 1986) LC50 = 0.014 mg/L (2) B6C3F1 mice (6/sex/group) were exposed whole-body to CASRN 624-83-9 (99.9% purity) as a vapor at concentrations of 0, 1, 2.4, 5.4, 10.5 or 20.4 ppm (0, 0.00233, 0.0056, 0.0126, 0.0245 and 0.0476 mg/L) for 6 hours and observed for 14 days following exposure. No mortalities were observed in animals exposed to 1.0 or 2.4 ppm. Percent mortality of males and females combined was ~ 8%, 33% and 83% for the 5.4, 10.5 and 20.4 ppm groups, respectively. (Dodd et al., 1986) LC50 = 0.028 mg/L 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 (3) Hartley albino guinea pigs (6/sex/group) were exposed whole-body to CASRN 624-83-9 (99.9% purity) as a vapor at concentrations of 0, 1, 2.4, 5.4 or 10.5 ppm (0, 0.00233, 0.0056, 0.0126 and 0.0245 mg/L) for 6 hours and observed for 14 days following exposure. No mortalities were observed in animals exposed to 1.0 or 2.4 ppm. Percent mortality of males and females combined was ~ 42% and 100% for the 5.4 and 10.5 ppm groups, respectively. (Dodd et al., 1986) LC50 = 0.013 mg/L (4) Fischer 344 rats (6/sex/group) were exposed whole-body to CASRN 624-83-9 (99.9% purity) as a vapor at concentrations of 5.2, 15.2, 25.6 or 36.1 ppm (0.0121, 0.0355, 0.0597 or 0.0842 mg/L) for 4 hours and observed for 14 days following exposure. Percent mortality in males was 0%>, 83%), 100%) and 100% for the 5.2, 15.2, 25.6 and 36.1 ppm groups, respectively. Percent mortality in females was 17%, 67%, 83% and 100% for the 5.2, 15.2, 25.6 and 36.1 ppm groups, respectively. (Dodd et al., 1986) LC50 = 0.026 mg/L (5) Hartley guinea pigs (6/sex/group) were exposed whole-body to CASRN 624-83-9 (99.9% purity) as a vapor at concentrations of 5.2, 15.2, 25.6 or 36.1 ppm (0.0121, 0.0355, 0.0597 or 0.0842 mg/L) for 4 hours and observed for 14 days following exposure. Percent mortality in males was ~ 67%, 100%, 100% and 100% for the 5.2, 15.2, 25.6 and 36.1 ppm groups, respectively. Percent mortality in females was 50%, 100%, 83% and 100%) for the 5.2, 15.2, 25.6 and 36.1 ppm groups, respectively. (Dodd et al., 1986) LC50 < 0.012 mg/L Repeated-Dose Toxicity Although the studies in this section do not meet the minimum exposure duration of 28 days as specified in standard test guidelines, the endpoint is considered to be satisfied in light of: 1) the high toxicity that is observed in relatively short exposures, 2) the highly irritating nature of the substance which may preclude longer term testing, 3) the reactive nature of the substance (rapid rate of hydrolysis), 4) the likelihood that the substance is only used as a chemical intermediate and 5) evidence that the reproductive and developmental toxicity LOAELs are lower than those for the repeated-dose endpoint. Methane, isocyanato- (CASRN 624-83-9) (1) Fischer 344 rats (10/sex/group) were exposed whole-body to CASRN 624-83-9 (99.9% purity) as a vapor at mean measured concentrations of 0, 0.15, 0.58 or 3.1 ppm (0, 0.00035, 0.0014 and 0.0072 mg/L) for 6 hours/day for 8 days (two 4-day sessions separated by a 2-day rest) and sacrificed the morning following the last exposure. Animals were observed daily for mortality and clinical signs of toxicity; body weight and food consumption were measured periodically during the study. A modified Irwin screen to assess neurological function was performed on 5 rats/sex of the 0, 0.58 and 3.1 ppm groups prior to the first exposure and after the last exposure. Gross ophthalmoscopic examinations were done on control and high- concentration animals prior to exposure initiation and prior to sacrifice. At study termination, blood was collected for hematology, and the lungs, liver, kidneys and testes were weighed. The entire respiratory tract, lymph nodes, kidneys, liver, testes and gross lesions were preserved from 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 all animals and these tissues from the control and high-concentration animals were examined microscopically. Additionally, the respiratory tract of rats of the intermediate and low- concentration groups was examined microscopically. All animals survived to scheduled termination. No adverse effects were observed in animals exposed to 0.15 or 0.58 ppm. Treatment-related effects observed in high-concentration males and females included a higher incidence of reddish crust or clear nasal discharge than controls, impaired gait, arched back, significant (p < 0.001) weight loss and decreased food consumption. Increased hemoglobin concentration and decreased oxygen saturation were observed in males only in the high-concentration group. Observations in high-concentration animals at necropsy included significantly (p < 0.001) increased absolute and relative lung weights and reddening of the lungs. Absolute weights of liver, kidney and testes were decreased in animals exposed to 3.1 ppm; however, weight changes in these organs were considered to be due to the loss of body weight in animals, as no corresponding gross or microscopic lesions were observed. Microscopic findings were confined to the lungs and respiratory tract of animals exposed to 3.1 ppm. Statistically significantly (p < 0.05) increased incidences of rhinitis and squamous metaplasia were observed in the nasal passages of both sexes; the incidence of epithelial cell degeneration in the olfactory mucosa of the nasal cavity was increased in males, but not to a statistically significant