U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 SCREENING-LEVEL HAZARD CHARACTERIZATION SPONSORED CHEMICAL l,2-Dimethyl-4-nitrobenzene CASRN 99-51-4 SUPPORTING CHEMICALS 2,4-Dimethyl-l-nitrobenzene CASRN 89-87-2 CASRN 88-72-2 CASRN 99-08-1 CASRN 99-99-0 2-Nitrotoluene 3-Nitrotoluene 4-Nitrotoluene 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. 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 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. 2 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Chemical Abstract Service Registry Number (CASRN) SDonsored Chemical 99-51-4 Supporting Chemical 89-87-2 88-72-2 99-08-1 99-99-0 Chemical Abstract Index Name Sponsored Chemical Benzene, l,2-dimethyl-4-nitro- SuDDorting Chemical Benzene, 2,4-dimethyl-l-nitro- Benzene, l-methyl-2-nitro- Benzene, l-methyl-3-nitro- Benzene, l-methyl-4-nitro- Structural Formula See Appendix Summary l,2-Dimethyl-4-nitrobenzene is a pale, straw yellow-colored liquid with moderate vapor pressure and moderate water solubility. It is expected to have moderate mobility in soil. Although no biodegradation data are available for this compound, based on close structural analogs (2,4-dimethyl-l -nitrobenzene, 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene), l,2-dimethyl-4-nitrobenzene is not expected to be readily biodegradable. Volatilization of l,2-dimethyl-4-nitrobenzene is expected to be moderate. The rate of hydrolysis is negligible. l,2-Dimethyl-4-nitrobenene is not expected to bioconcentrate in aquatic organisms based on bioconcentration data measured in carp for 2-nitrotoluene. The rate of atmospheric photooxidation is negligible. l,2-Dimethyl-4-nitrobenzene is expected to have low persistence (PI) and low bioaccumulation potential (Bl). The acute oral and dermal toxicity of l,2-dimethyl-4-nitrobenzene is low in rats and rabbits, respectively. Dietary 13-week repeated-dose toxicity studies in rats with the supporting chemicals, 2-, 3- and 4-nitrotoluene, revealed reduced body weight gain and toxic effects to the liver, spleen and kidney (e.g., increased liver weights and clinical chemistry changes in these organs) at 42- 47 mg/kg-bw/day; the NOAEL is not established. Impaired testicular function (e.g., decreased sperm density and motility) and increased estrous cycle length were also seen at >375 mg/kg-bw/day. Similar systemic effects were seen in rats at 300 mg/kg-bw/day in a 28- day dietary study with the supporting chemical 2,4-dimethyl-4-nitrobenzene; the NOAEL is 60 mg/kg-bw/day. In mice, in 13-week dietary studies, decreased body weights were seen with 4-nitrotoluene at 813/1075 mg/kg-bw/day (males/females); the NOAEL is 439/625 mg/kg- 3 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 bw/day (males/females). 3-Nitrotoluene showed effects on relative liver weights 101 mg/kg- bw/day; the NOAEL was not established. In mice, 2-Nitrotoluene showed increased relative liver weights in females and nasal cavity lesions in both sexes at 187 mg/kg-bw/day; the NOAEL is 101 mg/kg-bw/day. No specific reproductive toxicity studies are available for 1,2- dimethyl-4-nitrobenzene. An oral gavage combined reproductive/developmental toxicity screening test in rats with the supporting chemical 4-nitrotoluene showed mortality in both sexes at 400 mg/kg-day and reduced pup weights at 100 mg/kg-day; the NOAEL for reproductive and developmental toxicity is 25 mg/kg-day. Based on decreasing body weights at all doses, the NOAEL for maternal toxicity is not established. In the 13-week dietary repeated-dose studies in rats, effects were observed on the reproductive systems of both sexes. Similar studies in mice showed no effects. l,2-Dimethyl-4-nitrobenzene and the supporting chemical, 2,4-dimethyl-l- nitrobenzene, induced gene mutations in bacteria in vitro and the supporting chemicals, 3- and 4-nitrotoluene, induced chromosomal aberrations in mammalian cells in vitro. 4-Nitrotoluene induced sister chromatid exchange in vitro. The supporting chemicals, 2- and 4-nitrotoluene increased the incidence of tumors in rats and mice. l,2-Dimethyl-4-nitrobenzene is not irritating to rabbit eyes or skin. The fish 96-h LCso for l,2-dimethyl-4-nitrobenzene ranges between 10-18 mg/L, based on the supporting chemical 2,4-dimethyl-l-nitrobenzene. The aquatic invertebrate 48-h EC so for l,2-dimethyl-4-nitrobenzene is 16 mg/L. The algal 72-h EC so for l,2-dimethyl-4-nitrobenzene is 8.9 mg/L. No data gaps were identified under the HPV Challenge Program. 4 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 The sponsor, Arcadis G&M, Inc., submitted a test plan and robust summaries to EPA for 1,2- dimethyl-4-nitrobenzene (CASRN 99-51-4; CA Index name: benzene, l,2-dimethyl-4-nitro-) dated November 29, 2006. EPA posted the submission on the ChemRTK HPV Challenge Web site on February 20, 2007 (http://www.epa.gov/chemrtk/pubs/summaries/12dim4nt/cl6493tc.htm). EPA comments on the submission were posted to the website on June 26, 2009. Public comments were also received and posted to the website. The sponsor also provided data on several proposed supporting chemicals: 2,4 dimethyl-1-nitrobenzene (4-nitro-w-xylene, CASRN 89-87-2), 2-nitrotoluene (CASRN 88-72-2), 3-nitrotoluene (CASRN 99-08-1) and 4-nitrotoluene (CASRN 99-99-0). In October 2009, EPA received revised test plan and robust summary documents for 1,2-dimethyl- 4-nitrobenzene (CASRN 99-51-4) and its proposed supporting chemicals. Justification for Supporting Chemicals The sponsor proposed to use data for the following four supporting chemicals to address the aquatic toxicity and most human health effects endpoints: 2,4-dimethyl-l -nitrobenzene (4-nitro- w-xylene, CASRN 89-87-2), 2-nitrotoluene (CASRN 88-72-2), 3-nitrotoluene (CASRN 99-08-1) and 4-nitrotoluene (CASRN 99-99-0). Based on available data, EPA agrees that use of the supporting chemical data is appropriate to address particular endpoints. For ecological and health effects endpoints, 2,4-dimethyl-l-nitrobenzene is the most appropriate analog. Additionally, for health effects endpoints, EPA considers data for 2-, 3- and 4-nitrotoluene are also appropriate based on close structural similarities. For environmental fate endpoints, data for the close structural analogs, 2,4-dimethyl-l- nitrobenzene, 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene are considered appropriate for the biodegradation endpoint. For the aquatic toxicity endpoints, data for the sponsored chemical and the most appropriate supporting chemical, 2,4-dimethyl-l-nitrobenzene, were available and are sufficient to characterize these endpoints for the purposes of the HPV Challenge program. Therefore, aquatic toxicity data supplied for the three other supporting chemicals (2-, 3- and 4-nitrotoluene) were not considered in this hazard characterization. 