United States Environmental Protection 1=1 m m Agency EPA/690/R-13/016F Final 7-26-2013 Provisional Peer-Reviewed Toxicity Values for Nitromethane (CASRN 75-52-5) Superfund Health Risk Technical Support Center National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGERS Harlal Choudhury, DVM, PhD, DABT National Center for Environmental Assessment, Cincinnati, OH Dan D. Petersen, PhD, DABT National Center for Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY ICF International 9300 Lee Highway Fairfax, VA 22031 PRIMARY INTERNAL REVIEWERS Dan D. Petersen, PhD, DABT National Center for Environmental Assessment, Cincinnati, OH Suryanarayana V. Vulimiri, BVSc, PhD, DABT National Center for Environmental Assessment, Washington, DC This document was externally peer reviewed under contract to Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421-3136 Questions regarding the contents of this document may be directed to the U.S. EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). li ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS iv BACKGROUND 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVs 1 INTRODUCTION 2 REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) 4 HUMAN STUDIES 10 Oral Exposures 10 Inhalation Exposures 10 ANIMAL STUDIES 11 Oral Exposures 11 Inhalation Exposures 12 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 22 Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity 27 Other Toxicity Studies (Exposures Other Than Oral or Inhalation) 27 Short-Term Studies 27 Metabolism/Toxicokinetic Studies 27 Mode-of-Action/Mechanistic Studies 28 Immunotoxicity 28 Neurotoxicity 28 DERIVATION 01 PROVISIONAL VALUES 28 DERIVATION OF ORAL REFERENCE DOSES 29 Derivation of Subchronic and Chronic Provisional RfD (p-RfD) 29 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 29 Derivation of Subchronic Provisional RfC (Subchronic p-RfC) 29 Derivation of Chronic Provisional RfC (Chronic p-RfC) 34 CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR 40 MODI: OI -ACTION DISCI SSION 40 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 40 Derivation of Provisional Oral Slope Factor (p-OSF) 40 Derivation of Provisional Inhalation Unit Risk (p-IUR) 40 APPENDIX A. PROVISIONAL SCREENING VALUES 43 APPENDIX B. DATA TABLES 44 APPENDIX C. BMD OUTPUTS 63 APPENDIX D. REFERENCES 73 in ------- COMMONLY USED ABBREVIATIONS BMC benchmark concentration BMCL benchmark concentration lower bound 95% confidence interval BMD benchmark dose BMDL benchmark dose lower confidence limit HEC human equivalent concentration HED human equivalent dose IUR inhalation unit risk LOAEL lowest-observed-adverse-effect level LOAELadj LOAEL adjusted to continuous exposure duration LOAELhec LOAEL adjusted for dosimetric differences across species to a human NOAEL no-ob served-adverse-effect level NOAELadj NOAEL adjusted to continuous exposure duration NOAELhec NOAEL adjusted for dosimetric differences across species to a human NOEL no-ob served-effect level OSF oral slope factor p-IUR provisional inhalation unit risk POD point of departure p-OSF provisional oral slope factor p-RfC provisional inhalation reference concentration p-RfD provisional oral reference dose RfC inhalation reference concentration RfD oral reference dose UF uncertainty factor UFa interspecies uncertainty factor UFC composite uncertainty factor UFd database uncertainty factor UFh intraspecies uncertainty factor UFl LOAEL-to-NOAEL uncertainty factor UFS subchronic-to-chronic uncertainty factor WOE weight of evidence iv ------- FINAL 7-26-2013 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR NITROMETHANE (CASRN 75-52-5) BACKGROUND A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant scientific literature using established Agency guidance on human health toxicity value derivations. All PPRTV assessments receive internal review by a standing panel of National Center for Environment Assessment (NCEA) scientists and an independent external peer review by three scientific experts. The purpose of this document is to provide support for the hazard and dose-response assessment pertaining to chronic and subchronic exposures to substances of concern, to present the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to characterize the overall confidence in these conclusions and toxicity values. It is not intended to be a comprehensive treatise on the chemical or toxicological nature of this substance. The PPRTV review process provides needed toxicity values in a quick turnaround timeframe while maintaining scientific quality. PPRTV assessments are updated approximately on a 5-year cycle for new data or methodologies that might impact the toxicity values or characterization of potential for adverse human health effects and are revised as appropriate. It is important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current information available. When a final Integrated Risk Information System (IRIS) assessment is made publicly available on the Internet (http://www.epa.eov/iris). the respective PPRTVs are removed from the database. DISCLAIMERS The PPRTV document provides toxicity values and information about the adverse effects of the chemical and the evidence on which the value is based, including the strengths and limitations of the data. All users are advised to review the information provided in this document to ensure that the PPRTV used is appropriate for the types of exposures and circumstances at the site in question and the risk management decision that would be supported by the risk assessment. Other U.S. Environmental Protection Agency (EPA) programs or external parties who may choose to use PPRTVs are advised that Superfund resources will not generally be used to respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program. QUESTIONS REGARDING PPRTVs Questions regarding the contents and appropriate use of this PPRTV assessment should be directed to the EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). 1 Nitromethane ------- FINAL 7-26-2013 INTRODUCTION Nitromethane, CAS No. 75-52-5, is a nitroparaffin. Nitromethane is primarily used in rocket and racing fuel as a gasoline additive and is also widely used as an industrial solvent. It can be produced by high-temperature, vapor-phase nitration of propane or methane using nitric acid in a free-radical reaction or by the interaction of sodium nitrite and sodium chloroacetate (HSDB, 2010). A table of physicochemical properties is provided below (see Table 1). The molecular formula for nitromethane is CH3NO2 (see Figure 1). ch3—no2 Figure 1. Nitromethane Structure Table 1. Physicochemical Properties of Nitromethane (CASRN 75-52-5)a Property (unit) Value Boiling point (°C) 101.1 Melting point (°C) -28.5 Relative density (g/cm3 at 20°C) 1.1371 Vapor pressure (mmHg at 25°C) 35.8 pH (unitless, 0.01M aqueous solution) 6.12 Solubility in water (mg/L at 25°C) 1.11 x 105 Relative vapor density (air =1) 2.11 Molecular weight (g/mol) 61.04 aHSDB (2010). A summary of the available toxicity values for Nitromethane from U.S. EPA and other agencies/organizations is provided below (see Table 2). 2 Nitromethane ------- FINAL 7-26-2013 Table 2. Summary of Available Toxicity Values for Nitromethane (CASRN 75-52-5) Source/Parameter" Value (Applicability) Notes Reference Date Accessed Noncancer ACGIH 8-hr TLV-TWA = 20 ppm NA ACGIH (2008) NA ATSDR NV NA ATSDR 7-22-2013 Cal/EPA NV NA Cal/EPA (2012) NA NIOSH NV NIOSH did submit comments to OSHA regarding its "Proposed Rule on Air Contaminants" (29 CFR 1910, Docket No. H-020) questioning whether the OSHA PEL for nitromethane was "adequate to protect workers from recognized health hazards" (NIOSH, 2011b) NIOSH (2011a) NA OSHA 8-hr PEL-TWA = 100 ppm NA OSHA (2006) NA IRIS NV NA U.S. EPA 7-22-2013 Drinking water NV HEEP declined to derive noncancer toxicity values due to inadequate data on noncancer effects and potential carcinogenic effects of the chemical U.S. EPA (2011a) NA HEAST NV NA U.S. EPA (2011b) NA CARA HEEP NV NA U.S. EPA (1994, 1985) NA WHO NV NA WHO 7-22-2013 Cancer IRIS NV NA U.S. EPA 7-22-2013 HEAST NV NA U.S. EPA (2011b) NA IARC Group 2B, "Possibly Carcinogenic to Humans " Based on sufficient animal carcinogenicity evidence IARC (2000) NA NTP "Reasonably Anticipated to be a Human Carcinogen" In the key study that supports this classification, increased mammary gland tumors in female rats, increased benign and malignant tumors in the Harderian gland and lung of male and female mice, and liver tumors in female mice were observed (NTP, 1997). NTP (2011) NA 3 Nitromethane ------- FINAL 7-26-2013 Table 2. Summary of Available Toxicity Values for Nitromethane (CASRN 75-52-5) Source/Parameter" Value (Applicability) Notes Reference Date Accessed Cal/EPA NSRL =39 ng/day Cancer Potency = 0.18 mg/kg-dayb Cal/EPA (2007) NA aSources: American Conference of Governmental Industrial Hygienists (ACGIH); Agency for Toxic Substances and Disease Registry (ATSDR); California Environmental Protection Agency (Cal/EPA); National Institute for Occupational Safety and Health (NIOSH); Occupational Safety and Health Administration (OSHA); Chemical Assessments and Related Activities (CARA); Health and Environmental Effects Profile (HEEP); World Health Organization (WHO); Integrated Risk Information System (IRIS); Health Effects Assessment Summary Tables (HEAST); International Agency for Research on Cancer (IARC); National Toxicology Program (NTP). bThis value was derived from NTP (1997) using "Route to Route" extrapolation. IDLH= immediately dangerous to life or health; NA = not applicable; NSRL = no significant risk level; NV = not available; PEL-TWA = permissible exposure level-time weighted average; REL-TWA = recommended exposure level-time weighted average; TLV-TWA = threshold limit value-time weighted average. Literature searches were conducted on sources published from 1900 through June 2013, for studies relevant to the derivation of provisional toxicity values for nitromethane, CAS No. 75-52-5. Searches were conducted using EPA's Health and Environmental Research Online (HERO) database of scientific literature. HERO searches the following databases: AGRICOLA; American Chemical Society; BioOne; Cochrane Library; DOE: Energy Information Administration, Information Bridge, and Energy Citations Database; EBSCO: Academic Search Complete; GeoRef Preview; GPO: Government Printing Office; Informaworld; IngentaConnect; J-STAGE: Japan Science & Technology; JSTOR: Mathematics & Statistics and Life Sciences; NSCEP/NEPIS (EPA publications available through the National Service Center for Environmental Publications [NSCEP] and National Environmental Publications Internet Site [NEPIS] database); PubMed: MEDLINE and CANCERLIT databases; SAGE; Science Direct; Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network): ANEUPL, CCRIS, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search, NIOSH, NTIS, PESTAB, PPBIB, RISKLINE, TRI; and TSCATS; Virtual Health Library; Web of Science (searches Current Content database among others); World Health Organization; and Worldwide Science. The following databases outside of HERO were searched for toxicity values: ACGIH, ATSDR, Cal/EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, U.S. EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS. REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) Table 3 provides an overview of the relevant database for nitromethane and includes all potentially relevant repeated short-term-, subchronic-, and chronic-duration studies. NOAELs, LOAELs, and BMDL/BMCLs are provided in HED/HEC units for comparison except that oral noncancer values are not converted to HEDs and are identified in parentheses as (Adjusted) 4 Nitromethane ------- FINAL 7-26-2013 rather than HED/HECs. Principal studies are identified in bold. Following the table, important aspects of all the studies in the table are provided in the same order as the table. Reference can be made to details provided in Table 3. The phrase "statistical significance," used throughout the document, indicates a value of <0.05. 