degree. Statistically significantly (p < 0.05) increased incidences of tracheitis and squamous metaplasia were observed in the tracheas of both sexes. Statistically significantly (p < 0.05) increased incidences of bronchiolitis and pneumonitis were observed in both sexes. An increased incidence of squamous metaplasia was observed in the bronchioles of both sexes, but the change was only statistically significant (p < 0.05) in females. An increased incidence of submucosal fibroplasias was observed in the lungs of males. Regenerative hyperplasia was observed in the trachea of one male and one female, as well as in the lungs/bronchioles of one female. A statistically significantly (p < 0.05) increased incidence of reactive hyperplasia of the bronchial lymph nodes was observed in both sexes. With the exception of rhinitis, none of these microscopic findings was observed in the control, 0.15 or 0.58 ppm animals. (Dodd et al., 1987) LOAEC = 0.0072 mg/L/day (based on decreased food consumption, body weight loss, clinical signs of toxicity [including nasal crust/discharge, impaired gait, and arched back], increased hemoglobin concentration [males only], decreased blood oxygen saturation [males only], increased absolute and relative lung weights, and gross and microscopic lesions of the respiratory tract) NOAEC = 0.0014 mg/L/day (2) Fischer 344 rats (28/sex for control and low-concentration groups; 56/sex for high- concentration groups) were exposed whole-body to CASRN 624-83-9 (> 99% purity) as a vapor at mean measured concentrations of 0, 1.13 or 2.98/2.79 (males/females) ppm (0, 0.0026 and 0.0070/0.0065 mg/L) for 6 hours/day for 4 days and observed for up to 91 days following exposure. Five animals/sex/group were sacrificed on days 7, 28 and 49, and all survivors were sacrificed on day 91. All animals were observed once or twice daily for mortality, morbidity and clinical signs throughout the study, and twenty animals per group were weighed periodically. All animals were subjected to gross necropsy and tissues examined microscopically. Organs and tissues examined microscopically included three levels of nasal passage, trachea at the level of 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 the thyroid, major bronchi at the bifurcation of the trachea, all lobes of the lung including major airways, liver, thyroids, parathyroids, esophagus, peribronchial lymph node, brain, both kidneys, eyes, thymus, spleen, heart, and glandular and nonglandular stomach. No exposure-related mortalities or adverse effects were observed in animals in the low concentration group. In the high-concentration group, 49 males and 51 females died during post- exposure days 9 to 28; therefore, while 5/rats/sex were killed at the day-7 sacrifice, only two males remained for sacrifice on day 28. Prior to death, both sexes had significantly (p < 0.05) decreased body weight beginning on day 3 for males and day 1 for females. Histopathologic effects observed in high-concentration animals included: respiratory epithelial regeneration in nasal passages and trachea; olfactory epithelial regeneration in the meatus and dorsal ethmoid recess; inflammation, epithelial erosion and/or fibrosis in the bronchi and bronchioles; alveolar inflammation and fibrosis; presence of hyaline membranes in alveolar ducts, and alveoli and pulmonary atelectasis. Lymphocytic necrosis of the thymus and atrophy of the spleen were observed in animals that died during days 8 to 14. Atrophy of the thymus, sometimes with increased extramedullary hematopoiesis of the spleen, was observed in animals that died after day 15. Centrilobular necrosis of the liver and thrombosis of the left cardiac atrium were observed together in animals with severe injuries in the pulmonary alveoli, often with concomitant multifocal hemorrhage without inflammatory reaction in the brain. (Mitsumori et al., 1987) LOAEC (males/females) = 0.0070 / 0.0065 mg/L/day (based on mortality, decreased body weight, respiratory and olfactory lesions, atrophy of the spleen, lymphocytic necrosis and atrophy of the thymus, atrial thrombosis and hepatocellular necrosis) NOAEC (both sexes) = 0.0026 mg/L/day (3) B6C3F1 mice (28/sex/group) were exposed whole-body to CASRN 624-83-9 (> 99% purity) as a vapor at mean measured concentrations of 0, 1.13 or 2.98/2.79 (males/females) ppm (0, 0.0026 and 0.0070/0.0065 mg/L) for 6 hours/day for 4 days and observed for up to 91 days following exposure. Five animals/sex/group were sacrificed on days 7, 28 and 49, and all survivors were sacrificed on day 91. All animals were observed once or twice daily for morbidity, mortality and clinical signs throughout the study, and twenty animals per group were weighed periodically. All animals were subjected to gross necropsy. The organs and tissues examined histopathologically included three levels of nasal passage, trachea at the level of the thyroid, major bronchi at the bifurcation of the trachea, and all lobes of the lung including major airways, liver, gallbladder, thyroids, parathyroids, esophagus, peribronchial lymph node, brain, both kidneys, eyes, thymus, spleen, heart, and glandular and nonglandular stomach. No exposure-related mortalities or adverse effects were observed in animals exposed at the low concentration. One male exposed in the high concentration group died on day 16 post-exposure; no further mortalities at