4-Nitrotoluene has been assessed in the OECD HPV program and the data can be viewed at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-44b8-8d66-411f4a3cel3c 2-Nitrotoluene has been assessed in the OECD HPV program and the European Union Risk Assessment Report can be viewed at: http://esis.irc.ec.europa.eu/doc/risk assessment/REPORT/2nitrotoluenereport403.pdf. 1. Chemical Identity 1.1 Identification and Purity l,2-Dimethyl-4-nitrobenzene is a pale, straw yellow-colored liquid with moderate vapor pressure and moderate water solubility. 5 ------- U.S. Environmental Protection Agency September, 2014 Hazard Characterization Document 1.2 Physical-Chemical Properties The physical-chemical properties of l,2-dimethyl-4-nitrobenzene are summarized in Table 1. Table 1. Physical-Chemical Properties of l,2-Dimethyl-4-nitrobenzene and Supporting Chemicals1 Property SPONSORED CHEMICAL l,2-Dimcthvl-4- nitrobcn/cnc SUPPORTING CHEMICAL 2,4-Dimethyl-l- nitrobenzene SUPPORTING CHEMICAL 2-Nitrotoluene SUPPORTING CHEMICAL 3-Nitrotoluene SUPPORTING CHEMICAL 4-Nitrotoluene CASRN 99-51-4 89-87-2 88-72-2 99-08-1 99-99-0 Molecular Weight 151.17 151.17 137.14 137.14 137.14 Physical State Pale, straw yellow liquid Liquid Yellow liquid Yellow liquid Colorless to light yellow solid Melting Point 30.5°C (measured) 9°C (measured) -10°C (measured) 15.5°C (measured) 51.6°C (measured) Boiling Point 251°C (measured) 247.8°C (measured) 222°C (measured) 232°C (measured) 238.3°C (measured) Vapor Pressure 3.59><10"3 mmHg at 25°C (measured) 50 mm Hg at 153.7°C (measured); 0.042 mm Hg at 25 °C (estimated)2 0.12 mmHg at 20°C (measured) 7.5 mmHg at 89.7°C (measured); 0.098 mmHg at 25°C (estimated)2 0.098 mmHg at 20°C (measured) Dissociation Constant Not applicable Not applicable Not applicable Not applicable Not applicable Henry's Law Constant 5.1xl0"5atm- m3/mole at 25°C (estimated)3 8.6><10"5atm- m3/mole at 25°C (estimated)3 1.25><10"5atm- m3/mole at 25°C (measured)4 9.3xl06atm- m3/mole at 25°C (measured)4 5.4xl06atm- m3/mole at 25°C (measured)4 Water Solubility 111.8 mg/L at 25°C (estimated)3 133 mg/L at 20°C (measured) 650 mg/L at 30°C (measured) 500 mg/L at 30°C (measured) 442 mg/L at 30°C (measured) Log Kow 2.91 (measured) 2.91 (estimated)3 2.3 (measured) 2.45 (measured) 2.37 (measured) 'BASF Corporation. 2009. Revised Test Plan and Robust Summary for l,2-dimethyl-4-nitrobenzene. Available online at http://www.epa.gov/chemrtk/pubs/summaries/12dim4nt/cl6493tc.htm as of June 22, 2012. 2N0M05. 1987. Programs to Enhance PC-Gems Estimates of Physical Properties for Organic Compounds. The Mitre Corp. 3U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as 0f June 22, 2012. 4SRC. 2012. The Physical Properties Database (PHYSPROP). Syracuse, NY: Syracuse Research Corporation. Available online at http://www.svrres.com/esc/phvsprop.htm as of June 22, 2012. 2. General Information on Exposure 2.1 Production Volume and Use CASRN 99-51-4 had an aggregated production and/or import volume in the United States between 10 to 50 million pounds during calendar year 2005. 6 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Non-confidential information in the IUR indicated that the industrial processing and uses of the chemical include pesticide and other agricultural chemical manufacturing as intermediates. No commercial and consumer uses were reported for the chemical. 2.2 Environmental Exposure and Fate The environmental fate characteristics of l,2-dimethyl-4-nitrobenzene are summarized in Table 2. l,2-Dimethyl-4-nitrobenzene is expected to possess moderate mobility in soil. No biodegradation studies were available for this compound; however, data from several structural analogs indicates that it should not be persistent in the environment. 4-Nitrotoluene (CASRN 99-99-0) was not readily biodegradable using an activated sludge inoculum and the modified MITI (OECD 301C) test and it did not degrade over the course of 20 days using a method similar to the closed bottle (OECD 301D) test, but it was completely degraded in 21 days (10-day acclimation period) and 98% degraded within 5 days using a method similar to the Zahn-Wellens (OECD 302B) test. Therefore, it is reasonable to conclude this substance is not readily biodegradable but is inherently biodegradable. 3-Nitrotoluene (CASRN 99-08-1) achieved 2% of its theoretical biochemical oxygen demand (BOD) after 14 days using an activated sludge inoculum and the MITI test. However, it degraded 93% over the course of 28 days using mixed microbial cultures amended with the sludge in a semi-continuous activated sludge (SCAS) system. 2-Nitrotoluene (CASRN 88-72-2) did not pass a MITI test degrading, <1% after 14 days; however, it was readily biodegradable (>70% loss after 20 days) using an adapted sludge as inoculum and the closed bottle (OECD 301D) test. 2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2) was reported to be inherently biodegradable as measured by the Zahn- Wellens test, although apparently there was significant loss due to air stripping during the test. The data from the structural analogs suggest that l,2-dimethyl-4-nitrobenzene may not be readily biodegradable, but is likely to be inherently biodegradable. Volatilization is expected to be moderate for l,2-dimethyl-4-nitrobenzene given its Henry's Law constant. The rate of hydrolysis of l,2-dimethyl-4-nitrobenzene is negligible. The rate of atmospheric photooxidation is negligible. No measured bioconcentration data are available for l,2-dimethyl-4-nitrobenzene; however, structural analog 2-nitrotoluene had measured BCF values of <100 measured in carp at 2 different concentrations. The overall weight of evidence suggests that l,2-dimethyl-4- nitrobenzene is expected to have low persistence (PI) and low bioaccumulation potential (Bl). Table 2. Environmental Fate Properties of l,2-Dimethyl-4-nitrobenzene and Supporting Chemicals' Property SPONSORED CHEMICAL l,2-Dimethyl-4- nitrobenzene SUPPORTING CHEMICAL 2,4-Dimethyl-l- nitrobenzene SUPPORTING CHEMICAL 2-Nitrotoluene SUPPORTING CHEMICAL 3-Nitrotoluene SUPPORTING CHEMICAL 4-Nitrotoluene CASRN 99-51-4 89-87-2 88-72-2 99-08-1 99-99-0 Photodegradation Half-life 10.2 days (estimated)2 5.5 days (estimated)2 13.8 days (estimated)2 18.4 days (estimated)2 13.8 days (estimated)2 7 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Table 2. Environmental Fate Properties of l,2-Dimethyl-4-nitrobenzene and Supporting