5 Nitromethane ------- FINAL 7-26-2013 Table 3. Summary of Potentially Relevant Data for Nitromethane (CASRN 75-52-5) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL" BMDL/ BMCLa LOAEL' Reference (Comments) Notesb Human 1. Oral (mg/kg-d)a Acute0 1/0, age 18, oral ingestion, case study, single ingestion NR Neurological symptoms NDr DU NDr Fernandez et al. (2008) PR Short-termd ND Long-term6 ND Chronicf ND 2. Inhalation (mg/m3)a Acute0 ND Short-termd 1/1, age 23 and 26 yr, inhalation, occupational case study, 1-2 mo 25-50 (mean =32) Neurological symptoms NDr DU NDr Page et al. (2001) PR Long-term0 ND Chronicf ND Reproductive ND Carcinogenicity ND Animal 1. Oral (mg/kg-d)a Subchronic 10/0, albino rat, drinking water, 15 wk 0, 131,280s (Adjusted) Mortality, reduced body weight, and reduced water consumption NDr DU 131 (FEL) Weatherby (1955) PR Chronic ND Developmental ND Reproductive ND 6 Nitromethane ------- FINAL 7-26-2013 Table 3. Summary of Potentially Relevant Data for Nitromethane (CASRN 75-52-5) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL" BMDL/ BMCLa LOAEL' Reference (Comments) Notesb Carcinogenicity ND 2. Inhalation (mg/m3)a Short-term 5/5, F344 rat, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 16 d (12 exposures) M: 0, 5.7, 11.2, 22.1,43.5, 86.lh F: 0,4.5,8.9, 17.7,35.3,69.7 Degeneration of the olfactory epithelium of the nasal turbinates1 M: 11.2 F: 8.9 DU M: 22.1 F: 17.7 NTP (1997) PR 5/5, B6C3F, mouse, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 16 d (12 exposures) M: 0, 6.6, 13.0, 25.6, 52.1, 103.7h F: 0, 5.1, 10.2, 20.2, 40.9, 81.9 Degeneration of the olfactory epithelium of the nasal turbinates1 M: 13.0 F: 10.2 DU M: 25.6 F: 20.2 NTP (1997) PR Subchronic 10/10, F344 rat, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 13 wk M: 0,6.6, 13.9, 27.1,53.0, 100. lh F: 0, 4.8, 9.7, 19.2, 38.1,72.1 Degeneration of the olfactory epithelium of the nasal turbinates1 M: 13.9 F: 9.7 DU M: 27.1 F: 19.2 NTP (1997) PR 10/0, Sprague-Dawley rat, whole body inhalation, 7 hr/d, 5 d/wk, 3 mo 0, 51, 388J Decreased hematocrit and hemoglobin levels 51 NCk 388 Lewis et al. (1979) PR 10/10, B6C3Fi mouse, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 13 wk M: 0, 7.1,14.1, 28.0,55.7, 112.7h F: 0,5.9,12.2, 24.9,48.0, 93.9 Degeneration of the olfactory epithelium and hyaline droplets in the respiratory epithelium of the nasal turbinates' 5.9 (F) 1.31 12.2 (F) NTP (1997) PS, PR 7 Nitromethane ------- FINAL 7-26-2013 Table 3. Summary of Potentially Relevant Data for Nitromethane (CASRN 75-52-5) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL" BMDL/ BMCLa LOAEL' Reference (Comments) Notesb Subchronic 5/0, New Zealand white rabbit, whole body inhalation, 7 hr/d, 5 d/wk, 3 mo 0, 51, 388J Decreased thyroxin levels 51 NCg 388 Lewis et al. (1979) PR Chronic 10/0, Sprague-Dawley rat, whole body inhalation, 7 hr/d, 5 d/wk, 6 mo 0, 51, 388J Increased relative thyroid weight 51 NCk 388 Lewis et al. (1979) PR 50/50, F344 rat, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 103 wk 0, 43, 87, 173J No noncancer effects observed 173 DU NDr NTP (1997) PR 40/40, Long-Evans rat, whole body inhalation, 7 hr/d, 5 d/wk, 2 yr 0, 45.6, 89.01 No noncancer effects observed 89.0 DU NDr Griffin et al. (1996) PR 50/50, B6C3Fi mouse, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 103 wk M: 0,22.2, 44.9,91.6h F: 0,21.9,43.2, 87.9 Increased nonneoplastic nasal lesions NDr 1.60 M: 22.2 F: 21.9 NTP (1997) PS, PR 5/0, New Zealand white rabbit, whole body inhalation, 7 hr/d, 5 d/wk, 6 mo 0, 51, 388J Decreased thyroxin levels NDr NCk 51 Lewis et al. (1979) PR Developmental ND Reproductive ND Carcinogenicity 50/50, F344 rat, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 103 wk 0,43, 87,173j Increased incidence of mammary gland tumors NA NA NA NTP (1997) PS, PR 8 Nitromethane ------- FINAL 7-26-2013 Table 3. Summary of Potentially Relevant Data for Nitromethane (CASRN 75-52-5) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL3 BMDL/ BMCLa LOAEL3 Reference (Comments) Notesb Carcinogenicity 40/40, Long-Evans rat, whole body inhalation, 7 hr/d, 5 d/wk, 2 yr 0, 45.6, 89.01 No cancer effects observed NA NA NA Griffin et al. (1996b) PR 50/50, B6C3F, mouse, whole body inhalation, 6 hr 12 min/d, 5 d/wk, 103 wk 0, 87, 173, 346J Increased incidence of Harderian gland and liver tumors; BMD based Harderian gland adenomas or carcinomas in males NA NA NA NTP (1997) PR ""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects and a human equivalent concentration (HEC in mg/m3) for inhalation noncancer and carcinogenic effects. All long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous exposure. bNotes: IRIS = utilized by IRIS, date of last update; PS = principal study; PR = peer reviewed; NPR = not peer reviewed; NA = not applicable. 0 Acute = Exposure for 24 hours or less (U.S. EPA, 2002). dShort-term = Repeated exposure for >24 h <30 d (U.S. EPA, 2002). "Long-term = Repeated exposure for >30 d <10% lifespan (based on 70 years typical lifespan) (U.S. EPA, 2002). fChronic = Repeated exposure for >10% lifespan (U.S. EPA, 2002). 8Daily doses were estimated from digitizing the data in the figure of weekly intakes provided in the study report. hHECRESp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x regional gas dose ratio. 'Extra-respiratory effects were also observed, but the respiratory effects provided the lowest NOAEL/LOAEL. jHECExresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood:gas partition coefficient. kBMD was not conducted on the data because the effects occurred at concentrations several fold higher than the critical effect in the principal study. 'HECexresp = mg/m3 x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood:gas partition coefficient. DU = data unsuitable; NA = not applicable; NC = not conducted; ND = no data; NDr = not determinable; NR = not reported. 9 Nitromethane ------- FINAL 7-26-2013 HUMAN STUDIES Oral Exposures The effects of oral exposure of humans to nitromethane have been evaluated in one case study of an acute exposure (Fernandez et al., 2008). An 18-year-old male ingested an undetermined amount of nitromethane fuel. He had generalized tonic-clonic seizures that progressed to partial motor status epilepticus and was also hypertensive. A neurological exam showed him to have mild left dysmetria in finger-to-nose testing, low frequency intention tremor, broad-based gait, and inability to tandem-walk. An MRI revealed bilateral and symmetric lesions in cerebellar white matter, tonsils, uvula, and colliculi. Initial therapy included valproic acid, P-blockers, and clonidine to treat the symptoms. Valproic acid treatment continued. Eight months after exposure there were no abnormalities observed during a clinical exam or in an MRI. There were no other oral studies in humans identified. Inhalation Exposures The effects of subchronic inhalation exposure of humans to nitromethane have been evaluated in two case reports on individuals exposed for 1 or 2 months (Page et al., 2001). An investigation was initiated due to severe peripheral neuropathy in two workers in a headlight subassembly plant. OSHA was called in to conduct an industrial hygiene inspection at the plant. In the plant, nitromethane was used to clean off excess glue on the headlights. Nitromethane was sprayed on, and because it was wiped off with a rag without the use of gloves, dermal exposure occurred as well as inhalation exposure. However, in a separate study, Coulston International Inc. (1990) found that very little nitromethane was absorbed dermally in rhesus monkeys. As part of the investigation by Page et al. (2001), the air was sampled for nitromethane and ethyl cyanoacrylate over a 2-day period. Nitromethane was measured in the personal breathing zone of four workers cleaning headlights. Nitromethane levels ranged from 10-20 ppm (25-50 mg/m3) with a mean of 12.75 ppm (32 mg/m3) as the 8-hour TWA. A 26-year-old woman who cleaned the headlights with nitromethane began to note weakness in her hands, legs, and feet within a month of beginning the job. She stopped working but felt worse 2 weeks later and was admitted to the hospital. A neurological exam indicated that gastroscoleus and brachioradialis reflexes were absent with weakness more severe distally and in the lower extremities. An MRI did not reveal any abnormalities in the spinal cord. Laboratory tests did not find any abnormalities, but serum immunoelectrophoresis showed an increased gamma component, and a lumbar puncture showed elevated protein levels with a normal cell count. She was eventually diagnosed with severe peripheral neuropathy and had only slight improvement within 8 months. Four to five months later, a 23-year-old male began complaining about foot numbness within 1.5 weeks of beginning work, which progressed over the next few weeks to pain and swelling in both legs and feet. After about 6 weeks of employment as a headlight cleaner, the man was admitted to the hospital. Peripheral neuropathy of the lower extremities with normal testing on the upper extremities was observed. The individual demonstrated diminished lower extremity reflexes, slightly decreased muscle strength on dorsiflexon of both feet, and decreased sensation to pinprick up to the middle of the shin. Laboratory tests for this individual were unremarkable, but symptoms persisted. Follow-up electrodiagnostic studies found progressive denervation and severe axonal peripheral neuropathy. This individual had significant, but not complete, improvement. No other inhalation studies in humans were identified. 10 Nitromethane ------- FINAL 7-26-2013 ANIMAL STUDIES Oral Exposures The effects of oral exposure in animals to nitromethane have been evaluated in one subchronic study (Weatherby, 1955). Subchronic Studies Weatherby (1955) Weatherby (1955) exposed young male albino rats (10/treatment group) to 0, 0.1%, 0.25%, 0.5%), P/o, or 2% nitromethane (purity not reported) via drinking water for 15 weeks. There is no indication that the dose formulations were measured for homogeneity, stability, or concentration. It was stated that due to the slight solubility of nitromethane, emulsification with methylcellulose was used. During the first week, animals did not drink the water with concentrations >0.5%, and these groups were discontinued. However, several of the exposed animals did die during this time (number not specified), and survivors were sacrificed for microscopic examination of the tissues. The failure in these groups was attributed to refusal of the water. Fluid intake was recorded daily, and the intake of nitromethane was calculated. Intake levels were apparently measured on a per-cage basis, but the study report did not specify how many rats were maintained per cage. The weekly intakes (mg nitromethane/kg-week) were presented in the study report as a figure only and have been estimated by digitizing the data. The weekly intakes divided by seven provide estimated daily intakes of 131 mg/kg-day for the 0.1% group and 280 mg/kg-day for the 0.25% group. Body weights were measured weekly. Body weight and body-weight gain were lower than that of control animals in both nitromethane groups, but this observation did not appear to dose related and may have been a function of reduced fluid intake. Animals were stated to be in moderately good health at study termination. Four of the 131-mg/kg-day animals and three of the 280-mg/kg-day animals died during treatment. Due to destruction by their cage mates, the dead animals were not examined. At study termination, all survivors were sacrificed, and tissues (heart, lungs, liver, spleen, kidney, testes, adrenal gland, and small intestines) were examined microscopically. In the 131-mg/kg-day group, two of the six survivors had large hepatic cells with prominent nuclei. In the 280-mg/kg-day group, two of the seven survivors had Malpighian corpuscles that appeared more prominent than the normal spleens, and six of the survivors had a granular appearance in the cytoplasm of the hepatic cells, less deeply stained hepatocytes, more prominent nuclei, and lymphocytes in the periportal region. One of the 10 surviving controls had large hepatic cells with prominent nuclei. No NOAEL or LOAEL can be determined because the lowest dose was a frank effect level (FEL) due to mortality. Chronic Studies There is no suitable information to provide in this regard. Developmental Studies There is no suitable information to provide in this regard. Reproductive Studies There is no suitable information to provide in this regard. Carcinogenicity Studies There is no suitable information to provide in this regard. 11 Nitromethane ------- FINAL 7-26-2013 Inhalation Exposures The effects of inhalation exposure of animals to nitromethane have been evaluated in a short-term study in both rats and mice (NTP 1997), two subchronic studies in both rats and mice (NTP, 1997; Lewis, 1979), chronic studies in rats and mice (NTP, 1997; Lewis, 1979) and rats only (Griffin, 1996), and carcinogenicity studies in rats and mice (NTP, 1997) and rats only (Griffin, 1996). All the reports provided information on multiple studies and are individually discussed in this section. Short-Term Studies NTP (1997) In the various substudies conducted by NTP (1997) summarized below, a small particle detector (Type CN, Gardner Associates, Schenectady, NY) was used with and without animals in the exposure chambers to ensure that nitromethane vapor, and not aerosol, was produced. The purity of the chemical was analyzed by gas chromatography and carefully monitored during exposures (details are provided on pages 21-23 of the study report). For the short-term study, individually housed F344 rats (5/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0; 94; 188; 375; 750; or 1,500 ppm via whole body inhalation for 6 hours and 12 minutes (12 minutes was the time it took to achieve 90% target concentration) a day, 5 days a week, for 16 days (total of 12 exposures). Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity. Animals were weighed at study initiation and on Days 8 and 16. At study termination, animals were necropsied, and the heart, right kidney, liver, lungs, right testis, thymus, and thyroid glands were weighed. Histopathology was conducted on respiratory tissues, brain, and sciatic nerve. Typical NTP statistical methods were applied and were appropriate for the data. All rats survived until study termination. Clinical signs were observed in the high-dose group only and included increased preening, rapid breathing, hyperactivity early in the study, and hypoactivity and loss of coordination near the end of the study in all animals of both sexes. Although there were no statistically significant (p < 0.05) effects on body weight in any of the groups, high-dose males had a significantly (p < 0.05) lower body-weight gain compared with the controls (see Table B. 1). Necropsy body weight was significantly reduced in the high-dose males (see Table B.2). Absolute and relative liver weights were significantly increased and >10%) different from controls in female rats at concentrations >750 ppm. Relative liver weights were significantly increased in all male treatment groups but were only >10% different from controls at concentrations of 375 and 1,500 ppm (see Table B.2). Relative kidney weight was significantly increased by >10% in high-dose males and females. In all males and all but one female administered >375 ppm, there was degeneration of the olfactory epithelium of the nasal turbinates with minimal to mild severity and degeneration of the sciatic nerve with severity (minimal to moderate) increasing with exposure concentration (see Table B.3). Because respiratory and extra-respiratory effects were observed, human equivalent concentrations (HECs) are calculated for both effects. The average of initial and final body weight is used in the calculation of HECs for respiratory effects. The HECs for respiratory effects are 5.7, 11.2, 22.1, 43.5, and 86.1 mg/m3 in males, respectively, and 4.5, 8.9, 17.7, 35.3, and 69.7 mg/m3 in females, respectively, after adjusting for duration and multiplying by the regional gas dose ratio (RGDR) for extra-thoracic effects. The RGDR is the ratio of the minute volumes to the applicable surface areas of the lung in the two species respectively (see U.S. EPA 1994b, pages 4-44 through 4-64). 12 Nitromethane ------- FINAL 7-26-2013 This ratio is used to adjust the observed gas exposure level for interspecies dosimetric differences. The NOAEL for respiratory effects is 11.2 mg/m3 in males and 8.9 mg/m3 in 3 3 females. The LOAEL for respiratory effects is 22.1 mg/m in males and 17.7 mg/m in females based on degeneration of the olfactory epithelium in nasal turbinates. HECs for extra-respiratory effects calculated by adjusting for duration and using the default blood:gas partition coefficient of 1 since chemical-specific information is unavailable (see U.S.EPA 1994b). The "3 corresponding values are 46, 91, 181, 363, and 726 mg/m in both sexes. The NOAEL for systemic effects is 91 mg/m3, and the LOAEL is 181 mg/m3 based on degeneration of the sciatic nerve in both sexes. NTP (1997) Individually housed male and female B6C3Fi mice (5/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0; 94; 188; 375; 750; or 1,500 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 16 days (total of 12 exposures). Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity. Animals were weighed at study initiation and on Days 8 and 16. At study termination, animals were necropsied, and the heart, right kidney, liver, lungs, right testis, thymus, and thyroid glands were weighed. Histopathology was conducted on respiratory tissues, brain, and sciatic nerve. Typical NTP statistical methods were applied and were appropriate for the data. All mice survived until study termination. Clinical signs were observed in the high-dose group only and included hypoactivity and tachypnea of both sexes at the end of the study. There were no treatment-related effects on body weight or gross pathology. Absolute liver weight was >10% different from controls at all concentrations in both sexes, but relative liver weights were statistically significant and >10% different from controls at concentrations >375 ppm in males and >188 ppm in females (see Table B.4). All males and females administered >375 ppm had degeneration of the olfactory epithelium of the nasal turbinates with minimal to mild severity. Because respiratory and extra-respiratory effects were observed, HECs are calculated for both effects. The average of initial and final body weight is used for the calculation of HECs for respiratory effects. HECs for respiratory effects are 6.6, 13.0, 25.6, 52.1, and 103.7 mg/m in males, respectively, and 5.1, 10.2, 20.2, 40.9, and 81.9 mg/m3 in females, respectively, after adjusting for duration and multiplying by the RGDR for extra-thoracic effects. The NOAEL for respiratory effects is 13.0 mg/m in males and 10.2 mg/m3 in females. The LOAEL for 3 3 respiratory effects is 25.6 mg/m in males and 20.2 mg/m in females based on degeneration of the olfactory epithelium in nasal turbinates. HECs for extra-respiratory effects calculated by adjusting for duration and using the default blood:gas partition coefficient of 1 (chemical- specific information was unavailable) are 46, 91, 181, 363, and 726 mg/m3 in both sexes. The 3 3 NOAEL for systemic effects is 46 mg/m , and the LOAEL is 91 mg/m based on increased relative liver weights in females. Subchronic Studies NTP (1997) Individually housed male and female F344 rats (10/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0; 94; 188; 375; 750; or 1,500 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 13 weeks. Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity. Animals were weighed at study 13 Nitromethane ------- FINAL 7-26-2013 initiation, weekly, and at study termination. Blood was collected on Days 3 and 23 and at study termination for hematology (hematocrit, hemoglobin, erythrocyte counts, nucleated erythrocyte counts, mean cell volume [MCV], mean cell hemoglobin [MCH], mean cell hemoglobin concentration [MCHC], platelet counts, total leukocyte count and differentials, and methemoglobin) and clinical chemistry (blood urea nitrogen [BUN], creatinine, total protein, albumin, globulin, alanine aminotransferase [ALT], alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, bile acid, thyroid-stimulating hormone [TSH], triiodothyronine, and total and free thyroxine concentrations). At study termination, animals were necropsied, and the heart, right kidney, liver, lungs, right testis, thymus, and thyroid glands were weighed. Complete histopathology was conducted on control and high-dose animals. In addition, the following tissues were examined in the lower concentrations until a no-effect level (NOEL) was reached: bone marrow, lung, and nose in both sexes; and cecum, larynx, and testis in male rats. Sperm samples were collected from the 0; 375-; 750-; and 1,500-ppm groups at study termination and evaluated for sperm count and motility. The left cauda, epididymis, and testis were weighed. Vaginal samples were collected for seven consecutive days before study termination and evaluated for the relative frequency of estrous stages and for estrous cycle length. Neurobehavioral tests (forelimb and hindlimb grip strength, tail flick latency, and startle response) were conducted during Week 11. Typical NTP statistical methods were applied and were appropriate for the data. All rats survived until study termination. Clinical signs included hindlimb paralysis in all high-dose males and females beginning on Day 21, and one male and four females in the 750-ppm group beginning on Day 63. There was a statistically significant decrease in terminal body weight and body-weight gain in high-dose males (see Table B.5). There were several changes in hematology in all treatment groups (see Table B.6). These animals developed microcytic responsive anemia as indicated by mild-to-moderate decreases in hematocrit and hemoglobin and minimal-to-moderate decreases in mean cell volume (see Table B.6). MCH levels were also consistently decreased. The results were dose dependent but were most prominent with concentrations >375 ppm. Although the increases in MCHC were less than 5% from the control, the change was consistent. The study authors also reported several changes in erythrocyte morphology related to damage and anemia (e.g., Heinz bodies, schistocytes, polychromasia), but data were not provided. Hyperplasia in the bone marrow was observed at concentrations of 750 and 1,500 ppm, supporting the hematology findings. There were also consistent increases in platelets and methemoglobin mainly at concentrations of 750 and 1,500 ppm. Although there were changes in thyroid hormone levels on Day 23, this was a transient effect in both males and females. Thyroid weight was increased in 1,500-ppm males only, but only the relative weight was statistically significant, which is likely an effect of the lower body weight in this group. There were no other biologically significant changes in organ weights. At concentrations >375 ppm, there was degeneration of the olfactory epithelium of the nasal turbinates with minimal-to-mild severity and degeneration of the sciatic nerve and spinal cord with severity increasing with exposure concentration (see Table B.7). Grip strength was only significantly reduced in high-dose males (hind and forelimb) and females (hindlimb) (see Table B.8). There was a statistically significant decrease in absolute left cauda, epididymis, and testis weight in high-dose males. Although this finding may be due to reduced body weight, there also was a 15% decrease in sperm concentration, and sperm motility was significantly decreased with concentrations of 750 and 1,500 ppm (see Table B.9). There were no treatment-related changes in estrous cycle length or stages. Because respiratory and extra-respiratory effects were observed, HECs are calculated for both effects. Because only 14 Nitromethane ------- FINAL 7-26-2013 initial and final body weights were provided, the average is used to estimate average body weights for the calculation of HECs for respiratory effects. The HECs for respiratory effects are 6.6, 13.9, 27.1, 53.0, and 100.1 mg/m3 in males, respectively, and 4.8, 9.7, 19.2, 38.1, and 72.1 mg/m3 in females, respectively, after adjusting for duration and multiplying by the RGDR for extra-thoracic effects. HECs for extra-respiratory effects calculated by adjusting for duration and using the default blood:gas partition coefficient of 1 are 43, 87, 173, 346, and 691 mg/m3 in both sexes. The NOAEL is 188 ppm, and the LOAEL is 375 ppm based on anemia accompanied by changes in the bone marrow and histopathology of the nasal turbinates, sciatic nerve, and spinal cord. Because the respiratory effects relay a lower HEC value, the study NOAEL is 13.9 mg/m3 in males and 9.7 mg/m in females, and the LOAEL is 27.1 mg/m in males and "3 19.2 mg/m in females. Lewis etal. (1979) Male Sprague-Dawley rats (10/treatment group) were administered nitromethane (96.5% pure with 1.5% nitroethane and 1.4% 2-nitropropane) at analytical concentrations of 0, 98 ± 5, or 745 ± 34 ppm via whole body inhalation, 7 hours a day, 5 days a week, for 3 months (Lewis et al., 1979a). While Lewis et al. (1979) provided a peer-reviewed publication of the data, the proprietary data were also available for review (Huntingdon Research Center, 1989). Additional groups of 10 animals/concentration group were sacrificed at 2 days, 10 days, and 1 month. Animals were housed in groups of 10 in wire mess stainless-steel cages, which were part of the exposure chamber. Chamber concentrations were measured hourly and recorded twice a day. Animals were observed daily for overt signs of toxicity. Body weights were obtained at regular intervals. At sacrifice, blood was collected for hematology (erythrocyte counts [RBC], hematocrit, hemoglobin, prothrombin time, and methemoglobin) and clinical chemistry (ALT, ornithine carbamyl transferase [OCT], and thyroxin). Animals were necropsied, and select organs (brain, liver, kidneys, lungs, and thyroid) were weighed. Tissue samples were obtained from the brain and lungs. The samples were weighed, dried, and weighed again to determine the percent of water. Adrenals, bronchi, cerebellum, cerebral hemispheres, eyes, kidneys, liver, lung, spleen, thyroid, and trachea were examined histologically. Appropriate statistical methods (Bartlett's test for homogeneity of variance; one-way analysis of variance followed by Student's /-test) were conducted on the data. Not all the data were provided in Lewis et al. (1979). Data in the Huntingdon Research Center (1989) report were not provided in any sort of order, were hard to find (tables were not in order), and, in some cases, were not reported. Both sources were used for evaluation. There were no clinical signs of toxicity noted and no treatment-related effects on mortality. There was no difference in body weight between the low-dose group and controls. However, high-dose rats were stated to have a slower weight gain that was statistically significant from the control beginning around Week 8 (except during Week 13). Body-weight data were only provided in a figure (even in the proprietary data), but the body weights do not appear to differ by more than 10%) at any time point during the 3 months. There was a statistically significant decrease in hematocrit and hemoglobin levels in the high-dose group that began after 10 days of treatment (see Table B. 10). There were no treatment-related changes in clinical chemistry. Additionally, there were no treatment-related changes in organ weights, lung or brain edema, gross pathology, or organ histopathology. Based on the absence of respiratory toxicity noted, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects "3 adjusted for duration and a blood:gas partition coefficient of 1 are 51 and 388 mg/m , 15 Nitromethane ------- FINAL 7-26-2013 3 3 respectively. Therefore, the NOAEL is 51 mg/m , and the LOAEL is 388 mg/m based on consistent decreases in hematocrit and hemoglobin levels. NTP (1997) The 13-week component of the peer-reviewed mouse study by NTP (1997) is selected as the principal study for the derivation of the subchronic p-RfC. Individually housed male and female B6C3Fi mice (10/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0; 94; 188; 375; 750; or 1,500 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 13 weeks. Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity. Animals were weighed at study initiation, weekly, and at study termination. At study termination, animals were necropsied, and the heart, right kidney, liver, lungs, right testis, and thymus glands were weighed. Complete histopathology was conducted on control and high-dose animals. In addition, tissues of the nose and spleen in both sexes were examined in the lower concentrations until a NOEL was reached. Sperm samples were collected from the 0-; 375-; 750-; and 1,500-ppm groups at study termination and evaluated for sperm count and motility. The left cauda, epididymis, and testis were weighed. Vaginal samples were collected for seven consecutive days before study termination and evaluated for the relative frequency of estrous stages and for estrous cycle length. Typical NTP statistical methods were applied and were appropriate for the data. All mice survived until study termination. There were no treatment-related clinical signs or changes in body weight. There were statistically significant increases in the absolute and/or relative kidney weight in all treatment groups except in low-dose females (see Table B.l 1). Although the relative kidney weights in exposed males were >10% relative to controls at all concentrations tested, relative kidney weight changes were not dose dependent and were only >10%) in females at the highest concentration tested. Relative liver weights were also significantly increased in males at concentrations >375 ppm. However, this response was not dose dependent and was >10% only at concentrations of 750 and 1,500 ppm. Liver weights were unaffected in female mice at any dose. There were no treatment-related changes in male reproductive tissue weight or in sperm concentration, but there was a statistically significant decrease in sperm motility at all concentrations examined (i.e., >375 ppm) (see Table B.12). There was a dose-related increase in estrous cycle length at all concentrations examined (see Table B. 12). At concentrations >375 ppm, there was degeneration of the olfactory epithelium of the nasal turbinates with minimal to moderate severity and hyaline droplets in the respiratory epithelium in all mice (see Table B. 13). Female mice also had a statistically significant increase in the incidence of these lesions at 188 ppm. There was a significant increase in extramedullary hematopoiesis in the spleens of high-dose males and females (see Table B. 13). Although respiratory and extra-respiratory effects were observed, the NOAEL and LOAEL are based on respiratory effects because they occurred at lower concentrations. Because only initial and final body weights were provided, the average is used to calculate body weights for the calculation of HECs for respiratory effects. The HECs for respiratory effects are 7.1, 14.1, 28.0, 55.7, and 112.7 mg/m3 in males, respectively, and 5.9, 12.2, 24.9, 48.0, and 93.9 mg/m3 in females, respectively, after adjusting for duration and multiplying by the RGDR for extra-thoracic effects. The NOAEL is 5.9 mg/m3 (94 ppm), and the LOAEL is 12.2 mg/m3 (188 ppm) based on increased histopathology of the nasal turbinates in female mice. 16 Nitromethane ------- FINAL 7-26-2013 Lewis etal. (1979) Male New Zealand white rabbits (5/treatment group) were administered nitromethane (96.5% pure with 1.5% nitroethane and 1.4% 2-nitropropane) at analytical concentrations of 0, 98 ± 5, or 745 ± 34 ppm via whole body inhalation, 7 hours a day, 5 days a week, for 3 months (Lewis et al., 1979b). While Lewis et al. (1979) provides a peer-reviewed publication of the data, the proprietary data were also available for review (Huntingdon Research Center, 1989). An additional group of 5 animals/concentration group was sacrificed after 1 month of treatment. Animals were housed individually in wire mess stainless steel cages, which was part of the exposure chamber. Chamber concentrations were measured hourly and recorded twice a day. Animals were observed daily for overt signs of toxicity. Body weights were obtained at regular intervals. At sacrifice, blood was collected for hematology (RBC, hematocrit, hemoglobin, prothrombin time, and methemoglobin) and clinical chemistry (ALT, OCT, and thyroxin). Animals were necropsied, and select organs (brain, liver, kidneys, lungs, and thyroid) were weighed. Tissue samples were obtained from the brain and lungs. The samples were weighed, dried, and weighed again to determine the percent of water. Adrenals, bronchi, cerebellum, cerebral hemispheres, eyes, kidneys, liver, lung, spleen, thyroid, and trachea were examined histologically. A Kruskal-Wallis one-way analysis of variance followed by a Mann-Whitney U test was used on the data due to the small number of animals used. Not all the data were provided in Lewis et al. (1979). Data in the Huntingdon Research Center (1989) report were not provided in any sort of order, were hard to find (tables were not in order), and, in some cases, were not reported. There were no clinical signs of toxicity noted and no treatment-related effects on mortality or body weight. There was a statistically significant increase in methemoglobin in high-dose rabbits at the 3-month time point only, and a statistically significant decrease in hemoglobin in the high-dose rabbits at the 1-month time point only. There were increases in OCT at both 1 and 3 months (see Table B. 14); however, this was not noted at 6 months (see chronic studies below). Thyroxin levels were lower than the control values at both 1 and 3 months but only achieved statistical significance in the high-dose group at 1 month (see Table B.14). Lungs of rabbits at 1 month had focal areas of moderate-to- moderately severe hemorrhage and congestion of the alveolar and alveolar duct walls. This was not stated to occur at 3 months. There were no other treatment-related changes in organ weights, lung or brain edema, gross pathology, or organ histopathology. Based on the absence of respiratory toxicity noted, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects adjusted for duration and a blood:gas partition coefficient of 1 are 51 and 388 mg/m3, respectively. Therefore, the NOAEL is 51 mg/m3, and "3 the LOAEL is 388 mg/m based on consistent decreases in thyroxin levels. Chronic Studies Lewis etal. (1979) Male Sprague-Dawley rats (10/treatment group) were administered nitromethane (96.5% pure with 1.5% nitroethane and 1.4% 2-nitropropane) at analytical concentrations of 0, 98 ± 5, or 745 ± 34 ppm via whole body inhalation, 7 hours a day, 5 days a week, for 6 months (Lewis et al., 1979c). While Lewis et al. (1979) provides a peer-reviewed publication of the data, the proprietary data were also available for review (Huntingdon Research Center, 1989). This study was conducted in conjunction with the 3-month subchronic study discussed above, and materials and methods were the same as those previously detailed. 17 Nitromethane ------- FINAL 7-26-2013 There were no treatment-related effects on mortality. There was no difference in body weight between the low-dose group and the controls. High-dose rats had a slower weight gain, with body weights stated to be statistically significantly different from the control beginning around Week 8 (except during Week 13), but by study termination, body weight was similar to the controls according to the figure in the study report. There was a statistically significant decrease in hematocrit and hemoglobin levels in the high-dose group (see Table B.10). There were no treatment-related changes in clinical chemistry and no treatment-related changes in lung or brain edema, gross pathology, or organ histopathology. The only change in organ weight was a statistically significant increase in absolute and relative thyroid weight in the high-dose group that was dose related (see Table B.15). Based on the absence of respiratory toxicity noted, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects adjusted for duration and a blood:gas partition coefficient of 1 are 51 3 3 and 388 mg/m , respectively. Therefore, the NOAEL is 51 mg/m , and the LOAEL is 388 mg/m3 based on an increase in relative thyroid weights. NTP (1997) Individually housed male and female F344 rats (50/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0, 94, 188, or 375 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 103 weeks. Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity and weighed at study initiation, weekly through Week 12, monthly from Week 15 through Week 91, then every 2 weeks until study termination, and at study termination. At study termination, animals were necropsied. Complete histopathology was conducted on all animals except that spinal cord and sciatic nerve were only examined in control and high-dose animals (15/sex). Typical NTP statistical methods were applied and were appropriate for the data. There was no treatment-related effect on mortality. Besides tumor masses, there were no clinical signs observed. There were no statistically significant changes in body weight compared with the controls. However, the study authors noted that the high-dose group had a slightly higher body weight than the control beginning around Week 23. The differences were not statistically significant nor did they exceed a 10% difference from controls. The only noncancer effect observed in rats was a slight increase in severity of the nephropathy that occurred in all male rats (2.8, 2.9, 3.1, and 3.2 at 0, 94, 188, and 375 ppm, respectively). Based on the absence of significant noncancer effects, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects adjusted for duration and a blood:gas partition 3 3 coefficient of 1 are 43, 87, and 173 mg/m , respectively. Therefore, the NOAEL is 173 mg/m , the highest concentration tested. Griffin et al. (1996) Male and female Long-Evans rats (40/sex/treatment group) were administered nitromethane (96.26%) pure with 2.79% nitroethane and 0.62% 2-nitropropane) at concentrations of 0, 100, or 200 ppm via whole body inhalation, 7 hours a day, 5 days a week, for 2 years (Griffin et al., 1996). While Griffin et al. (1996) provided a peer-reviewed publication of the study, the proprietary data were also available for review (Coulston International Inc., 1990). Animals were individually housed in stainless-steel cages except during exposure periods when they were placed in exposure chambers. Chamber concentrations were measured 3-4 times per day, with average analytical results of 99.5 ppm (stated by the study authors to be equivalent to 18 Nitromethane ------- FINAL 7-26-2013 3 3 219 mg/m ) and 199.3 ppm (stated by the study authors to be equivalent to 427 mg/m ). Animals were observed daily for overt signs of toxicity. Body weights were obtained weekly for the first 6 months and every 2 weeks thereafter. At terminal sacrifice, blood was collected from 10 rats/sex/treatment group for hematology (RBC, leukocyte counts [WBC], MCV, hematocrit, hemoglobin, and platelet counts) and serum clinical chemistry (aspartate aminotransferase [AST], ALT, bilirubin, total protein, BUN, creatinine, sodium, and potassium). Animals were necropsied, and select organs (brain, liver, kidneys, lungs, and heart) were weighed. Thirty-three different tissues/organs were examined histologically in both sexes of rat. Appropriate statistical methods were conducted on the data. There were no clinical signs of toxicity noted. There were no treatment-related effects on mortality. There were no treatment-related changes in body weight in male rats. Although female rats in both treatment groups had statistically significant lower body weights compared to the controls during the second half of the study, the results were not dose dependent and did not exceed a 10% difference from the control. There were no treatment-related effects on hematology. The only change noted in clinical chemistry was an increase in creatinine in the 200-ppm dose group. However, the increase was associated with the method used (i.e., Jaffe reaction) as nitromethane in the plasma has been found to increase the reaction (De Leacy et al., 1989). There were no treatment-related changes in organ weights, gross pathology, or organ histopathology. Due to the absence of toxicity, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects adjusted for duration and a "3 blood:gas partition coefficient of 1 are 45.6 and 89.0 mg/m , respectively. Therefore, the NOAEL is 89.0 mg/m3, the highest concentration tested. NTP (1997) The 2-year component of the peer-reviewed mouse study by NTP (1997) is selected as the principal study for the derivation of the chronic p-RfC. Individually housed male and female B6C3Fi mice (50/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0, 188, 375, or 750 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 103 weeks. Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity. Animals were weighed at study initiation, weekly through Week 12, monthly from Week 15 through Week 91, then every 2 weeks until study termination, and at study termination. At study termination, animals were necropsied. Complete histopathology was conducted on all animals except that spinal cord and sciatic nerve were not examined. Typical NTP statistical methods were applied and were appropriate for the data. There was no treatment-related effect on mortality. Clinical signs observed included swelling around the eyes and exophthalmos. This was stated to be consistent with Harderian gland neoplasms, which occurred at a greater incidence in treated animals (see carcinogenicity studies below). There were no statistically significant changes in body weight compared with the controls. There were increases in liver lesions in female mice and nasal lesions in both sexes (see Table B.16). The HECs for respiratory effects are 22.2, 44.9, and 91.6 mg/m in males, -3 respectively, and 21.9, 43.2, and 87.9 mg/m in females, respectively, after adjusting for duration and multiplying by the RGDR for extra-respiratory effects. No NOAEL can be determined. The 3 3 LOAEL is 22.2 mg/m in males and 21.9 mg/m in females based on increased incidence of nasal lesions in both sexes of mice. 19 Nitromethane ------- FINAL 7-26-2013 Lewis etal. (1979) Male New Zealand white rabbits (5/treatment group) were administered nitromethane (96.5% pure with 1.5% nitroethane and 1.4% 2-nitropropane) at analytical concentrations of 0, 98 ± 5, or 745 ± 34 ppm via whole body inhalation for 7 hours a day, 5 days a week, for 6 months (Lewis et al., 1979d). While Lewis et al. (1979) provides a peer-reviewed publication of the data, the proprietary data were also available for review (Huntingdon Research Center, 1989). This study was conducted in conjunction with the 3-month subchronic study discussed above, and the materials and methods were the same as those previously detailed. There were no treatment-related effects on mortality, body weight, hematology, lung or brain edema, gross pathology, or organ histopathology. Thyroxin levels at both concentrations were statistically significantly (p < 0.05) lower than the control (see Table B. 14). This was accompanied by an increase in absolute and relative thyroid weight that did not achieve statistical significance (see Table B.17). Based on the absence of respiratory toxicity noted, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects adjusted for duration and a blood:gas partition coefficient of 1 are 51 and 388 mg/m3, respectively. Therefore, the LOAEL is 51 mg/m3 based on decreases in thyroxin levels, which were accompanied by changes in organ weight. No NOAEL can be determined from the data. Developmental Studies There are no developmental studies via oral or inhalation exposure to nitromethane. However, Whitman et al. (1977) intraperitoneally injected 0.5 mL of a 1.5-M solution of nitromethane in saline every third day beginning prior to pregnancy through gestation to albino rats. Few details were provided on the dosing procedure. Controls were similarly treated with saline. No differences were observed in the number of pups (dead or alive), pup body weight, or dam behavior. Pups were maintained with mothers until weaning and behavioral maze tests were conducted when pups were 2.5 months old. Nitromethane-exposed pups had significantly more errors to criterion compared with the control animals. Reproductive Studies There are no multigeneration reproductive studies with nitromethane by either oral or inhalation routes of exposure. However, the two 13-week studies by NTP (NTP, 1997) examined some reproductive endpoints after inhalation exposure. In rats (NTP, 1997), there were statistically significant decreases in absolute left cauda, epididymis, and testis weight at 1,500 ppm. Although this may be due to reduced body weight, there was a 15% decrease in sperm concentration in high-dose males, and sperm motility was significantly decreased with concentrations of 750 and 1,500 ppm (see Table B.9; NTP, 1997). Mice also had reduced sperm motility (NTP, 1997), but there was no effect on reproductive tissue weight or sperm concentration (see Table B. 12). There were no treatment-related changes in estrous cycle length or estrous stages in female rats (NTP, 1997), but there was a dose-related increase in estrous cycle length at all concentrations examined in female mice (see Table B.12; NTP, 1997). Carcinogenicity Studies NTP (1997) The carcinogenicity component of the chronic peer-reviewed rat study by NTP (1997) is selected as the principal study for the derivation of the p-IUR. Individually housed male and female F344 rats (50/sex/treatment group) were administered nitromethane 20 Nitromethane ------- FINAL 7-26-2013 (purity >98%) at concentrations of 0, 94, 188, or 375 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 103 weeks. Chamber concentrations were routinely checked over the course of exposure. Animals were observed twice a day for clinical signs of toxicity. Animals were weighed at study initiation, weekly through Week 12, monthly from Week 15 through Week 91, then every 2 weeks until study termination, and at study termination. At study termination, animals were necropsied. Complete histopathology was conducted on all animals except that spinal cord and sciatic nerve were only examined in control and high-dose animals (15/sex). Typical NTP statistical methods were applied and were appropriate for the data. There was no treatment-related effect on mortality. Besides tumor masses, there were no clinical signs of toxicity observed. There were no statistically significant changes in body weight compared with controls. However, the study authors noted that the high-dose group had a slightly higher body weight than the control beginning around Week 23. The differences were not statistically significant nor did they exceed a 10% difference from the control. There were no treatment-related increases in tumors in male rats. There was an increase in tumors of the mammary gland in females with concentrations of 188 and 375 ppm (see Table B. 18). The incidence of mammary gland tumors in the 188- and 375-ppm groups also exceeded the historical control data from inhalation studies. There were concerns raised that the slight increase in body weight in the 375-ppm females was related to the increase in tumors observed in the female rat. However, it was concluded that this was not the case. Based on the increase in body weights, it would be predicted that there would be a 51% incidence of mammary gland tumors, but the incidence was 82%. NTP determined that there was no evidence of carcinogenicity in male rats, but there was clear evidence of carcinogenicity in female rats. The HECs for extra-respiratory carcinogenic effects adjusted for duration and a blood:gas partition -3 coefficient of 1 are 43, 87, and 173 mg/m , respectively. Griffin et al. (1996) Male and female Long-Evans rats (40/sex/treatment group) were administered nitromethane (96.26%) pure with 2.79% nitroethane and 0.62% 2-nitropropane) at concentrations of 0, 100, or 200 ppm via whole body inhalation for 7 hours a day, 5 days a week, for 2 years (Griffin et al., 1996b). While Griffin et al. (1996) provides a peer-reviewed publication of the data, the proprietary data were also available for review (Coulston International Inc., 1990). See the chronic studies discussed above for details on the materials and methods. There was no treatment-related effect on mortality. There were no statistically significant differences in neoplastic lesions. However, two high-dose females had adenocarcinomas of the uterus, which were not observed in either of the other groups. Because the incidence is low and there is no dose response, it is not clear if this is related to treatment. Based on the absence of either systemic or respiratory carcinogenicity, the NOAEL and LOAEL are selected based on extra-respiratory effects. The HECs for extra-respiratory effects adjusted for duration and a blood:gas partition coefficient of 1 are 45.6 and 89.0 mg/m , respectively. There is no evidence of carcinogenicity. NTP (1997) Individually housed male and female B6C3Fi mice (50/sex/treatment group) were administered nitromethane (purity >98%) at concentrations of 0, 188, 375, or 750 ppm via whole body inhalation for 6 hours and 12 minutes a day, 5 days a week, for 103 weeks. Chamber concentrations were routinely checked over the course of exposure. Animals were observed 21 Nitromethane ------- FINAL 7-26-2013 twice a day for clinical signs of toxicity. Animals were weighed at study initiation, weekly through Week 12, monthly from Week 15 through Week 91, then every 2 weeks until study termination, and at study termination. At study termination, animals were necropsied. Complete histopathology was conducted on all animals except that spinal cord and sciatic nerve were not examined. Typical NTP statistical methods were applied and were appropriate for the data. There was no treatment-related effect on mortality. Clinical signs observed included swelling around the eyes and exophthalmos. This was stated to be consistent with Harderian gland neoplasms, which occurred at a greater incidence in treated animals. There were no statistically significant changes in body weight compared with controls. There were increases in the incidence of Harderian gland tumors in both sexes, liver tumors in females, and alveolar/bronchiolar carcinomas in males (see Table B.19). Respiratory and systemic tumors developed; therefore, HECs were calculated using both the respiratory and extra-respiratory methods respectively, for respiratory tumors (adjusted for duration and multiplied by a RGDR) and for extra-respiratory tumors (duration-adjusted and multiplied by a blood:gas partition coefficient of 1) (U.S.EPA, 1994b). The HECs for respiratory carcinogenic effects are 359; 727; and 1,484 mg/m3 in males and 355; 700; and 1,423 mg/m3 in females, respectively, based on the area of the thoracic region and average body weights provided in the study report. The HECs for extra-respiratory carcinogenic effects are 87, 173, and 346 mg/m3, respectively. The study authors concluded that there was clear evidence of carcinogenicity in both sexes of mice. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) The genotoxicity of nitromethane has been studied in several in vitro test systems (see Table 4A). Little information on the toxicokinetics of nitromethane is available (Coulston International Inc., 1990; Sakurai et al., 1980) and is presented in Table 4B. Further in-depth details follow Tables 4A and 4B. 22 Nitromethane ------- FINAL 7-26-2013 Table 4A. Summary of Nitromethane Genotoxicity Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation Genotoxicity studies in prokaryotic organisms Reverse mutation Salmonella typhimurium strains TA1537, TA1535, TA100, TA1538, and/or TA98 with or without S9 metabolic activation 50,000 ng/plate Dellarco and Prival (1989) also used flavin mononucleotide in a modified preincubation assay to facilitate nitro reduction and found negative results; some pages of the Haskell Laboratories study illegible; 0.5% stated to be half the 50% survival concentration (Dow Chemical, 1975a) Dow Chemical (1975a); Haskell Laboratories (2000); Chiuetal. (1978); Lofroth et al., (1986) Mortelmans et al. (1986); Dellarco and Prival (1989); NTP (1997); Gockeetal. (1981a) Reverse mutation Preincubation modification of Salmonella typhimurium strains TA98, TA100, TA102 200 |imol/platc Concentrations >500 |imol/platc were cytotoxic and could not be used Dayal et al. (1989a) Reverse mutation Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 with or without S9 activation 50 |iL/platc + + Mutagenic in TA1535 only Ong et al. (1980) SOS repair induction ND Genotoxicity studies in nonmammalian eukaryotic organisms Mutation Saccharomyces cerevisiae strain D4 with and without metabolic activation 5% - - 5% stated to be half the 50% survival concentration Dow Chemical (1975b) Recombination induction ND 23 Nitromethane ------- FINAL 7-26-2013 Table 4A. Summary of Nitromethane Genotoxicity Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation Chromosomal aberration The Base test using Drosophila melanogastex Berlin K (wild type) and Base strains 125 mM 2% ethanol used as the