the high-concentration level were observed. In the high-concentration group, modest decreases in body weight (7-11% compared to control, p < 0.05) were observed in males on post-exposure days 3 and 6 and in females on day 1, with absolute body weight recovering to control levels by post-exposure day 14 and day 6 post-exposure in males and females, respectively. Histopathology effects observed in high-concentration animals at necropsy included: acute inflammation of the mucosa of the nasal passages, characterized by a mild influx of granulocytes, on day 7 (resolved by day 28); respiratory epithelial regeneration in 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 the nasal passages, trachea and bronchi on day 7 (resolved by day 28 in nasal passages and trachea and by day 49 in bronchi); olfactory epithelial regeneration in the dorsal meatus and ethmoturbinates on day 7 (resolved by day 28) and mural and/or intraluminal fibrosis of the bronchi at sacrifices on days 7, 28, 49, and 91. (Mitsumori et al., 1987) LOAEC (males/females) = 0.0070 / 0.0065 mg/L/day (based on olfactory and respiratory tract lesions) NOAEC (both sexes) = 0.0026 mg/L/day Reproductive/Developmental Toxicity Although these studies do not meet the minimum exposure duration specified in standard test guidelines, the endpoint is considered to be satisfied in light of: 1) the high toxicity that is observed in relatively short exposures, 2) the highly irritating nature of the substance which may preclude longer term testing, 3) the reactive nature of the substance (rapid rate of hydrolysis) and 4) the likelihood that the substance is only used as a chemical intermediate. Methane, isocyanato- (CASRN 624-83-9) (1) Charles Foster rats (5 females/group; number of males/group not specified) were exposed to CASRN 624-83-9 (> 98% purity) as a vapor at 0, 0.212, 0.265 or 0.353 ppm (0, 0.000495, 0.000618 and 0.000824 mg/L) for 30 minutes and then mated. Mating occurred between animals exposed at the same concentration level. Dams were killed on gestation day 20 and all fetuses were examined for external abnormalities. Observations were made for the following: number of resorptions, implantations and implantation sites; number of live and still births; size, weight, sex and gross abnormality of each fetus and viability, growth and deformity of newborns; visceral abnormalities (half of newborns) and skeletal abnormalities (half of newborns). No mortality was observed in dams and there were no effects on maternal food and water intake; results for males were not reported. No information on statistical analyses/significance was reported for any observation. All groups of dams gained weight after exposure; however, the amount of weight gained was inversely related to the exposure concentration. Final body weight on gestation day 20 in the low-, mid- and high-concentration groups was reduced 10%, 19% and 24%), respectively, relative to controls. Fetal weight, length and width were decreased compared to control at the low-concentration (10%>, 12%> and 8%>, respectively), mid-concentration (22%>, 3% and 7%>, respectively) and high-concentration (20%>, 15%> and 10%>, respectively). The percentages of total resorptions were 3%>, 12%>, 20%> and 32%> in the control, low-, mid- and high-concentration groups, respectively; however, data were not reported on a per litter basis. The number of implantations was reduced by 24%>, 32%> and 24%> in the low-, mid- and high- concentration groups, respectively, compared to the control group. The total number of live births was reduced by 31%>, 44%> and 47%> in the low-, mid- and high-concentration groups, respectively, compared to the control group. In each group, the numbers of corpora lutea and implantations were equivalent, indicating no evidence of pre-implantation loss. The percentages of fetuses (litter incidence not given) with limb defects and skeletal and visceral malformations were increased in the treated groups in a concentration-related manner. The following gross abnormalities were increased in exposure groups by at least 5%> compared to the control: everted and inverted claw, valgus deformity, blood clot formation (in neck, limbs and 12 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 tail), and wrinkling of skin (low-, mid- and high-concentration); kinking of tail, wrist drop and knee and shoulder joint defects (mid- and high-concentration) and clubbing of hind limbs (high- concentration). The following visceral and skeletal abnormalities were increased in exposure groups by at least 5% compared to the control: cleft palate formation, partial or non-ossification of skull bones, ribs attached to sternum, absence of ribs, unequal ribs and ribs bent downward (low-, mid- and high-concentration) and liver enlargement, immovable humerus, ribs bent upwardly and partial ossification of ribs (mid- and high-concentration). (Singh et al., 1994) LOAEC (maternal toxicity) = 0.00050 mg/L (based on decreased maternal body weight) NOAEC (maternal toxicity) = Not established LOAEC (reproductive/developmental toxicity) = 0.00050 mg/L (based on reduced fetal body weight, length and width, increased percentage of total resorptions, reduced numbers of implantations and live births, increased incidences of gross abnormalities [everted and inverted claw, valgus deformity, blood clot formation in neck, limbs, and