Chemicals1 Property SPONSORED CHEMICAL l,2-Dimcthvl-4- nitrobenzene SUPPORTING CHEMICAL 2,4-Dimethyl-l- nitrobenzene SUPPORTING CHEMICAL 2-Nitrotoluene SUPPORTING CHEMICAL 3-Nitrotoluene SUPPORTING CHEMICAL 4-Nitrotoluene Hydrolysis Half- life Stable Stable Stable Stable; 18.1% loss after 8 days at pH 7.4 and 25°C Stable Biodegradation No data 95% after 27 days (inherently biodegradable) >90% after 15 days (inherently biodegradable); >70% after 20 days (readily biodegradable)5; 0.5-0.8% after 14 days (not readily biodegradable)3 2% after 14 days (not readily biodegradable); 93% after 28 days (inherently biodegradable) 0.8% after 14 days (not readily biodegradable); 100% after 20 days (inherently biodegradable); 98% after 5 days (inherently biodegradable); 0% after 20 days (not readily biodegradable) Bioaccumulation Factor BAF = 51.4 (estimated)2 BAF = 50.9 (estimated)2 BCF = 12.5-29.9 (measured in carp at 0.1 ppm)3; BCF = 6.6-29.7 (measured in carp at 0.01 ppm)3; BAF = 18.3 (estimated)2 BAF = 19.8 (estimated)2 BAF = 14.4 (estimated)2 Log Koc 2.8 (estimated)2 2.8 (estimated)2 2.6 (estimated)2 2.6 (estimated)2 2.6 (estimated)2 Fugacity (Level III Model)2 Air (%) Water (%) Soil (%) Sediment (%) 3.1 16.3 80.0 0.5 3.0 15.4 81.1 0.5 2.8 18.9 77.8 0.4 2.6 19.1 77.9 0.4 2.6 19.2 77.8 0.4 Persistence4 PI (low) PI (low) PI (low) PI (low) PI (low) Bioaccumulation4 B1 (low) B1 (low) B1 (low) B1 (low) B1 (low) 1 BASF Corporation. 2009. Revised Test Plan and Robust Summary for 1,2-dimethyl-4-nitrobenzene. Available online at http://www.epa.gov/chemrtk/pubs/summaries/12dim4nt/cl6493tc.htm as of June 22, 2012. 2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of June 22, 2012. 3National Institute of Technology and Evaluation. 2002. Chemical Risk Information Platform (CHIRP). Available online at http://www.safe.nite.go.ip/english/db.html as of June 22, 2012. 4Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances. Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204. 5Used adapted sludge as inoculum. 8 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Conclusion: l,2-Dimethyl-4-nitrobenzene is a pale, straw yellow-colored liquid with moderate vapor pressure and moderate water solubility. It is expected to have moderate mobility in soil. Although no biodegradation data are available for this compound, based on close structural analogs, (2,4-dimethyl-l -nitrobenzene, 2-nitrotoluene, 3-nitrotoluene and 4-nitrotoluene), 1,2- dimethyl-4-nitrobenzene is not expected to be readily biodegradable. Volatilization of 1,2- dimethyl-4-nitrobenzene is expected to be moderate. The rate of hydrolysis is negligible. 1,2- Dimethyl-4-nitrobenene is not expected to bioconcentrate in aquatic organisms based on bioconcentration data measured in carp for 2-nitrotoluene. The rate of atmospheric photooxidation is negligible. l,2-Dimethyl-4-nitrobenzene is expected to have low persistence (PI) and low bioaccumulation potential (Bl). 3. Human Health Hazard A summary of health effects data for SIDS and other endpoints is provided in Table 3. The table also indicates where data for the supporting chemicals are read-across (RA) to the sponsored substance. Acute Oral Toxicity l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) Male Wistar rats (10/dose) were administered neat l,2-dimethyl-4-nitrobenzene orally at 712, 1424, 2848 or 5695 mg/kg (length of post-dosing observation period was not specified). Mortality (9/10 rats) was observed at 5695 mg/kg within 24 hours. No mortality was observed at 712 mg/kg. LDso = 2636 mg/kg 2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical) Wistar rats of both sexes (number not specified) were administered 2,4-dimethyl-l-nitrobenzene (> 99% purity) orally at unspecified dose levels. No additional information provided. LD50 = 1690/2240 mg/kg (male/female) Acute Dermal Toxicity l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) Five male New Zealand White rabbits received a single dermal application of an unspecified amount of neat l,2-dimethyl-4-nitrobenzene (purity not provided) to clipped, intact skin for 24 hours under an occlusive wrap. Animals were observed for 14 days post-exposure. One animal died. LD50 = 5695 mg/kg Repeated-Dose Toxicity l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) 9 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 No Data. 2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical) In a 28-day dietary repeated-dose toxicity study, Wistar rats (5/sex/dose) were fed 0, 100, 600 or 3000 ppm (approximately 0, 10, 60 or 300 mg/kg-bw/day) 2,4-dimethyl-l-nitrobenzene (purity not provided) daily and observed for 14 days post exposure. There were no effects on general health, behavior, water consumption and on urinary parameters. At 3000 ppm, reduced weight gain (significance not stated) was observed both during the treatment period and during the recovery period. This reduction in body weight gain was accompanied by a reduction in feed consumption. Hematological effects in the 3000 ppm group included a decreased concentration of hemoglobin and red blood cells and an increase in platelet count. In addition, decreased serum protein levels were observed. Increased liver and spleen weights were observed; however, no test substance-related gross pathological or histopathological effects occurred. Additional information not provided. LOAEL ~ 300 mg/kg-bw/day (based on reduced body weight gain, hematological and clinical chemistry effects, increased liver and spleen weights) NOAEL ~ 60 mg/kg-bw/day 4-Nitrotoluene (CASRN 99-99-0, supporting chemical) (1) In a 13-week National Toxicology Program (NTP) dietary study, F334/N rats (10/sex/concentration) were administered 4-nitrotoluene (>96% purity) in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm [approximately 0, 42, 82, 165, 342 or 723 mg/kg-bw/day (males) or 0, 44, 82, 164, 335 or 680 mg/kg-bw/day (females)]. There were no mortalities. Decreased body weights were seen at > 342/335 mg/kg-bw/day. There were many organ weight changes. Absolute heart weight was reduced at > 82 mg/kg-bw/day (males) and 680 mg/kg-bw/day (females). In males, absolute kidney weights were reduced at 723 mg/kg-bw/day and relative kidney weights were increased at > 342 mg/kg-bw/day; in females, absolute kidney weights were reduced at > 335 mg/kg-bw/day and relative kidney weights were increased at 680 mg/kg- bw/day. Absolute liver weights were reduced in males at 723 mg/kg-bw/day and relative liver weights were increased in males at > 342 mg/kg-bw/day and in females at 680 mg/kg-bw/day. Absolute thymus weights were decreased in males at 