solvent Gocke et al. (1981b) Chromosomal malsegregation ND Mitotic arrest ND Genotoxicity studies in mammalian cells—in vitro Mutation ND Chromosomal aberration Micronucleus assay of Syrian hamster embryo cells In DMSO: 6.0 ng/mL In media: 5,000 ng/mL Reduction in cell numbers in DMSO at 5.5 and 6.0 ng/mL; no increased frequency in micronuclei Gibson etal. (1997); Hazelton Washington (1996) Chromosomal aberration Transformation assay using Syrian hamster embryo cells 4,000 iig/mL + + Dose-related increase in the number of morphological transformed colonies; cells incubated for 24 hr prior to application of nitromethane; incubation with nitromethane lasted 24 hr Kerckaert et al. (1996); Brauninger (1995) Chromosomal aberration Abs test in Chinese hamster ovary cells with or without S9 metabolic activation 5,000 |ig/mL — — No induction of chromosome aberrations NTP (1997) Sister chromatid exchange (SCE) Chinese hamster ovary cells with or without S9 metabolic activation 5,000 |ig/mL - - No induction of SCE NTP (1997) DNA damage ND DNA adduct ND 24 Nitromethane ------- FINAL 7-26-2013 Table 4A. Summary of Nitromethane Genotoxicity Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation Genotoxicity studies in mammals—in vivo Chromosomal aberration Subchronic inhalation exposure of male and female mice 1,500 ppmfor 13 wk NA No increased frequency of micronucleated erythrocytes in peripheral blood samples NTP (1997) Chromosomal aberration Micronucleus test on mouse bone marrow 1,830 mg/kg at 0 and 24 hr - NA All treated mice survived; animals sacrificed at 30 hr Gocke et al. (1981c) Sister chromatid exchange (SCE) ND DNA damage ND DNA adduct ND Mouse biochemical or visible specific locus test ND Dominant lethal ND Genotoxicity studies in subcellular systems DNA binding ND aLowest effective dose for positive results, highest dose tested for negative results. b+ = positive; ± = equivocal or weakly positive; ~ = negative; T = cytotoxicity; NA = not applicable; ND = no data; NDr = not determined; NR = not reported; NR/Dr = not reported but determined from data. 25 Nitromethane ------- FINAL 7-26-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Carcinogenicity other than oral/inhalation ND Other toxicity studies (exposures other than oral or inhalation) Male and female BALB/c mice, hepatoxicity test via intraperitoneal injection at 4.5, 6.7, or 9.0 mmol/kg; control mice injected with NaCl; animals terminated at 24, 48, 72, or 96 hr after dosing; blood assays for sorbitol dehydrogenase, alanine aminotransferase, and aspartate aminotransferase No abnormalities found in the enzyme activities for mice dosed with 9 mmol/kg Not toxic at 9 mmol/kg after 24, 48, 72, or 96 hr Dayal et al. (1989b) Short-term studies Rabbit, acute exposure (80 min) at 8 mg; no information regarding control or study methods provided No significant effect on blood pressure or respiration No significant effect on blood pressure or respiration Machle and Scott (1943) Metabolism/ toxicokinetic Two adult female rhesus monkeys; single dermal exposure (300 |iL ether/ethanol solution, 5.5% 14C-nitromethane) for 12 h. No signs of toxicity reported; levels of nitromethane recovered in urine, feces, blood; absorption in skin considered to be low Not hazardous to human skin Coulston International Inc. (1990) Nitromethane aerobically incubated with liver microsomes isolated from male Sprague-Dawley rat livers treated with phenobarbital or 3-methylcholanthrene Formaldehyde produced from nitromethane via NADPH-independent reaction; nitromethane did not result in a substrate-binding difference spectrum in the oxidized rat liver microsomal suspension, but peaked at 437 nm, indicating cytochrome P450-NO complex production Nitrite, formaldehyde, and cyclohexanone are the major reaction products of nitromethane in activated rat liver microsomes in the presence of NADPH and dioxygen; study authors speculated that a special form of cytochrome P450 was necessary to cleave the C-N bond Sakurai et al. (1980) Mode of action/ mechanistic ND Immunotoxicity ND Neurotoxicity ND ND = no data. 26 Nitromethane ------- FINAL 7-26-2013 Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity As seen in Table 4A, the majority of the genotoxicity (e.g., clastogenicity, mutagenicity) studies were negative. There was only one study (Ong et al., 1980) using the Ames assay that found a positive result in one strain (TA 1535) of Salmonella typhimurium. Negative results were also observed in Saccharomyces cerevisiae (Dow Chemical, 1975) and Drosophila melanogaster (Gocke et al., 1981). Chromosomal aberrations were not observed in Syrian hamster embryo cells using the micronucleus assay (Gibson et al., 1997; Hazleton Washington, 1996) but were observed as a dose-related increase in morphological-transformed colonies in the transformation assay (Kerkaert et al., 1996; Brauninger, 1995). Chromosomal aberrations were not observed in Chinese hamster ovary cells using either an Abs test or sister chromatid exchange test (NTP, 1997). Chromosomal aberrations were also not observed in mouse bone marrow using the micronucleus test (NTP, 1997; Gocke et al., 1981). Other Toxicity Studies (Exposures Other Than Oral or Inhalation) Dayal et al. (1989b) compared the potential hepatotoxicity of 2-nitropropane, nitromethane, and nitroethane following intraperitoneal injection. Biochemical and histopathological effects in male and female BALB/c mice were assessed following intraperitoneal injection of nitromethane at doses 0, 4.5, 6.7, or 9.0 mmol/kg in saline. Animals were sacrificed 24, 48, 72, or 96 hours after dosing, and blood was collected via cardiac puncture. Assays were performed for sorbitol dehydrogenase, alanine aminotransferase, and aspartate aminotransferase enzymes. Livers were investigated for biochemical effects. No liver abnormalities or other toxicity were reported in mice after treatment with 9-mmol/kg nitromethane. Results were not reported for the other doses. Short-Term Studies Machle and Scott (1943) examined the blood pressure and respiration of rabbits when exposed to nitromethane and other mononitroparaffins at 8 mg for 80 minutes. Although details of the study were lacking, the study authors reported no significant effect on rabbit blood pressure or respiration. Metabolism/Toxicokinetic Studies In a toxicokinetic study, Coulston International Inc. (1990) conducted a skin absorption study on two female rhesus monkeys. Animals were administered a single dose of 5.5% 14C-nitromethane (in a 300-|iL ether/ethanol solution) to shaved intact skin on the back for 12 hours. The dosed area was occluded with a plastic foil patch and taped until removal at the end of the 12-hour dosing period. Upon removal of the application patch, the skin was cleaned with acetone and soap to remove any remaining test material. The patch and swab were extracted with ethanol and acetone, respectively, and then the extracts were assayed for radioactivity. Blood, urine, and feces were collected while animals were held in metabolism cages for 72 hours after dosing and assayed for radioactivity. Skin and subcutaneous fat were removed and assayed for radioactivity. Skin samples were examined histologically. No toxicity was reported by the study authors. Feed and water consumption as well as feces and urine output were low during the first 12 hours but returned to normal after animals were returned to their cages. No change in body weight was reported. Skin samples did not reveal any sign of skin damage or irritation. The study authors considered the level of radioactivity detected in blood plasma (37.8 ng/mL and 40.3 ng/mL average maximum per 1 mL blood plasma) and 27 Nitromethane ------- FINAL 7-26-2013 erythrocytes (40.8 ng/g and 44.3 ng/g maximum) of the two monkeys to be low. Due to the low level of absorption after a high concentration level, study authors concluded that nitromethane is not hazardous to human skin. Sakurai et al. (1980) investigated the metabolism of nitromethane in liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and dioxygen (O2). Male Sprague-Dawley rats were pretreated with either phenobarbital (80 mg/kg for 3 days) or 3-methylcholanthrene (20 mg/kg for 2 days). The microsomal fraction was then isolated from the livers of the pretreated rats and incubated in the presence of NADPH and dioxygen. The results of the incubation indicated a linear production of nitrite and formaldehyde. Cyclohexanone was also identified as a reaction product of nitromethane. The specific activity (±S.E.M) for denitrification of nitromethane by rat liver microsomal monooxygenases was reported as 0.2 ±0.1 nmol/mg protein-min. The study authors reported that the addition of nitromethane to oxidized rat liver microsomal suspensions resulted in a peak of 437 nm, which is outside the normal substrate-binding spectrum. Further, the study authors explained the peak as the formation of the cytochrome P450-NO complex. Additionally, the study authors also reported that the oxidized rat liver microsomes produced formaldehyde from nitromethane in an independent NADPH reaction. Mode-of-Action/Mechanistic Studies There is insufficient information to determine the mode of action. Immunotoxicity There is no suitable information to provide in this regard. Neurotoxicity There is no suitable information to provide in this regard. DERIVATION OF PROVISIONAL VALUES Tables 5 and 6 present a summary of noncancer reference and cancer values, respectively, for nitromethane. IRIS data are indicated in the tables, if available. 28 Nitromethane ------- FINAL 7-26-2013 Table 5. Summary of Noncancer Reference Values for Nitromethane (CASRN 75-52-5) Toxicity Type (Units) Species/Sex Critical Effect p-Reference Value POD Method POD UFC Principal Study Subchronic p-RfD (mg/kg-d) NDr Chronic p-RfD (mg/kg-d) NDr Subchronic p-RfC (mg/m3) Mouse/F Hyaline droplets in the respiratory epithelium 4 x 10"3 BMCL10 1.31 300 NTP (1997) Chronic p-RfC (mg/m3) Mouse/F Hyaline degeneration in the respiratory epithelium 5 x 10"3 BMCL10 1.60 300 NTP (1997) NDr = not determinable. Table 6. Summary of Cancer Values for Nitromethane (CASRN 75-52-5) Toxicity Type Species/Sex Tumor Type Cancer Value Principal Study p-OSF NDr p-IUR Rat/F Combined mammary gland fibroadenoma, adenoma, and carcinoma 8.8 x HP' (mg/m3)-1 NTP (1997) NDr = not determinable. DERIVATION OF ORAL REFERENCE DOSES Derivation of Subchronic and Chronic Provisional RfD (p-RfD) No subchronic or chronic p-RfD value can be derived because no adequate, well-described studies are available. Justification There is a single subchronic oral study available for nitromethane (Weatherby, 1955). The lowest dose administered (i.e., 0.1%) caused mortality in 4 of the 10 animals tested. Due to cannibalism of the dead animals, it was not possible to determine cause of death. Because the lowest dose administered was considered an FEL, it is not possible to use this study to derive a p-RfD. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS Derivation of Subchronic Provisional RfC (Subchronic p-RfC) There are several studies available for the derivation of the subchronic p-RfC (see Table 7). The study by NTP (1997) in mice is selected as the principal study. This study was peer reviewed and GLP compliant. NTP (1997) examined numerous endpoints in both sexes in 29 Nitromethane ------- FINAL 7-26-2013 two species (i.e., rats and mice). Details are provided in the "Review of Potentially Relevant Data" section. Among the available and acceptable studies, the NTP (1997) subchronic study in mice represents the lowest POD for developing a subchronic p-RfC. The critical endpoint is hyaline droplets in the respiratory epithelium of the nasal turbinates in female mice. The NTP (1997) studies included histopathologic evaluations of the upper respiratory tract, additionally, histopathological lesions of the nasal tract were observed in rats and mice after 16 days (NTP, 1997) and 13 weeks (1997), with possible progression noted in the 2-year studies (NTP, 1997). The hyaline droplets in the respiratory epithelium are selected as the critical effect because, in addition to being the most sensitive effect in the most sensitive species (mice), it was a lesion of progressing severity. Hyaline droplets of the respiratory epithelium were not noted after 16 days of exposure but were observed after 13 weeks of exposure and progressed to degeneration of the respiratory epithelium after 2 years of exposure. In addition, the severity of the hyaline droplets was stated to increase with concentration, ranging from mild in the lower concentrations to moderate at the highest concentration. Degeneration of the olfactory epithelium also occurred at the same concentrations as hyaline droplets in the respiratory epithelium. Benchmark dose (BMD) analysis has been conducted on both endpoints (hyaline droplets and degenerative lesions) only in mice; these same nasal lesions were also observed in the rats but occurred at greater concentrations. The hyaline droplets in the respiratory epithelium have a lower BMCL. However, this endpoint models better when the highest two concentrations are dropped due to maximal