tail, and wrinkling of skin], visceral abnormalities [cleft palate formation], and skeletal abnormalities [partial ossification of skull bones, non-ossification of skull bones, ribs attached to sternum, absence of ribs, unequal ribs, and ribs bent downwardly]) NOAEC (reproductive/developmental toxicity) = Not established (2) Charles Foster rats (5 females/group) were exposed to CASRN 624-83-9 (> 90% purity) as a vapor at 0, 0.212, 0.265 or 0.353 ppm (0, 0.000495, 0.000618 and 0.000824 mg/L) for 30 minutes and then mated with untreated males. Animal body weight was measured periodically throughout the study. Dams were sacrificed on gestation day 20 and all fetuses were examined for external abnormalities. Observations were made for the following: number of resorptions, implantations and implantation sites; number of live and still births; size, weight, sex and gross abnormality of each fetus and viability, growth and deformity of newborns. One-half of the fetuses were examined for visceral abnormalities and the remaining one-half were examined for skeletal abnormalities. No information on statistical analyses/significance was reported for any observation. No mortality or effects on food and water intake were observed during the study. Final maternal body weight on gestation day 20 was decreased 6%, 6% and 10% in the low-, mid- and high- concentration groups, respectively, compared to the control group. Compared to the control group, fetal weight was decreased 3%, 23% and 6% in the low-, mid- and high-concentration groups, respectively, and fetal length was decreased by 2%, 5% and 5% in the low-, mid- and high-concentration groups, respectively. Fetal width, compared to the control group, was decreased by 2%, 4% and 4% in the low-, mid- and high-concentration groups, respectively. The percentages of total resorption in the control, low-, mid- and high- concentration groups were 3%, 4%, 6% and 9%, respectively. A 5% difference in the percentage of total resorptions between treatment and control groups is considered to be toxicologically significant; therefore a toxicologically significant effect on total resorptions occurred in the high- concentration group. The following gross abnormalities were increased in exposure groups by at least 5% compared to the control: everted claw and blood clot formation (low-, mid- and high-concentration); wrist drop, inverted claw, knee and shoulder joint defects and wrinkling of skin (mid- and high- concentration) and kinking of tail and valgus deformity (high-concentration). The following 13 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 visceral and skeletal abnormalities were increased in exposure groups by at least 5% compared to the control: non-ossification of skull bones, ribs attached to sternum and absence of ribs (low-, mid- and high-concentration); liver enlargement, cleft palate formation, partial ossification of the skull bone, unequal ribs, ribs bent upwardly or downwardly and partial ossification of the ribs (mid- and high-concentration) and immovable humerus (high-concentration). (Singh et al., 1996) LOAEC (maternal toxicity) = 0.00082 mg/L (based on decreased maternal body weight) NOAEC (maternal toxicity) = 0.00062 mg/L LOAEC (developmental toxicity) = 0.00050 mg/L (based on increased gross abnormalities [everted claw and blood clot formation] and increased skeletal abnormalities [absence of ribs, non-ossification of skull bones and ribs attached to sternum]) NOAEC (developmental toxicity) = Not established LOAEC (reproductive toxicity) = 0.00082 mg/L (based on increased percentage of total resorptions) NOAEC (reproductive toxicity) = 0.00062 mg/L (3) Pregnant female Swiss (CD-I) mice (39-44 females/group) were exposed whole-body to CASRN 624-83-9 as a vapor at 0, 1 or 3 ppm (0, 0.0023 and 0.0070 mg/L; analytical concentrations were within 10% of nominal) for 6 hours/day on gestation days 14 through 17. Dams were allowed to litter naturally and offspring were evaluated for survival and development. Maternal body weight was measured on gestation days 14 and 18 and on day 21 of lactation. Offspring were weighed, counted and sexed upon delivery and on lactation days 4, 7, 14 and 21. The following developmental parameters were evaluated: number of days from mating to parturition; number of live and dead offspring at delivery; mean pup weights and the number of pregnant females with live pups. No maternal toxicity was observed. A statistically significant (p < 0.05) increase in the total number of dead fetuses at birth was observed in both exposure groups, and a statistically significant (p < 0.05) increase in pup mortality during lactation was observed in the high-concentration group only. Pup mortalities in the high- concentration group resulted in statistically significantly (p < 0.05) fewer pups per litter on lactation days 0 through 4. Pup body weight during lactation was not affected by treatment. (Schwetz et al., 1987) NOAEC (maternal toxicity) = 0.0070 mg/L/day (highest concentration tested) LOAEC (reproductive/developmental toxicity) = 0.0023 mg/L/day (based on a significant increase in the number of dead fetuses observed at birth) NOAEC (reproductive/developmental toxicity) = Not established (4) Pregnant female Swiss Webster mice (11-18 females/treatment group; 24 females/control group) were exposed whole-body to CASRN 624-83-9 as a vapor at 