723 mg/kg-bw/day and in females at > 335 mg/kg-bw/day. Absolute lung weights were reduced in both sexes at 723/680 mg/kg-bw/day; relative lung weights were increased in males at > 342 mg/kg-bw/day. Absolute testis weights were decreased in males at > 342 mg/kg-bw/day. Hyaline droplet nephropathy5 was seen in males at all dose levels and karyomegaly was seen in both males (> 82 mg/kg-bw/day) and females (all dose levels). Pigmentation in kidneys was also seen in males (723 mg/kg-bw/day) and females (all dose levels). Splenic hematopoiesis, hemosiderin pigment and congestion were seen in both sexes at all dose levels. Testicular degeneration was seen in males at 723 mg/kg- bw/day. Changes in hematology and clinical chemistry were seen in both sexes and included 5 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the nephropathy in the males may be occurring by an alpha2U-globulin-mediated mechanism, which appears to be unique to male rats and the response is probably not relevant to humans for purposes of risk assessment. EPA's Risk Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action (Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3 - 91/019F). A key event, alpha2U-globulin accumulation, has been demonstrated in the NTP study. 10 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 increased hematocrit in males (723 mg/kg-bw/day) and females (> 335 mg/kg-bw/day) at week 1 and decreased hematocrit in females at 680 mg/kg-bw/day at week 13; increased erythrocytes in males (723 mg/kg-bw/day) and females (> 335 mg/kg-bw/day) at week 1 and decreased erythrocytes in males (723 mg/kg-bw/day) and females (680 mg/kg-bw/day) at week 13; decreased creatinine in both sexes at > 165/164 mg/kg-bw/day; and increased methemoglobin and bile acid in both sexes at the high dose level. LOAEL ~ 42/44 mg/kg-bw/day (based on karyomegaly in females, kidney pigmentation in females, splenic hematopoiesis, hemosiderin pigment and congestion in both sexes) NOAEL ~ Not established (2) In a 13-week NTP dietary study, B6C3F1 mice (10/sex/concentration) were administered 4-nitrotoluene (purity >96%) daily in feed at 0, 675, 1250, 2500, 5000 or 10,000 ppm [approximately 0, 131, 212, 439, 813 or 1491 mg/kg-bw/day (males) or 0, 164, 320, 625, 1075 or 1634 mg/kg-bw/day (females)]. There were no mortalities. Decreased final body weights were seen in males at > 813 mg/kg-bw/day and in females at 1634 mg/kg-bw/day. Increased relative liver weights were seen at all dose levels in both sexes, but the summary indicated that since no treatment-related gross lesions and no histopathological liver lesions were observed, this weight increase was judged to be not treatment related. No effect on the reproductive system of either sex was observed. LOAEL ~ 813/1075 mg/kg-bw/day (male/female) (based on decreased body weights) NOAEL ~ 439/625 mg/kg-bw/day (male/female) (3) See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb- 44b8-8d66-411f4a3cel3c 3-Nitrotoluene (CASRN 99-08-1, supporting chemical) (1) In a 13-week NTP dietary study, F334/N rats (10/sex/concentration) were administered 3-nitrotoluene (>96% purity) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm [approximately 0, 47, 94, 188, 375 or 750 mg/kg-bw/day]. There were no mortalities. Decreased body weight gain and increased relative liver weights were seen in both sexes at 750 mg/kg- bw/day. Increased relative kidney weights were seen at 750 mg/kg-bw/day in males and 375 mg/kg-bw/day in females. Other effects included increased bile acids in males at > 375 mg/kg- bw/day and in females at 750 mg/kg-bw/day; a mild increase in ALT in females at > 188 mg/kg- bw/day; hyaline droplet nephropathy6 in males at all dose levels; changes in hematology and clinical chemistry in both sexes at all dose levels, including increased methemoglobin at 375 mg/kg-bw/day (males only) and 750 mg/kg-bw/day (both sexes); and splenic hemosiderin and/or congestion in both sexes at > 375 mg/kg-bw/day. Testicular degeneration was seen in males at 750 mg/kg-bw/day, along with decreased epididymal sperm count and concentration. Estrous cycle length was increased and the number of cycling animals was decreased at > 375 mg/kg- 6 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the nephropathy in the males may be occurring by an alpha2U-globulin-mediated mechanism, which appears to be unique to male rats and the response is probably not relevant to humans for purposes of risk assessment. EPA's Risk Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action (Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3 - 91/019F). A key event, alpha2U-globulin accumulation, has been demonstrated in the NTP study. 11 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 bw/day. Additional information from NTP TR 23 available at: http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf LOAEL ~ 47 mg/kg-bw/day (based on hematological and clinical chemistry changes in both sexes and splenic hemosiderin and/or congestion in both sexes) NOAEL ~ Not established (2) In a 13-week NTP dietary study, male and female B6C3F1 mice (10/sex/group) were administered 3-nitrotoluene (purity >96%) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm [approximately 0, 101, 187, 375, 750 or 1500 mg/kg-bw/day]. There were no mortalities. Decreased food consumption and body weight gain were seen in both sexes at > 750 mg/kg- bw/day. Increased relative liver weights were seen in both sexes at all dose levels (there were no gross or microscopic liver lesions). Increased relative lung weights were seen in both sexes at 750 mg/kg-bw/day. No effect on the reproductive system of either sex was observed. Additional information from NTP TR 23 available at: http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf LOAEL ~ 101 mg/kg-bw/day (based on increased relative liver weights in both sexes) NOAEL ~ Not established 2-Nitrotoluene (CASRN 88-72-2, supporting chemical) (1) In a 13-week NTP dietary study, F334/N rats (10/sex/concentration) were administered 2- nitrotoluene (>96% purity) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm [approximately 0, 47, 94, 188, 375 or 750 mg/kg-bw/day]. There were no mortalities. There were decreases in final body weight at > 188 mg/kg-bw/day and food consumption at 750 mg/kg-bw/day. Other effects included increased relative liver weights in both sexes (all dose levels); increased relative kidney weights (> 188 mg/kg-bw/day in males, > 94 mg/kg-bw/day in females); toxic effects to the liver (cytoplasmic vacuolization and oval cell hyperplasia in males), spleen (increase in splenic hematopoiesis; hemosiderin