response observed in the top three concentrations. Also occurring at higher nitromethane concentrations was anemia noted by decreased hematocrit and hemoglobin levels as well as other hematological effects, effects to the thyroid (increased thyroid weight and decreased thyroxin levels), and other histological lesions in both species. Relative liver weights were increased in female mice after 16 days of exposure at the same concentration that caused the nasal effects (i.e., 188 ppm). BMD analyses have not been conducted on these endpoints. Histopathology in the 16-day rat and mouse studies and the 13-week rat study was not amenable to BMD analysis due to unsuitable dose-response data. 30 Nitromethane ------- FINAL 7-26-2013 Table 7. Summary of Relevant Inhalation Subchronic Toxicity Studies for Nitromethane Reference Species, #/Sex (M/F) Exposure (ppm) Frequency/ Duration NOAEW (mg/m3) LOAELAI)lb (mg/m3) Critical Endpoint NTP (1997) Rat, 5/5 0; 94; 188; 375; 750; 1,500 6.2 hr, 5 d/wk, 16 d 8.9 17.7 Degeneration of the olfactory epithelium of the nasal turbinates in females NTP (1997) Mouse, 5/5 0; 94; 188; 375; 750; 1,500 6.2 hr, 5 d/wk, 16 d 10.2 20.2 Degeneration of the olfactory epithelium of the nasal turbinates in females NTP (1997) Rat, 10/10 0; 94; 188; 375; 750; 1,500 6.2 hr, 5d/wk, 13 wk 9.7 19.2 Degeneration of the olfactory epithelium of the nasal turbinates in females NTP (1997) Mouse, 10/10 0; 94; 188; 375; 750; 1,500 6.2 hr, 5 d/wk, 13 wk 5.9 11.8 Degeneration of the olfactory epithelium and hyaline droplets in the respiratory epithelium of the nasal turbinates in females Lewis et al. (1979) Rat, 10/0 0, 98, 745 7 hr/d, 5 d/wk, 3 mo 51 388 Decreased hematocrit and hemoglobin levels Lewis (1979) Rabbit, 5/0 0, 98, 745 7 hr/d, 5 d/wk, 3 mo 51 388 Decreased thyroxin levels "NOAELA|,[ is the HEC value for respiratory or extra-respiratory effects; HECresp = ppm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed total days) x RGDR; RGDRs were based on average body weights from the study reports and the surface area of the respiratory area affected; HECexresp = PPm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed ^ total days) x blood:gas partition coefficient. YOAELadj is the HEC value for respiratory or extra-respiratory effects; HECresp = ppm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed total days) x RGDR; RGDRs were based on average body weights from the study reports and the surface area of the respiratory area affected; HECexresp = PPm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed ^ total days) x blood:gas partition coefficient. 31 Nitromethane ------- FINAL 7-26-2013 The following dosimetric adjustments have been made for inhalation with an EXPOSURE,,,,,,, for respiratory effects. The RGDR is the ratio of the respiratory minute volumes divided by the surface areas of the extra-thoracic pulmonary region (where the effect occurred) in mice and humans, respectively, and is used in calculating the HEC (U.S. EPA, 1994b). The example shown below is for the second lowest dose. (EXPOSUREhec, extra-thoracic)n = PPm x (ppm conversion) x (average daily dose) x RGDR = ppm x (MW ^ 24.45) x ([hours exposed ^ 24 hours] x [days exposed per week study days per week]) x RGDR = 188 x (61.04 -h 24.45) x (6.2 - 24) x (5 - 7) x 0.136 = 11.8 mg/m3 Where: RGDR = (Ve SA)a ^ (Ve ^ SA)h; Ve = minute volume (m3/day); SA = surface area of the extra-thoracic region (m ); calculated using average body weights from the study report = (0.0408 -h 0.0003) -h (20 - 0.02) = 0.136 All available dichotomous models in the EPA Benchmark Dose Software (BMDS version 2.1.2) have been fit to the data on hyaline droplets in the respiratory epithelium in female mice following exposure to nitromethane for 13 weeks (see Table 8). A benchmark response (BMR) of 10% extra risk above the control mean has been used to estimate the BMD (U.S. EPA, 2010). Table 8. Incidence Data for Female B6C3Fi Mice Treated with Nitromethane for 13 Weeks—Used for BMD Analysis" (PPM)n (EXPOSUREhec extra-thoracic)n (mg/m ) Number of Subjects Response 0 0 10 0 94 5.9 10 2 188 11.8 10 9** 375 24.9 10 10** 750 48.0 10 10** 1500 93.9 10 10** aNTP (1997). **Statistically significant difference from control (p < 0.01) using Fisher's exact test as performed by the study authors. Table 9 summarizes the BMDS model results for the respiratory epithelium hyaline droplet data in female mice. The curve and BMDS output for the selected model only are provided in Appendix C. The BMDS Multistage model with a benchmark concentration lower 32 Nitromethane ------- FINAL 7-26-2013 "3 bound 95% confidence interval BMCLiohec, extra-thoracic of 1.31 mg/m is selected. For the models that provided adequate fit (using goodness-of-fittest;p> 0.1), the Akaike Information Criterion (AIC) values are close, and the BMCLs are within 3-fold of each other; therefore, the model with the lowest AIC is selected. Table 10 summarizes the uncertainty factors applied. Table 9. Model Predictions for Respiratory Epithelial Hyaline Droplets in Female B6C3Fi Mice" Model Goodness-of-Fit p-V alueb AIC for Fitted Model BMC10 (mg/m3) BMCL10 (mg/m3) Conclusions Multistage 1.00 18.556 4.431 1.31 Selected as lowest AIC value for POD with a range of 1.314-3.126 Weibull 1.00 20.510 4.709 2.14 Not selected Probit 0.99 20.549 4.755 2.54 Not selected Logistic 0.96 20.659 4.825 2.73 Not selected Log-logistic 0.98 20.569 5.078 3.11 Not selected Log-probit 1.00 20.517 5.121 3.13 Not selected aNTP (1997). bChi-square p-valuc = /j-value from chi-square test for lack of fit. Values <0.1 fail to meet conventional goodness-of-fit criteria. AIC = Akaike Information Criteria; BMC = benchmark concentration; BMCL lower confidence limit (95%) on the benchmark concentration; BMCi0 = BMC at a response rate of 10% incidence, extra risk. -3 A POD of 1.31 mg/m is determined by BMD analysis. The subchronic p-RfC for nitromethane, based on a BMCLiohec, extra-thoracic of 1.31 mg/m3 in female mice, is derived as follows: Sllbchronic p-RfC BIVICLlOin.C, extra-thoracic ' UFc = 1.31 mg/m3 300 = 4 x 10 3 mg/m3 33 Nitromethane ------- FINAL 7-26-2013 Table 10. Uncertainty Factors for Subchronic p-RfC of Nitromethane UF Value Justification UFa 3 A UFa of 3 is applied for animal-to-human extrapolation to account for the toxicodynamic portion of the UFA because the toxicokinetic portion (100 5) has been addressed in dosimetric conversions. ufd 10 A UFd of 10 is applied because there are no acceptable two-generation reproductive studies or developmental studies by this route of exposure. UFh 10 A UFh of 10 is applied for intraspecies differences to account for potentially susceptible individuals in the absence of information on the variability of response to humans. ufl 1 A UFl of 1 is applied because the POD was developed using a BMCL. UFS 1 A UFS of 1 is applied because a subchronic study was utilized. UFC <3000 300 Composite uncertainty factor The confidence of the subchronic p-RfC for nitromethane is medium as explained in Table 11 below. Table 11. Confidence Descriptors for Subchronic p-RfC for Nitromethane Confidence Categories Designation" Discussion Confidence in study H The study by NTP (1997) provided sufficient details, used sufficient numbers of animals, examined both sexes, and examined numerous endpoints including both portal-of-entry and systemic endpoints. In addition, NTP (1997) examined two species (rats and mice) and conducted short-term studies to determine appropriate doses for the subchronic study. Confidence in database M Although there are several short-term, subchronic, chronic, and carcinogenicity studies available, there are no acceptable reproductive or developmental studies available for nitromethane. Confidence in subchronic p-RfCb M The overall confidence is medium because the confidence in the database is medium. "L = low; M = medium; H = high. bThe overall confidence cannot be greater than lowest entry in the table. Derivation of Chronic Provisional RfC (Chronic p-RfC) There are several studies available for the derivation of the chronic p-RfC (see Table 12). The study by NTP (1997) is selected as the principal study for derivation of the chronic p-RfC. This study was peer reviewed and GLP compliant. NTP (1997) examined numerous endpoints in both sexes of two species (i.e., rats and mice). Details are provided in the "Review of Potentially Relevant Data" section. Among the available and acceptable studies, this study represents the lowest POD for developing a chronic p-RfC. The critical effect is hyaline degeneration of the respiratory epithelium of the nasal turbinates in female mice. Only the NTP 34 Nitromethane ------- FINAL 7-26-2013 (1997) studies included histopathologic evaluations of the upper respiratory tract, but histopathological lesions of the nasal tract were observed in rats and mice after 16 days, 13 weeks, as well as in mice after 2 years (NTP, 1997). As stated previously, the hyaline degeneration of the respiratory epithelium is a progressive lesion with hyaline droplets observed after 13 weeks of exposure in mice. There was also a concentration-related increase in severity of the nasal lesions. Degeneration of the olfactory epithelium also occurred at the same concentration as the hyaline degeneration of the respiratory epithelium. BMD analysis has been conducted on both the degeneration in the olfactory epithelium and the hyaline degeneration in the respiratory epithelium (both in the nasal turbinates) in mice, but hyaline degeneration of the respiratory epithelium is the most sensitive effect. The NTP studies also found signs of anemia including decreased hematocrit and hemoglobin levels and effects on the thyroid (increased thyroid weight and decreased thyroxin levels), but these effects occurred at higher concentrations. Because these effects occurred at higher HECs than the nasal effects observed in the 2-year mouse study, BMD analyses have not been conducted on these endpoints. 35 Nitromethane ------- FINAL 7-26-2013 Table 12. Summary of Relevant Inhalation Chronic Toxicity Studies for Nitromethane Reference Species, #/Sex (M/F) Exposure (ppm) Frequency/ Duration NOAEW (mg/m3) LOAELAI)lb (mg/m3) Critical Endpoint NTP (1997) Rat, 50/50 0, 94, 188, 375 6.2 hr, 5 d/wk, 103 wk 173 NDr No noncancer effects observed, but tumors were observed at 87 mg/m3 NTP (1997) Mouse, 50/50 0, 188, 375, 750 6.2 hr, 5 d/wk, 103 wk NDr 21.9 Increase in nonneoplastic nasal lesions in females Griffin et al. (1996) Rat, 40/40 0, 100, 200 7 hr, 5 d/wk, 2 yr 89.0 NDr No noncancer effects observed Lewis et al. (1979) Rat, 10/0 0, 98, 745 7 hr/d, 5 d/wk, 6 mo 51 388 Increase in relative thyroid weight Lewis et al. (1979) Rabbit, 5/0 0, 98, 745 7 hr/d, 5 d/wk, 6 mo NDr 51 Decreased thyroxin levels :iNOAELA[,j is the HEC value for respiratory or extra-respiratory effects; HECresp = ppm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed total days) x RGDR; RGDRs were based on average body weights from the study reports and the surface area of the respiratory area affected; HECexresp = PPm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed ^ total days) x blood:gas partition coefficient. LO AEL U)[ is the HEC value for respiratory or extra-respiratory effects; HECresp = ppm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed ^ total days) x RGDR; RGDRs were based on average body weights from the study reports and the surface area of the respiratory area affected; HECexresp = PPm x (molecular weight ^ 24.45) x (hours exposed per day ^ 24) x (days dosed ^ total days) x blood:gas partition coefficient. NDr = not determinable. 36 Nitromethane ------- FINAL 7-26-2013 Based on its physical properties, nitromethane is a Category 2 gas. This is supported by the observation of both respiratory (direct contact) and extra-respiratory (systemic) effects in the studies that examined endpoints inclusive of both effects. Therefore, dosimetric conversions are based on the endpoint observed and differ for respiratory and extra-respiratory effects. Note that the RGDR used in dosimetric conversions is different from the subchronic-duration derivation because of the difference in average body weights at 13 weeks versus 103 weeks. The following dosimetric adjustments have been made for inhalation with an EXPOSURE,,,,,,, for respiratory effects. The example shown is for the lowest dose. (EXPOSUREhec, resp)ii = ppm x (ppm conversion) x (average daily dose) x RGDR = ppm x (MW ^ 24.45) x ([hours exposed ^ 24] x [days exposed ^ study duration]) x RGDR = 188 x (61.04 -h 24.45) x (6.2 - 24) x (5 - 7) x 0.254 = 22.0 m«/m3 Where: RGDR = (Ve SA)a ^ (Ve ^ SA)h; Ve = minute volume (m3/day); SA = Surface Area of the extra-thoracic region (m2); calculated using average body weights from the study report = (0.0763 -h 0.0003) - (20 - 0.02) = 0.254 The following dosimetric adjustments have been made for inhalation with an EXPOSUREppm for extra-respiratory effects. Data for calculating a specific blood:gas partition coefficient were not available; therefore, a value of 1 is employed. EXPOSUREhec, exresp — PPm x (PPm conversion) x (average daily dose) x blood:gas partition coefficient = ppm x (MW ^ 24.45) x ([hours exposed ^ 24] x [days exposed ^ study duration]) x blood:gas partition coefficient = 188 x (61.04 -h 24.45) x (6.2 - 24) x (5 - 7) x 1 = 86.6 mg/m3 All available dichotomous models in the EPA Benchmark Dose Software (BMDS version 2.1.2) have been fit to the hyaline degeneration of the respiratory epithelium data in female mice following exposure to nitromethane for 2 years (see Table 13). A BMR of 10% extra risk above the control mean is used to estimate the BMD (U.S. EPA, 2010). 