0, 2, 6, 9 or 15 ppm (0, 0.0047, 0.014, 0.021 and 0.035 mg/L) for 3 hours on gestation day 8 (GD 8). Additionally, groups of 5 and 3 animals were similarly exposed to 9 ppm and 15 ppm, respectively, on GD 14. A pre-mating exposure group consisting of 8 male and 12 virgin female mice was exposed to CASRN 624-83-9 at 9 ppm for 3 hours and then placed with control females and males, respectively, for approximately 2 weeks to determine their mating performance. All dams exposed during gestation were assessed for mortality and numbers of resorptions and dead and live fetuses; body, placenta and lung weights were measured daily in dams exposed on GD 8. All fetuses of animals from the 9 and 15 ppm dose groups exposed on 14 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 GD 8 and GD 14 were examined for external and visceral or skeletal abnormalities. For animals exposed during pre-mating, mating performance (presence of vaginal plugs and pregnancy rates) was assessed. Serum levels of corticosterone and progesterone were evaluated in all pregnant and nonpregnant females. Two dams in each of the 9- and 15-ppm GD-8 groups died, but the difference was not statistically significant compared to the control group. All three animals exposed to 15 ppm on GD 14 died within 24 hours. Mortality was observed in two of five animals exposed to 9 ppm on GD 14. A concentration-dependent decrease in maternal body weight on GD 18 was observed in mice exposed to 15 or 9 ppm on GD 8, with reductions of 43% and 41%, respectively, relative to controls. Relative lung weights of mice exposed to 2 or 6 ppm on GD 8 were significantly decreased compared to controls. Relative lung weights of mice exposed to 9 or 15 ppm on GD 8 were significantly increased; study authors speculate that this may be a reflection of substantially lower body weight of mice in these groups. Complete litter resorption occurred in 0%, 9%, 8%, 80% and 75% of females exposed on GD 8 at 0, 2, 6, 9 and 15 ppm, respectively. In the surviving animals exposed to 9 ppm on GD 14, 17%) of implants had resorbed. Decreased fetal and placental weights in animals that retained pregnancy were observed at all GD-8 exposure levels. In the 9 ppm/GD 14 group, fetal body weight was decreased by 13%. Meningocele was observed in 1 pup exposed on GD 8 (exposure group not specified); no additional external abnormalities were observed in fetuses of exposed animals. An increased incidence of visceral abnormalities was observed in fetuses of animals exposed to 9 or 15 ppm on GD 8; additionally, these exposure groups showed a significant (~ 20%) decrease in the length of fetal mandible, humerus, radius, ulna, femur, tibia and fibula. Changes in hormone levels were associated with toxicity in mice exposed on GD 8 or prior to mating. A statistically significant (p < 0.05) increase in serum corticosterone levels was observed in males and non-pregnant females exposed to 9 ppm in the pre-mating study. During a 12-day observation period, persistent diestrus was observed in eleven of twelve females exposed prior to mating, while control mice showed a regular estrus cycle. Nine of twelve nonpregnant females exposed to 9 ppm mated within 12 days, whereas all thirty control females mated within 6 days of cohabitation. The resulting pregnancy rate was 33% in exposed females and 77% in controls. Five of eight males exposed to 9 ppm mated control females within 12 days, while all ten control males mated at least one control female in less than 2 days and all thirty control females in 6 days; the pregnancy rates of the exposed and control male matings were 55% and 77%, respectively. Males and nonpregnant females exposed to 9 ppm showed statistically significant (p < 0.001) differences in mating performance compared to controls. (Varma et al., 1987) LOAEC (maternal) = 0.021 mg/L (based on decreased body weights) NOAEC (maternal) = 0.014 mg/L LOAEC (reproductive/developmental) = 0.0047 mg/L (based on increased fetal resorption and decreased placental and fetal weights) NOAEC (reproductive/developmental) = Not established 15 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Genetic Toxicity - Gene Mutation In vitro Methane, isocyanato- (CASRN 624-83-9) (1) In an NTP reverse mutation assay, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to CASRN 624-83-9 (> 99% purity) at 0 (solvent control), 3.3, 10, 33, 100 or 333 |ig/plate with metabolic activation and at 0 (solvent control), 0.3, 1, 3.3, 10, 33 or 100 |ig/plate without metabolic activation. The cytotoxic concentrations were 333 and 33 |ig/plate with and without metabolic activation, respectively. Positive and solvent controls were included and responded appropriately. No increase in revertants was observed at any concentration in any strain with or without metabolic activation. (Mason et al., 1987) CASRN 624-83-9 was not mutagenic in this assay. (2) In an NTP reverse mutation assay, Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were exposed to CASRN 624-83-9 (> 99% purity) at 0 (solvent control), 3.3, 10, 33, 100 or 333 |ig/plate with metabolic activation and at 0, 3.3, 10, 33, 100 or 200 |ig/plate without metabolic activation. The cytotoxic concentration was 100 |ig/plate with and without metabolic activation. Positive and solvent controls were included and responded appropriately. No increase in revertants was observed at any concentration in any strain with or without metabolic activation. (Mason et al., 1987) CASRN 624-83-9 was not mutagenic in this assay. 