deposition and/or congestion) and kidney (hyaline droplets in the renal tubular epithelial cells of males7) at > 94 mg/kg-bw/day; decreased spermatid count and an increase in testicular lesions in males (> 375 mg/kg-bw/day); increased estrous cycle length in females at 750 mg/kg-bw/day; and increases in mesothelial cell hyperplasia and mesothelioma in males at > 375 mg/kg-bw/day. Additional information from NTP TR 23 available at: http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf LOAEL ~ 47 mg/kg-bw/day (based on increased relative liver weights in both sexes) NOAEL ~ Not established (2) In 13-week NTP study, B6C3F1 mice (10/sex/concentration) were administered 2- nitrotoluene (>96% purity) daily in feed at 0, 625, 1250, 2500, 5000 or 10,000 ppm [approximately 0, 101, 187, 375, 750 or 1500 mg/kg-bw/day]. There were no mortalities. Lesions of the nasal cavity were seen at > 187 mg/kg-bw/day and decreased final body weights were noted at >375 mg/kg-bw/day. Increased relative liver weights were also seen at > 375 mg/kg-bw/day in males and >187 mg/kg-bw/day in females. Males at 750 mg/kg-bw/day had 7 The presence of nephropathy in association with the hyaline droplet accumulation in male rats suggests that the nephropathy in the males may be occurring by an alpha2U-globulin-mediated mechanism, which appears to be unique to male rats and the response is probably not relevant to humans for purposes of risk assessment. EPA's Risk Assessment Forum has outlined the key events and the data that are necessary to demonstrate this mode of action (Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Rat, EPA/625/3 - 91/019F). A key event, alpha2U-globulin accumulation, has been demonstrated in the NTP study. 12 ------- U.S. Environmental Protection Agency September, 2014 Hazard Characterization Document decreased epididymal sperm motility. Additional information from NTP TR 23 available at: http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf LOAEL ~ 187 mg/kg-bw/day (based on increased relative liver weights in females and nasal cavity lesions in both sexes) NOAEL ~ 101 mg/kg-bw/day (3) See human health data at: http://esis.irc.ec.europa.eu/doc/risk assessment/REPORT/2nitrotoluenereport403.pdf. Reproductive Toxicity l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) No Data. 4-Nitrotoluene (CASRN 99-99-0, supporting chemical) (1) In an oral gavage reproductive/developmental toxicity screening test, Wistar rats (12/sex/dose) were administered 0, 25, 100 or 400 mg/kg-day 4-nitrotoluene (purity 99%; vehicle: 1% methylcellulose). Males were dosed for 2 weeks prior to mating, during mating and up to day 36. Females were dosed for 2 weeks prior to mating, during mating and gestation and up to day 4 or 5 postpartum. Additional investigations included histopathological examinations of liver, spleen, kidney, pituitary gland, uterus, uterine cervix and vagina, mammary gland, epididymides and prostate. Three males and five females at 400 mg/kg-day died during the mating period and an additional female was sacrificed moribund on day 22. Significantly decreased food consumption (P<0.05 - <0.01) was seen at 400 mg/kg-day in both sexes. Females showed s significantly reduced (P< 0.01) body weight gain at 400 mg/kg-day during gestation and lactation. Clinical signs seen in both sexes at 400 mg/kg-day included piloerection, sunken flanks and increased water intake and urination. Mean absolute and relative liver weights in males were significantly increased (P<0,05 - 0.01) at 100 and 400 mg/kg-day, respectively. Mean absolute and relative spleen weights were significantly increased (P<0.01) at 400 mg/kg-day in both sexes. Splenic congestion was seen in males and females at 100 and 400 mg/kg-day. Histopathological findings included increased amounts of pigment in the spleen and increased amount of periportal pigment at 400 mg/kg-day. In a single male at 400 mg/kg-day, debris was observed in the epididymides together with exfoliation of spermatids. No histological changes in male accessory sexual glands, ovaries, female mammae with mamillae and pituitary gland were observed. Testicular atrophy and epididymal cellular debris were seen at 400 mg/kg- day. There was no effect on fertility index. The gestation index, the number of corpora lutea and mean number of pups delivered were decreased at 400 mg/kg-day; prenatal loss was increased at 400 mg/kg-day. Mean pup weights were significantly reduced at 100 mg/kg-day (P<0.05) and at 400 mg/kg-day (P<0.01). See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-44b8-8d66-411f4a3cel3c LOAEL (reproductive toxicity) = 100 mg/kg-day (based on reduced pup weights) NOAEL (reproductive toxicity) = 25 mg/kg-day (2) In the NTP study in F334/N rats described above, absolute testis weights were decreased in males at > 342 mg/kg-bw/day and testicular degeneration and decreased sperm count and 13 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 motility were seen in males at 723 mg/kg-bw/day. Increased estrous cycle length and increased proportion of rats in diestrus were seen in females at 680 mg/kg-bw/day. (3) In the NTP study in B6C3F1 mice described above, there were no effects on reproductive parameters. (4) In a 13-week oral gavage study (frequency of treatment not stated), Fischer 344 rats (10/sex/dose) were administered 0, 90, 180 or 360 mg/kg-day 4-nitrotoluene (vehicle: corn oil). Sperm morphology and vaginal cytology examinations were performed. Males at 360 mg/kg- day had decreased terminal body weights, decreased absolute cauda epididymis, epididymides and testes weights and decreased relative epididymis weights. There were no effects on sperm parameters. There were no effects on females. (5) In a 13-week oral gavage study (frequency of treatment not stated), B6C3F1 mice (10/sex/dose) were administered 0, 40, 80 or 160 mg/kg-day 4-nitrotoluene (vehicle: corn oil). Sperm morphology and vaginal cytology examinations were performed. There were no effects on reproductive organ weights, sperm parameters or estrous cycle length. (6) See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb- 44b8-8d66-411f4a3cel3c 3-Nitrotoluene (CASRN 99-08-1, supporting chemical) (1) In the NTP 13-week study in F334/N rats described previously, testicular degeneration was seen in males at 750 mg/kg-bw/day, along with decreased epididymal sperm count and concentration. Estrous cycle length was increased at > 375 mg/kg-bw/day. (2) In the NTP 13-week study in B3C6F1 mice described previously, there were no effects on reproductive parameters. 2-Nitrotoluene (CASRN 88-72-2, supporting chemical) (1) In the 13-week NTP study in F334/N rats described previously, males at 375 and 750 mg/kg- bw/day had marked degeneration of the seminiferous tubules and decreased epididymal sperm motility and density. At 750 mg/kg-bw/day, an increased proportion of female rats were in diestrus, and estrous cycle length was increased. (2) In the 13-week NTP study in B3C6F1 mice described previously, males at 750 mg/kg- bw/day had decreased epididymal sperm motility. No effect on the estrous cycle was observed. (3) See human health data at: http://esis.irc.ec.europa.eu/doc/risk assessment/REPORT/2nitrotoluenereport403.pdf. Developmental Toxicity l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) 14 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 No Data. 4-Nitrotoluene (CASRN 99-99-0, supporting chemical) In the reproductive/developmental toxicity screening test described previously, Wistar rats administered 4-nitrotoluene via gavage at 0, 25, 100 or 400 mg/kg-day (vehicle: 1% methylcellulose) mean pup weights were reduced at 100 and 400 mg/kg-day. Clear signs of maternal toxicity were observed at 400 mg/kg-day and included reduced feed intake, severe body weight loss, hypoactivity, alteration of gaits, piloerection, respiratory sounds, increased water intake and urination, sunken flanks, and reduced amount of feces with an increased incidence of soft and light colored feces. Six females were sacrificed in a moribund condition. Spleen weights were increased. At 100 and 25 mg/kg bw/day, feed intake and body weight gain during lactation were marginally reduced. Mean pup weights were significantly decreased during day 0-day 4 at 100 mg/kg-day (P<0.05) and 400 mg/kg-day (P<0.01). Two pups from two litters at 400 mg/kg-day had hematomas, an effect not seen in any other groups. See human health data at: http://webnet.oecd.org/hpv/UI/handler.axd?id=4cdf8442-3ecb-44b8- 8d66-411f4a3cel3c LOAEL (maternal toxicity) = 25 mg/kg-day (based on decreased body weight gain) NOAEL (maternal toxicity) = Not established LOAEL (developmental toxicity) = 100 mg/kg-day (based on decreased pup weights) NOAEL (developmental toxicity) = 25 mg/kg-day Genetic Toxicity — Gene Mutation l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) No Data. In vitro l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) (1) In a National Toxicology Program (NTP) reverse mutation assay, Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with l,2-dimethyl-4-nitrobenzene purity not provided) at concentrations of 0, 3, 10, 33, 100, 333, 1000 and 1666 |ig/plate with and without metabolic activation. Positive and vehicle controls were included and responded appropriately. Cytotoxicity was observed at > 333 |ig/plate. l,2-Dimethyl-4-nitrobenzene caused reproducible positive responses with TA100 with activation. Additional information from NTP website: http://tools.niehs.nih.gov/ntp tox/index.cfm?fuseaction=salmonella.salmonellaData&studv no= 589230&cas no=99%2D5l%2D4&endpointlist=SA l,2-Dimethyl-4-nitrobenzene was mutagenic in this assay. (2) In a reverse mutation assay, Salmonella typhimurium strains TA98 and TA100 were treated with l,2-dimethyl-4-nitrobenzene (purity not provided) at unspecified concentrations with and without metabolic activation. No information about controls or cytotoxic concentrations was provided in the summary. l,2-Dimethyl-4-nitrobenzene was not mutagenic in this assay. 15 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical) In a NTP reverse mutation assay, Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with 2,4-dimethyl-l-nitrobenzene (purity not provided) at concentrations of 0, 3, 10, 33, 100, 333 and 1000 |ig/plate with and without metabolic activation. Positive and vehicle controls were included and responded appropriately. Cytotoxicity was observed at > 100 |ig/plate. 2,4-Dimethyl-l-nitrobenzene caused reproducible positive responses with TA100 with activation. Additional information from NTP website: http://tools.niehs.nih.gov/ntp tox/index.cfm?fuseaction=salmonella.salmonellaData&studv no= 153433&cas no=89%2D87%2D2&endpointlist=SA 2,4-Dimethyl-l-nitrobenzene was mutagenic in this assay. Genetic Toxicity — Chromosomal Aberrations l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) No Data. In vitro 4-Nitrotoluene (CASRN 99-99-0, supporting chemical) In an in vitro test, Chinese hamster ovary (CHO) cells were treated with 4-nitrotoluene (> 96% purity) with and without metabolic activation at concentrations ranging from 300 to 600 |ig/mL. Positive and solvent controls were tested concurrently, but no information was provided on whether they responded appropriately. Cytotoxicity was observed at > 500 |ag/m L; chromosomal aberrations were induced both with and without metabolic activation. 4-Nitrotoluene induced chromosomal aberrations in this assay. 3-Nitrotoluene (CASRN 99-08-1, supporting chemical) (1) In an in vitro test, Chinese hamster ovary (CHO) cells were exposed to 3-nitrotoluene (>96% purity) with and without metabolic activation at concentrations of 150, 300, 398, 437, 460 or 483 |ig/mL, Positive and negative controls were tested concurrently, but no information was provided on whether they responded appropriately. It was not stated whether negative controls were included. The high concentration was estimated to reduce cell growth by 50%. 3-Nitrotoluene did not induce chromosomal aberrations in this assay. (2) In an in vitro test, Chinese hamster lung cells were exposed to 3-nitrotoluene (purity not stated) without metabolic activation at concentrations up to 250 |ig/mL. There was no information on cytotoxicity or positive and negative controls. 3-Nitrotoluene did not induce chromosomal aberrations in this assay. (3) Human peripheral lymphocytes were exposed to 3-nitrotoluene (purity not stated) without metabolic activation at concentrations of 0.002, 0.02, 0.1 and 0.5 mmol/L. There was no information on cytotoxicity or positive and negative controls. The summary noted that 3- nitrotoluene caused an increase in the ratio of the number of aberrant cells to the number of metaphase cells scored. 3-Nitrotoluene induced chromosomal aberrations in this assay. 16 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Genetic Toxicity - Other 4-Nitrotoluene (CASRN 99-99-0, supporting chemical) In an in vitro test, Chinese hamster ovary (CHO) cells were treated with 4-nitrotoluene (> 96% purity) with and without metabolic activation at concentrations ranging from 300 to 600 |ig/mL. Positive and solvent controls were tested concurrently, but no information was provided on whether they responded appropriately. Cytotoxicity was observed at > 500 |ag/m L; sister chromatid exchange were induced both with and without metabolic activation. 