16 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 (3) In an NTP study, mouse lymphoma L5178Y cells were exposed to CASRN 624-83-9 (> 99% purity) at 0 (solvent control), 0.5, 0.75, 1, 1.5, 2 or 3 nL/mL without metabolic activation. The cytotoxic concentration was 3 nL/mL. Positive and solvent (dimethyl sulfoxide; DMSO) controls were included and responded appropriately. Genetic mutations were observed at all concentrations tested. (Caspary and Myhr, 1986) CASRN 624-83-9 was mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations In vitro Methane, isocyanato- (CASRN 624-83-9) (1) In an NTP sister chromatid exchange (SCE) assay, Chinese hamster ovary (CHO) cells were exposed to CASRN 624-83-9 (> 99% purity) at 0 (solvent control), 0.3, 0.9, 3.1 or 9.2 |ig/mL with and without metabolic activation and 0 (solvent control), 0.7, 1.5, 3.0, or 6.0 ng/mL without metabolic activation. Cultures exposed to > 6.0 jag/m L were not scorable due to toxicity. Positive and solvent (DMSO) controls were included and responded appropriately. A concentration-related increase in SCE was observed with and without metabolic activation at 0.9, 3.0, and 3.1 |ig/mL, but not at 0.3 |ag/mL or in the presence of cytotoxicity. (Mason et al., 1987) CASRN 624-83-9 induced sister chromatid exchange in this assay. (2) In an NTP cytogenetics assay, CHO cells were exposed to CASRN 624-83-9 (> 99% purity) at 0 (solvent control), 10.1, 15.0, 20.1 or 25.0 |ag/mL without metabolic activation and at 0 (solvent control), 15.0, 20.1, 25.0 or 30.5 |ag/mL with metabolic activation. Cultures exposed to the highest test concentration, both with and without metabolic activation, were not scorable due to toxicity. Positive and solvent (DMSO) controls were included and responded appropriately. An increased incidence of chromosomal aberrations was observed at all non-toxic exposure concentrations. (Mason etal., 1987) CASRN 624-83-9 induced chromosomal aberrations in this assay. In vivo Methane, isocyanato- (CASRN 624-83-9) In an NTP cytogenetic assay, male B6C3F1 mice (number not specified) were exposed whole- body to CASRN 624-83-9 (> 99% purity) via inhalation at 0 (vehicle control), 3, 10 or 30 ppm (0, 0.007, 0.023 and 0.07 mg/L) for 2 hours. Male and female B6C3F1 mice (number not specified) were similarly exposed to CASRN 624-83-9 at 0, 1, 3 or 6 ppm (0, 0.0023, 0.007, and 0.014 mg/L) 6 hours a day for 4 consecutive days. Positive and vehicle (air) controls were included and responded appropriately. No significant increase in chromosomal aberrations, SCEs or micronuclei was observed in the bone marrow cells of animals exposed for 2 hours; however, a significant delay in cellular proliferation was observed in animals treated with this exposure regimen. Animals receiving the 4-day exposure regimen showed a delay in bone marrow cell proliferation at > 3 ppm for males and at 6 ppm for females; additionally, a depressed rate of erythropoiesis was observed, with males more strongly affected than females. The 4-day exposure regimen showed a weak but significant increase in chromosomal aberrations and SCEs in males (in one experiment) and females (in two experiments). A significant increase 17 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 in micronuclei was observed in males only at the 6 ppm exposure concentration. (Tice et al., 1987) CASRN 624-83-9 induced chromosomal aberrations, SCE and micronuclei in this assay. Genetic Toxicity - Other In vivo Methane, isocyanato- (CASRN 624-83-9) In a dominant lethal study, Swiss (CD-I) mice (30 males/group) were exposed whole-body to CASRN 624-83-9 (> 99% purity) as a vapor at 0, 1 or 3 ppm (0, 0.00233 and 0.007 mg/L) for 6 hours/day for 4 consecutive days. Following the final day of exposure, male mice were mated with untreated females for 8 weeks (different females each week). No treatment-related effect on the incidence or distribution of resorptions was observed. (Schwetz et al., 1987) CASRN 624-83-9 did not induce dominant lethal mutations in this assay. Additional Information Skin Irritation Methane, isocyanato- (CASRN 624-83-9) In four studies, rabbits or guinea pigs were exposed to CASRN 624-83-9 (purity not provided) via the dermal route. Observations included hyperemia, erythema, edema, exfoliation, capillary injection and necrosis. Data are from TSCATS (EPA Doc. No. 86-910000658D, Fiche No. OTS0529151; EPA Doc. No. 86-910000361S, Fiche No. OTS0530132; EPA Doc No. 88- 920009582, Fiche No. OTS0571239). CASRN 624-83-9 was irritating to rabbit and guinea pig skin in these studies. Eye Irritation Methane, isocyanato- (CASRN 624-83-9) In three studies, rabbits were exposed to CASRN 624-83-9 (purity not provided) via the ocular route. Observations included hazy cornea, injected iris, edema, erythema, constriction of the pupil, conjunctival swelling, corneal injury, complete loss of sight, severe eyelid injury, hemorrhage and necrosis. Data are from TSCATS (EPA Doc. No. 86-910000658D, Fiche No. OTS0529151; EPA Doc. No. 86-910000361S, Fiche No. OTS0530132; EPA Doc. No. 86- 910000056, Fiche No. OTS0528350). CASRN 624-83-9 was irritating to rabbit eyes in these studies. Sensitization Methane, isocyanato- (CASRN 624-83-9) In four studies, guinea pigs were exposed to CASRN 624-83-9 (purity not provided) via epidermal exposure, intradermal injections or inhalation. This induction exposure was followed by a challenge exposure via epidermal application or intradermal injection. A sensitization response was observed in each study. Data are from TSCATS (EPA Doc. No. 88-920010381, 18 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 FicheNo. OTS0555750; EPA Doc. No. 86-910000658D, FicheNo. OTS0529151; EPA Doc. No. 88-920009582, FicheNo. OTS0571239). CASRN 624-83-9 was sensitizing to guinea pigs in these studies. Conclusion: The acute inhalation toxicity of CASRN 624-83-9 is high in rats, mice and guinea pigs. The LC50 values for inhalation are 0.014, 0.028 and < 0.012 mg/L for rats, mice and guinea pigs, respectively. Although the available repeated-dose and reproductive/developmental toxicity studies do not meet the minimum exposure duration of standard test guidelines, these endpoints are considered to be satisfied due to: 1) the high toxicity that is observed in relatively short exposures, 2) the highly irritating nature of the substance which may preclude longer term testing, 3) the reactive nature of the substance (rapid rate of hydrolysis) and 4) the likelihood that the substance is only used as a chemical intermediate. In an 8-day repeated-exposure inhalation toxicity study, rats exposed to CASRN 624-83-9 as a vapor at 0.0072 mg/L/day showed body weight loss, decreased food consumption, and hematological and respiratory tract effects. The NOAEC is 0.0014 mg/L/day. In a 4-day repeated-exposure inhalation toxicity study, rats exposed to CASRN 624- 83-9 as a vapor at 0.0070 mg/L/day in males and 0.0065 mg/L/day in females showed mortality, decreased body weight and respiratory tract, spleen, thymus, heart and liver effects. The NOAEC is 0.0026 mg/L/day (males and females). In a 4-day repeated-exposure inhalation toxicity study in mice, respiratory tract lesions were observed at 0.0070 mg/L/day in males and 0.0065 mg/L/day in females. The NOAEC is 0.0026 mg/L/day (males and females). In a reproductive/developmental toxicity study in which male and female rats received a single 30-minute inhalation exposure to CASRN 624-83-9 as a vapor before mating, reproductive and developmental effects included reductions in numbers of implantations and live births, reduced fetal size, increased resorptions, and increased incidences of gross, visceral and skeletal abnormalities at 0.00050 mg/L; decreased maternal body weight was also observed at 0.00050 mg/L. The NOAECs for maternal and reproductive/developmental toxicity are not established. In another single-exposure reproductive/developmental toxicity study in which female rats exposed to CASRN 624-83-9 for a 30-minute period were mated with untreated males, increased resorptions and decreased maternal body weight were observed at 0.00082 mg/L and increased incidences of gross and skeletal abnormalities were observed at 0.00050 mg/L. The NOAEC for reproductive and maternal toxicity is 0.00062 mg/L, while the NOAEC for developmental toxicity is not established. In a prenatal inhalation developmental toxicity study in which mice were exposed to CASRN 624-83-9 as a vapor on gestation days 14 through 17, a significant increase in fetal deaths was observed at 0.0023 mg/L but no signs of maternal toxicity were evident. The NOAEC for developmental toxicity is not established, while the NOAEC for maternal toxicity is 0.0070 mg/L, the highest concentration tested. In an inhalation reproductive/developmental toxicity study in which mice were exposed for 3 hours to CASRN 624-83-9 as a vapor on gestation day 8, increased fetal resorptions and decreased fetal weights were observed at 0.0047 mg/L, while decreased maternal body weight was observed in mice at 0.021 mg/L. The NOAEC for maternal toxicity is 0.014 mg/L and the NOAEC for reproductive/developmental toxicity is not established. 19 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 CASRN 624-83-9 induced genetic mutations, sister chromatid exchange and chromosomal aberrations in mammalian cells in vitro, but did not induce genetic mutations in bacteria in vitro. CASRN 624-83-9 induced chromosomal aberrations, sister chromatid exchange and micronuclei in mice in vivo. CASRN 624-83-9 did not induce dominant lethal mutations in mice in vivo. CASRN 624-83-9 is irritating to the eyes of rabbits and skin of rabbits and guinea pigs. CASRN 624-83-9 is a dermal sensitizer in guinea pigs. Table 3. Summary of the Screening Information Data Set - Human Health Data Endpoints TEST RULE CHEMICAL Methane, isocyanato- (624-83-9) Acute Toxicity Inhalation LC50 (mg/L) 0.014 (rats) 0.028 (mice) < 0.012 (guinea pigs) Repeated-Dose Toxicity NOAEC/LOAEC Inhalation (mg/L/day) NOAEC = 0.0026 LOAEC (m/f) = 0.0070 / 0.0065 (rats and mice) Reproductive/Developmental Toxicity NOAEC/LOAEC Inhalation (mg/L/day) Maternal NOAEC = Not established LOAEC = 0.00050 (rats) NOAEC = 0.014 LOAEC = 0.021 (mice) Reproductive/Developmental NOAEC = Not established LOAEC = 0.00050 (rats) NOAEC = Not established LOAEC = 0.0023 (mice) Genetic Toxicity - Gene Mutation In vitro Positive 20 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 Table 3. Summary of the Screening Information Data Set - Human Health Data Endpoints TEST RULE CHEMICAL Methane, isocyanato- (624-83-9) Genetic Toxicity - Chromosomal Aberrations In