4-Nitrotoluene induced sister chromatid exchange in this assay. Additional Information Eye Irritation l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) l,2-Dimethyl-4-nitrobenzene (0.1 mL) was instilled into the conjunctival sac of one eye of each of six rabbits (strain and sex not indicated). The cornea, iris and conjunctivae were examined at 24, 48 and 72 hours post-instillation. No irritation was observed. l,2-Dimethyl-4-nitrobenzene was not irritating to rabbit eyes in this study. Dermal Irritation l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) Six rabbits (sex and strain not specified) were exposed to 0.5 mL neat l,2-dimethyl-4- nitrobenzene on shaved, intact or abraded skin under an impervious wrap for 24 hours. Skin reactions were scored according to Draize for up to 72 hours after exposure. The primary irritation score was 1.25 (no other results details were provided). l,2-Dimethyl-4-nitrobenzene was not irritating to rabbit skin in this study. Carcinogenicity 4-Nitrotoluene (CASRN 99-99-0, supporting chemical) (1) In a 105-week NTP study, F344/N rats (50/sex/concentration) were fed 0, 1250, 2500 or 5000 ppm [approximately 0, 55, 110 or 240 mg/kg-bw/day (males) or 0, 60, 125 or 265 mg/kg-bw/day (females)] 4-nitrotoluene (> 99% purity) in the diet for 105 - 106 weeks. In female rats, increased incidence of clitoral gland neoplasms over the control was seen at 125 mg/kg-bw/day and this also exceeded historical control ranges. The study authors indicated that there was no increase in the highest dose group, possibly because of lower body weights in that group. In male rats, incidences for fibromas and fibrosarcomas were seen at 55 and 110 mg/kg-bw/day, but not at 240 mg/kg-bw/day; therefore, the study authors indicated that the increased incidences were considered as an "uncertain finding." In male rats at 240 mg/kg-bw/day, incidences of germinal epithelial atrophy of the testes were increased. 4-Nitrotoluene increased the incidence of tumors in rats in this study. 17 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 (2) In a 105-week NTP study, B6C3F1 mice (50/sex/concentration) were fed 0, 1250, 2500 or 5000 ppm [approximately 0, 170, 345 or 690 mg/kg-bw/day (males) or 0, 155, 315 or 660 mg/kg-bw/day (females)] 4-nitrotoluene (> 99% purity) in the diet for 105 - 106 weeks. In male mice at 690 mg/kg-bw/day, the incidence of alveolar/bronchiolar adenomas or carcinomas was increased. In the liver, the incidences of hepatocyte syncytial alterations were increased in males in all test groups. This change was considered to be preneoplastic by the study authors. 4-Nitrotoluene increased the incidence of tumors in male mice in this study. 2-Nitrotoluene (CASRN 88-72-2, supporting chemical) (1) In a 105-week NTP study, F344/N rats (60 - 70/sex/group) were fed 0, 1250, 2500 or 5000 ppm [approximately 0, 25, 50 or 90 mg/kg-bw/day (males) or 0, 30, 60 or 100 mg/kg-bw/day (females)] 2-nitrotoluene (> 99% purity). In a 3-month, stop-exposure study, groups of 70 male rats were fed 125 or 315 mg/kg-bw/day in the diet for 13 weeks followed by undosed feed for the remainder of the study. Incidences of subcutaneous skin neoplasms were increased in males at all treatment levels and the incidences of fibroma or fibrosarcoma (combined) were increased in treated females. In all treated groups except 90 mg/kg-bw/day core study males, the incidences of mammary gland fibroadenoma were increased. Increased incidences of mesothelioma, skin neoplasms and mammary gland fibroadenoma were seen in the stop-exposure males. The incidences of mammary gland hyperplasia were increased in females at 30 and 60 mg/kg-bw/day (but not 100 mg/kg-bw/day). Liver weights of stop-exposure males at 315 mg/kg-bw/day were greater than those of the controls at 3 months. The incidences of hepatocellular adenoma in high-dose core study males and females and hepatocellular adenoma or carcinoma (combined) in high-dose core study and high-dose stop-exposure males were increased. Cholangiocarcinoma occurred in three high-dose stop-exposure males. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were increased in high- dose stop-exposure males, as were alveolar/bronchiolar hyperplasia in most exposed groups of males and females. 2-Nitrotoluene increased the incidences of tumors in rats in this study. (2) In a 105-week NTP study, B6C3F1 mice (60/sex/group) were fed 0, 1250, 2500 or 5000 ppm [approximately 0, 165, 360 or 700 mg/kg-bw/day (males) or 0, 150, 320 or 710 mg/kg-bw/day (females)] 2-nitrotoluene (> 99% purity) in the diet. The incidences of hemangiosarcoma in all exposed groups of males and in high-dose females were greater than those in the controls. Large intestine carcinomas were observed in all exposed groups except high-dose males. The incidences of hepatocellular neoplasms were increased in mid- and high-dose females. 2-Nitrotoluene increased the incidence of tumors in mice in this study. Conclusion: The acute oral and dermal toxicity of l,2-dimethyl-4-nitrobenzene is low in rats and rabbits, respectively. Dietary 13-week repeated-dose toxicity studies in rats with the supporting chemicals, 2-, 3- and 4-nitrotoluene, revealed reduced body weight gain and toxic effects to the liver, spleen and kidney (e.g., increased liver weights and clinical chemistry changes in these organs) at 42- 47 mg/kg-bw/day; the NOAEL is not established. Impaired testicular function (e.g., decreased sperm density and motility) and increased estrous cycle length were also seen at >375 mg/kg-bw/day. Similar systemic effects were seen in rats at 300 mg/kg- bw/day in a 28-day dietary study with the supporting chemical 2,4-dimethyl-4-nitrobenzene; the NOAEL is 60 mg/kg-bw/day. In mice, in 13-week dietary studies, decreased body weights were 18 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 seen with 4-nitrotoluene at 813/1075 mg/kg-bw/day (males/females); the NOAEL is 439/625 mg/kg-bw/day (males/females). 