vitro Positive Genetic Toxicity - Chromosomal Aberrations In vivo Positive Genetic Toxicity - Other Dominant Lethal Mutation, In vivo Negative Additional Information Skin Irritation Eye Irritation Sensitization Irritating Irritating Sensitizing Measured data in bold text 4. Hazard to the Environment A summary of aquatic toxicity data for SIDs endpoints is provided in Table 4. The table also indicates where data for the supporting chemical are read-across (RA) to the test rule chemical. Acute Toxicity to Fish Urea, N,N'-dimethyl- (CASRN 96-31-1, supporting chemical) Golden orfe (Leuciscus idus) were exposed to CASRN 96-31-1 (purity not specified) at nominal concentrations of 0, 1000, 2150, 4640 or 10,000 mg/L under static conditions for 96 hours. Analytical monitoring was not conducted. Exposures occurred at a pH of 7.3 - 7.5, a temperature of 20°C and a dissolved oxygen concentration of 8.1 - 8.6 mg/L. Mortality (60%) was observed only at the 10,000 mg/L concentration. Study summary available at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0249 as of December 4, 2012 (BASF AG, 1989). 96-h LC50 ~ 10,000 mg/L Acute Toxicity to Aquatic Invertebrates Urea, N,N'-dimethyl- (CASRN 96-31-1, supporting chemical) Water fleas (Daphnia magna) were exposed to CASRN 96-31-1 (purity not specified) at nominal concentrations of 0, 31.25, 62.5, 125, 250 or 500 mg/L under static conditions for 48 hours. Analytical monitoring was not conducted. Exposures occurred at a pH of 7.7 - 8.0, a temperature of 19 - 21°C and a dissolved oxygen concentration of 8.0 - 8.7 mg/L. No immobility of Daphnia was observed up to the highest test concentration. Study summary available at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0249 as of December 4, 2012 (BASF AG, 1988a). 21 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 48-h LC50 > 500 mg/L Toxicity to Aquatic Plants Urea, N,N'-dimethyl- (CASRN 96-31-1, supporting chemical) Green algae (Desmodesmus subspicatus) were exposed to CASRN 96-31-1 (purity not specified) at nominal concentrations of 0, 31.25, 62.5, 125, 250 or 500 mg/L under static conditions for 72 hours. Analytical monitoring was not conducted. Exposures occurred at a temperature of 20°C. The pH was 8.5 - 8.6 at 0 hours. Growth rate was inhibited by about 15% at 500 mg/L. Study summary available at http://www.regulati0ns.g0v/#!documentDetail;D=EPA-HQ-QPPT-2005- 0033-0249 as of December 4, 2012 (BASF AG, 1988b). 72-h EC50 (biomass) = 560 mg/L 72-h EC50 (growth) > 500 mg/L Conclusion: The 96-h LC50 of supporting chemical CASRN 96-31-1 for fish is -10,000 mg/L. The 48-h EC50 of CASRN 96-31-1 for aquatic invertebrates is > 500 mg/L. The 72-h EC50 values of CASRN 96-31-1 for aquatic plants are 560 and > 500 mg/L for biomass and growth rate, respectively. Table 4. Summary of the Screening Information Data Set - Aquatic Toxicity Data TEST RULE CHEMICAL SUPPORTING CHEMICAL Endpoint Methane, isocyanato- Urea, N,N'-dimethyl- (624-83-9) (96-31-1) Fish No data 96-h LC50 (mg/L) ~ 10,000 ~ 10,000 (RA) Aquatic Invertebrates No data 48-h EC50 (mg/L) >500 >500 (RA) Aquatic Plants 72-h EC50 (mg/L) No data (biomass) 560 560 (growth) >500 >500 (RA) Measured data in bold text; RA = Read Across 22 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 5. References BASF AG. (1989) Department of Toxicology. Project 10F0173/895165. December 29, 1989 (unpublished study). BASF AG. (1988a) Department of Ecology. Project 0383/88. April 20, 1988 (unpublished study). BASF AG. (1988b) Department of Ecology. Project 0383/88. August 12, 1988 (unpublished study). Caspary, WJ; Myhr, B. (1986) Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells. MutatRes 174:285-293. Dodd, DE; Fowler, EH; Snellings, WM; Pritts, IM; Baron, RL. (1986) Acute inhalation studies with methyl isocyanate vapor. I. Methodology and LC50 determinations in guinea pigs, rats, and mice. Fundam Appl Toxicol 6:747-755. Dodd, DE; Fowler, EH; Snellings, WM; Pritts, IM. (1987) Methyl isocyanate eight-day vapor inhalation study with Fischer 344 rats. Environ Health Perspect 72:117-123. Mason, JM; Zeiger, E; Haworth, S; Ivett, J; Valencia, R. (1987) Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary. Environ Mutagen 9:19-28. Mitsumori, K; Boorman, GA; Gupta, BN; Bucher, JR. (1987) Four-day repeated inhalation and recovery study of methyl isocyanate in F344 rats and B6C3F1 mice. Fundam Appl Toxicol 9:480-495. Schwetz, BA; Adkins, B, Jr.; Harris, M; Moorman, M; Sloane, R. (1987) Methyl isocyanate: reproductive and developmental toxicology studies in Swiss mice. Environ Health Perspect 72:149-152. Singh, RK; Dayal, R; Srivastava, AK; Sethi, N. (1994) Teratological studies on methyl isocyanate in Charles Foster rats (Part-I). Biol Memoirs 20:117-123. Singh, RK; Srivastava, A; Sethi, N; Dayal, R. (1996) Teratological studies on methylisocyanate in Charles Foster rats (Part-II). Biol Memoirs 22:21-25. Tice, RR; Luke, CA; Shelby, MD. (1987) Methyl isocyanate: an evaluation of in vivo cytogenetic activity. Environ Mutagen 9:37-58. Varma, DR; Ferguson, JS; Alarie, Y. (1987) Reproductive toxicity of methyl isocyanate in mice. J Toxicol Environ Health 21:265-275. 23 ------- U.S. Environmental Protection Agency Hazard Characterization Document December 2012 APPENDIX Sponsored Chemical Chemical Name CASRN Structure Methane, isocyanato- 624-83-9 0=N ch3 SMILES: 0=C=NC 24 ------- |