3-Nitrotoluene showed effects on relative liver weights 101 mg/kg-bw/day; the NOAEL was not established. In mice, 2-Nitrotoluene showed increased relative liver weights in females and nasal cavity lesions in both sexes at 187 mg/kg-bw/day; the NOAEL is 101 mg/kg-bw/day. No specific reproductive toxicity studies are available for 1,2- dimethyl-4-nitrobenzene. An oral gavage combined reproductive/developmental toxicity screening test in rats with the supporting chemical 4-nitrotoluene showed mortality in both sexes at 400 mg/kg-day and reduced pup weights at 100 mg/kg-day; the NOAEL for reproductive and developmental toxicity is 25 mg/kg-day. Based on decreasing body weights at all doses, the NOAEL for maternal toxicity is not established. In the 13-week dietary repeated-dose studies in rats, effects were observed on the reproductive systems of both sexes. Similar studies in mice showed no effects. l,2-Dimethyl-4-nitrobenzene and the supporting chemical, 2,4-dimethyl-l- nitrobenzene, induced gene mutations in bacteria in vitro and the supporting chemicals, 3- and 4- nitrotoluene, induced chromosomal aberrations in mammalian cells in vitro. 4-Nitrotoluene induced sister chromatid exchange in vitro. The supporting chemicals, 2- and 4-nitrotoluene increased the incidence of tumors in rats and mice. l,2-Dimethyl-4-nitrobenzene is not irritating to rabbit eyes or skin. 19 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoints SPONSORED CHEMICAL 1,2-Dimcthyl- 4-nitrobcnzcnc (99-51-4) SUPPORTING CHEMICAL 2,4-Dimethylbenzene- 1-nitrobenzene (89-87-2) SUPPORTING CHEMICAL 2-Nitrotoluene (88-72-2) SUPPORTING CHEMICAL 3-Nitrotoluene (99-08-1) SUPPORTING CHEMICAL 4-Nitrotoluene (99-99-0) Acute Oral Toxicity LDso (mg/kg) 2636 1690-2240 - - - Acute Dermal Toxicity LDso (mg/kg) 5695 - - - - Repeated-Dose Toxicity NOAEL/LOAEL Oral (mg/kg-bw/day) No Data LOAEL = 42 NOAEL = Not established (RA) LOAEL = 300 NOAEL = 60 LOAEL = 47 NOAEL = Not established LOAEL = 47 NOAEL = Not established LOAEL = 42/44 (m/f) NOAEL = Not established Reproductive Toxicity NOAEL/LOAEL Oral (mg/kg-day) Reproductive Toxicity No Data LOAEL = 100 NOAEL = 25 (RA) Effects on male and female reproductive system in males and females in 13-week dietary studies in rats and male mice Effects on male and female reproductive system in males and females in 13-week dietary studies in rats but not in mice LOAEL = 100 NOAEL = 25 Developmental Toxicity NOAEL/LOAEL Oral (mg/kg-bw/day) Maternal Toxicity Developmental Toxicity No Data LOAEL = 25 NOAEL = Not established LOAEL = 100 NOAEL = 25 (RA) LOAEL = 25 NOAEL = Not established LOAEL = 100 NOAEL = 25 Genetic Toxicity - Gene Mutation In vitro Positive Positive - - - ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Human Health Data Endpoints SPONSORED CHEMICAL 1,2-Dimcthyl- 4-nitrobcnzcnc (99-51-4) SUPPORTING CHEMICAL 2,4-Dimethylbenzene- 1-nitrobenzene (89-87-2) SUPPORTING CHEMICAL 2-Nitrotoluene (88-72-2) SUPPORTING CHEMICAL 3-Nitrotoluene (99-08-1) SUPPORTING CHEMICAL 4-Nitrotoluene (99-99-O) Genetic Toxicity - Chromosomal Aberrations In vitro No Data Positive (RA) Positive Positive Additional Information Eye Irritation Skin Irritation Carcinogenicity Not irritating Not irritating Positive Positive Bold = measured data; RA = read across; - indicates endpoint not addressed for this chemical; m = male; f=female 21 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 4. Hazard to the Environment A summary of ecotoxicity data for SIDS and other endpoints is provided in Table 4. The table also indicates where data for the supporting chemical are read-across (RA) to the sponsored substances. Since the submitted ecotoxicity studies were lacking relevant testing details, ECOSAR (v. 1.1) was used to support the endpoint values. Acute Toxicity to Fish l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) The ECOSAR-predicted 96-hour LCso value for fish was 19.1 mg/L. A measured log Kow value of 2.91 was input into the estimation program. 96-h LCso =19.1 mg/L (estimated) 2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical) Rainbow trout (Brachydanio rerio) were exposed to nominal concentrations of 0, 10, 18, and 25 mg/L of 2,4-dimethyl-l-nitrobenzene (> 99% purity) under static conditions for 96 hours. No further methods or results details were provided. 96-h LCso =10-18 mg/L Acute Toxicity to Aquatic Invertebrates l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) (1) Daphnia magna were exposed to unspecified measured concentrations of l,2-dimethyl-4- nitrobenzene (> 99% purity, dissolved in dimethyl sulfoxide) under static conditions for 48 hours. No details concerning mortality/effects at each test concentration were provided. 48-h ECso = 16 mg/L (2) The ECOSAR-predicted 48-hour LC50 value for daphnids was 11.8 mg/L. A measured log Kow value of 2.91 was input into the estimation program. 48-h LCso = 11.8 mg/L (estimated) 2,4-Dimethyl-l-nitrobenzene (CASRN 89-87-2, supporting chemical) Daphnia magna were exposed to unspecified nominal concentrations of 2,4-dimethyl-l- nitrobenzene (unspecified purity) under unspecified conditions for 48 hours. No further methods or results details were provided. 48-h ECso = 15 mg/L 22 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 Toxicity to Aquatic Plants l,2-Dimethyl-4-nitrobenzene (CASRN 99-51-4) (1) Green algae (Chlorellapyrenoidosa) were exposed to unspecified nominal concentrations of l,2-dimethyl-4-nitrobenzene (> 99% purity) for 96 hours. No details concerning biomass at each test concentration were provided. 96-h ECso (biomass) = 8.9 mg/L (2) The ECOSAR-predicted 96-hour EC50 value for algae was 8.44 mg/L. A measured log Kow value of 2.91 was input into the estimation program. 96-h EC50 = 11.2 mg/L (estimated) Conclusion: The fish 96-h LC50 for l,2-dimethyl-4-nitrobenzene ranges between 10-18 mg/L, based on the supporting chemical 2,4 dimethyl-1-nitrobenzene. The aquatic invertebrate 48-h EC50 for l,2-dimethyl-4-nitrobenzene is 16 mg/L. The algal 72-h EC50 for 1,2-dimethyl- 4-nitrobenzene is 8.9 mg/L. Summary of the Screening Information Data Set as Submitted under the U.S. HPV Challenge Program - Aquatic Toxicity Data Endpoints SPONSORED CHEMICAL l,2-Dimcthyl-4-nitrobcnzcnc (99-51-4) SUPPORTING CHEMICAL 2,4-Dimcthylbcnzcnc- l-nitrobcnzcnc (89-87-2) Fish 96-h LCso (mg/L) No Data 10-18 (RA) 10-18 Aquatic Invertebrates 48-h ECso (mg/L) 16 15 Aquatic Plants 72-h ECso (mg/L) (growth) (biomass) 8.9 - Bold = experimentally-derived test data; RA = read across; - indicates endpoint not addressed for this chemical 23 ------- U.S. Environmental Protection Agency Hazard Characterization Document September, 2014 APPENDIX Sponsored Chemical Chemical Name CASRN Structure l,2-Dimethyl-4- nitrobenzene 99-51-4 CH, 6r no2 SMILES: 0=N(=0)c(ccc(c 1 C)C)c 1 Supporting Chemicals Chemical Name CASRN Structure 2,4-Dimethyl-l- nitrobenzene 89-87-2 CH, £ T CH3 no2 SMILES: 0=N(=0)c(c(cc(cl)C)C)c 1 2-Nitro toluene 88-72-2 ch3 cr SMILES: N(=0)(=0)c(c(cccl)C)c 1 3-Nitro toluene 99-08-1 CH, & ^no2 SMILES: N(=0)(=0)c(cccclC)c 1 4-Nitrotoluene 99-99-0 CH, no2 SMILES: N(=0)(=0)c(ccc(cl)C)c 1 24 ------- |