37 Nitromethane ------- FINAL 7-26-2013 Table 13. Incidence Data for Hyaline Degeneration of the Respiratory Epithelium in Female B6C3Fi Mice Treated with Nitromethane for 2 Years—Used for BMDS Analysis3 (PPM)n (EXPOSUREhec,resp) (mg/m3) Number of Subjects Response 0 0 50 16 188 22.0 49 39** 375 43.9 50 50** 750 87.8 50 50** aNTP (1997). **Statistically significant difference from control (p < 0.01) using Fisher's exact test as performed by the study authors. Table 14 summarizes the BMDS model results for the data in female mice. The curve and BMDS output for the selected model only are provided in Appendix C. The BMDS Multistage model with a BMCLiohec, resp of 1.60 mg/m3 is selected. Following EPA guidance (U.S. EPA, 2012), for the models that provided an adequate fit (using goodness-of-fit test; p > 0.1), the AIC values are close, but the BMCLs are not within 3-fold of each other; therefore, the model with the lowest BMCL is selected. Table 15 summarizes the uncertainty factors applied. Table 14. Model Predictions for Hyaline Degeneration of the Respiratory Epithelium in Female B6C3Fi Mice" Model Goodness-of-Fit p-V alueb AIC for Fitted Model BMC10 (mg/m3) BMCL10 (mg/m3) Conclusion Multistage 1.00 116.283 9.734 1.60 Selected as lowest BMCL value for POD with a range of 1.602-8.373 Logistic 0.47 118.672 2.810 2.19 Not selected Probit 0.66 117.579 2.885 2.33 Not selected Weibull 1.00 118.276 11.437 2.90 Not selected Log-probit 1.00 118.276 17.188 6.85 Not selected Log-logistic 1.00 116.276 18.523 8.37 Not selected aNTP (1997). bChi-square p value = p-valuc from chi-square test for lack of fit. Values <0.1 fail to meet conventional goodness- of-fit criteria. AIC = Akaike Information Criteria; BMC = benchmark concentration; BMCL lower confidence limit (95%) on the benchmark concentration; BMCio = BMC at a response rate of 10% incidence, extra risk. 38 Nitromethane ------- FINAL 7-26-2013 The POD in this study is a BMCLiohec, resp of 1.60 mg/m3. The chronic p-RfC for nitromethane, based on a BMCLiohec, resp of 1.60 mg/m3 in female mice, is derived as follows: Chronic p-RfC = BMCLiohec, resp UFc = 1.60 mg/m3 300 = 5 x 10 3 mg/m3 Table 15. Uncertainty Factors for Chronic p-RfC of Nitromethane UF Value Justification ufa 3 A UFa of 3 is applied for animal-to-human extrapolation to account for the toxicodynamic portion of the UFA because the toxicokinetic portion (100 5) has been addressed in dosimetric conversions. ufd 10 A UFd of 10 is applied because there are no acceptable two-generation reproductive studies or developmental studies by this route of exposure. UFh 10 A UFh of 10 is applied for intraspecies differences to account for potentially susceptible individuals in the absence of information on the variability of response to humans. ufl 1 A UFl of 1 is applied for using a POD based on a BMCL. UFS 1 A UFS of 1 is applied because a chronic study was utilized. UFC <3000 300 Composite uncertainty factor The confidence of the chronic p-RfC for nitromethane is medium as explained in Table 16 below. Table 16. Confidence Descriptors for Chronic p-RfC for Nitromethane Confidence Categories Designation" Discussion Confidence in study M The study by NTP (1997) provided sufficient details, used sufficient numbers of animals, examined both sexes, and examined numerous endpoints including both portal-of-entry and systemic endpoints. In addition, NTP (1997) examined two species (rats and mice) and selected doses based on shorter duration studies. However, the lowest dose tested was a LOAEL. Confidence in database M Although there are several short-term, subchronic, chronic, and carcinogenicity studies available, there are no reproductive or developmental studies available for nitromethane. Confidence in chronic p-RfCb M The overall confidence is medium because the confidence in the database is medium. aL = low; M = medium; H = high. bThe overall confidence cannot be greater than the lowest entry in table. 39 Nitromethane ------- FINAL 7-26-2013 CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR Table 17 identifies the cancer WOE descriptor for nitromethane. As there are statistically significant increases in tumors in more than one species (rats and mice) and in both sexes of mice, the cancer WOE descriptor is "Likely to Be Carcinogenic to Humans " (U.S. EPA, 2005) for the inhalation route of exposure. Because no carcinogenicity studies on nitromethane via the oral route of exposure have been identified, the cancer WOE descriptor for the oral route is "Inadequate Information to Assess Carcinogenic Potential". Table 17. Cancer WOE Descriptor for Nitromethane Possible WOE Descriptor Designation Route of entry (oral, inhalation, or both) Comments "Carcinogenic to Humans " NS NA No human data has been identified. "Likely to Be Carcinogenic to Humans" Selected Inhalation NTP (1997) determined evidence of carcinogenicity in female F344 rats and both sexes of B6C3F, mice. "Suggestive Evidence of Carcinogenic Potential" NS NA Evidence points more towards likely to be carcinogenic. "Inadequate Information to Assess Carcinogenic Potential" Selected Oral No carcinogenicity studies via the oral route of exposure have been identified. "Not Likely to Be Carcinogenic to Humans " NS NA Animal data indicate this is not true. NS = not selected; NA = not applicable. MODE-OF-ACTION DISCUSSION There are insufficient data to clearly define a mutagenic mode of action. Therefore, a linear approach is applied. DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of Provisional Oral Slope Factor (p-OSF) The lack of oral data on the carcinogenicity of nitromethane precludes the derivation of quantitative estimates for oral (p-OSF) exposure. Derivation of Provisional Inhalation Unit Risk (p-IUR) The study in rats by NTP (1997) is selected as the principal study for derivation of the p-IUR. The cancer endpoint is combined mammary gland fibroadenoma, adenoma, and carcinoma in females. While the NTP study in mice provided similar data, the dose response was clearer in rats. This study was peer reviewed, GLP compliant, and otherwise met the standards of study design and performance. Details are provided in the "Review of Potentially Relevant Data" section. Table 18 provides the data that have been applied to the BMDS model. 40 Nitromethane ------- FINAL 7-26-2013 Among the available, acceptable studies, this study represents the highest IUR from the relevant studies in the database. The following dosimetric adjustments have been made for inhalation treatment in adjusting exposures for cancer analysis. Since the endpoint (mammary gland tumors) is outside the respiratory tract, the HEC is calculated based upon extra-respiratory effects. Because the rat/human blood gas partition coefficient ratio was not available, the default value of 1 was used (U.S. EPA 1994b). The example shown is the for the low dose. (EXPOSUREhec, exresp)ii = PPm x (ppm conversion) x (average daily dose) x blood:gas partition coefficients = ppm x (MW ^ 24.45) x ([hours exposed ^ 24] x [days exposed ^ study duration]) x blood:gas partition coefficient) = 94 x (61.04 -h 24.45) x (6.2 - 24) x (5 - 7) x 1 = 43.3 mg/m3 Table 18. Incidence Data for Female Sprague-Dawley Rats Treated with Nitromethane for 2 Years—Used for BMD Analysis" (PPM)n (EXPOSUREhec, exresp)ii (mg/m3) Number of Subjects Response 0 0 50 21 94 43.3 50 25 188 86.6 50 34** 375 172.8 50 41** aNTP (1997). **Statistically significant difference from control (p < 0.01) using Fisher's exact test as performed by study authors. Table 19 shows the modeling results. Adequate model fit is obtained for the combined mammary gland fibroadenoma, adenoma, and carcinoma data in female rats using the BMDS version 2.1.2 multistage-cancer model (U.S. EPA, 2010). A BMCiohec exresp of 20.4 mg/m3 "3 ' and a BMCLiohec, exresp of 11.3 mg/m are calculated from BMD modeling with 10% extra risk for combined mammary gland fibroadenoma, adenoma, and carcinoma (see Table 19). The curve and BMDS output for the selected model only is provided in the BMD supplement (see Appendix C); Table C. 1 provides the IURs for the various endpoints modeled. 41 Nitromethane ------- FINAL 7-26-2013 Table 19. Model Predictions for Combined Mammary Gland Fibroadenoma, Adenoma, and Carcinoma in Females Rats a Goodness-of-Fit AIC for Fitted BMClOHEC, EXRESP BMCLiOHEC, EXRESP Model p-V alueb Model (mg/m3) (mg/m3) Conclusions Multistage- 0.49 253.652 20.4 11.3 Lowest BMCL Cancer aNTP (1997). bValues <0.10 fail to meet conventional goodness-of-fit criteria. AIC = Akaike's Information Criterion; BMC = benchmark concentration; BMCL = lower confidence limit (95%) on the benchmark concentration; BMCio = BMC at a response rate of 10% incidence, extra risk. HEC = Human Equivalent Concentration; EXRESP = HEC determined using the extra-respiratory method (U.S. EPA, 1994b) p-IUR - 0.1a BMCLiOHEC, EXRESP = 0.1 -h 11.3 m«/m3 = 8.8 x 10 3 (mg/m3)"1 aThe value 0.1 is the benchmark response/concentration (U.S. EPA 1994b, 2012). 42 Nitromethane ------- FINAL 7-26-2013 APPENDIX A. PROVISIONAL SCREENING VALUES No screening values for nitromethane are derived. 43 Nitromethane ------- FINAL 7-26-2013 APPENDIX B. DATA TABLES Table B.l. Mean Body Weights of Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 16 Days3 Parameter Exposure Group, ppm (HEC, mg/m3)b Male 0 94 (46) 188 (91) 375 (181) 750 (363) 1500 (726) Sample size 5 5 5 5 5 5 Weight0 Initial 145 ±4 147 ±4 (101) 146 ±4 (101) 145 ± 3 (100) 144 ± 3 (99) 146 ±3 (101) (g) Final 182 ±4 189 ± 5 (104) 187 ±5 (103) 182 ±6 (100) 177 ± 4 (97) 171 ±4 (94) Change 38 ±2 43 ±2 (113) 41 ±2 (108) 37 ± 3 (97) 34 ± 1 (89) 25 ±3** (66) Female 0 94 (46) 188 (91) 375 (181) 750 (363) 1500 (726) Sample size 5 5 5 5 5 5 Weight0 Initial 116 ± 2 116 ±2 (100) 116 ±2 (100) 116 ±2 (100) 117 ±2 (101) 117 ±2 (101) (g) Final 134 ±3 135 ±3 (101) 133 ±2 (99) 133 ±2 (99) 132 ± 1 (99) 128 ± 2 (96) Change 17 ± 1 18 ±3 (106) 17 ± 1 (100) 18 ±2 (106) 15 ± 2 (88) 11 ± 1 (65) "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Weights expressed as mean ± SE (% of control). Percent change from control in brackets. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by the study authors. 44 Nitromethane ------- FINAL 7-26-2013 Table B.2. Liver and Kidney Weights of Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 16 Days" Parameter Exposure Group, ppm (HEC, mg/m3)b Male 0 94 (46) 188 (91) 375 (181) 750 (363) 1500 (726) Sample size 5 5 5 5 5 5 Necropsy body weight0 (g) 218 ±3 225 ± 5 (103) 223 ± 6 (102) 215 ±7 (99) 206 ± 4 (94) 192 ±4 (88)** Liver weight0 Absolute (g) 8.922 ± 0.201 9.950 ±0.250 (112) 9.794 ±0.303 (110) 9.988 ±0.393 (112) 9.028 ±0.144 (101) 9.322 ±0.346 (104) Relative (mg/g) 40.98 ± 0.55 44.29 ±0.71 (108)** 43.87 ±0.66 (107)** 46.50 ±0.68 (113)** 43.84 ±0.63 (107)** 48.49 ±0.99 (118)** Right kidney weight0 Absolute (g) 0.852 ± 0.014 0.878 ±0.011 (103) 0.904 ± 0.029 (106) 0.870 ±0.037 (102) 0.854 ±0.016 (100) 0.880 ± 0.022 (103) Relative (mg/g) 3.91 ± 0.02 3.91 ±0.08 (100) 4.05 ±0.05 (104) 4.05 ±0.07 (104) 4.14 ±0.02 (106)* 4.58 ±0.08 (117)** Female 0 94 (46) 188 (91) 375 (181) 750 (363) 1500 (726) Sample size 5 5 5 5 5 5 Necropsy body weight0 (g) 146 ±2 148 ±3 (101) 147 ±2 (101) 146 ± 2 (100) 143 ± 2 (98) 137 ± 3 (94) Liver weight0 Absolute (g) 5.240 ± 0.182 5.472 ±0.193 (104) 5.578 ±0.187 (106) 5.750 ±0.160 (110)* 5.832 ±0.067 (111)* 6.204 ±0.118 (118)** Relative (mg/g) 35.95 ± 0.86 36.87 ±0.62 (103) 37.88 ±0.82 (105) 39.28 ± 1.09 (109)** 40.72 ± 0.22 (113)** 45.30 ±0.53 (126)** Right kidney weight0 Absolute (g) 0.612 ± 0.022 0.616 ±0.019 (101) 0.634 ±0.013 (104) 0.614 ±0.007 (100) 0.636 ±0.007 (104) 0.660 ± 0.009 (108) Relative (mg/g) 4.20 ± 0.13 4.15 ±0.05 (99) 4.31 ±0.08 (103) 4.19 ±0.04 (100) 4.44 ± 0.06 (106) 4.82 ±0.10 (115)** "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Weights expressed as mean ± SE. Percent change from control in brackets. * Significantly different from control (p < 0.05); Williams' or Dunnett's test performed by the study authors. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by the study authors. 45 Nitromethane ------- FINAL 7-26-2013 Table B.3. Histopathological Findings for Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 16 Days" Parameter Exposure Group, ppm (HEC, mg/m3) Male 0 94 (5.7)b 188 (11.2) 375 (22.1) 750 (43.5) 1500 (86.1) Nose/Turbinates, degeneration, olfactory epithelium0 0/5 (0) 0/5 (0) 0/ 5 (0) 5/5** (100; 1.0) 5/5** (100; 2.0) 5/5** (100; 2.0) Male 0 94 (46)d 188 (91) 375 (181) 750 (363) 1500 (726) Sciatic nerve degeneration0 0/4 (0) 0/5 (0) 0/5 (0) 5/5** (100; 1.0) 5/5** (100; 2.0) 5/5** (100; 3.0) Female 0 94 (4.5)b 188 (8.9) 375 (17.7) 750 (35.3) 1500 (69.7) Nose/Turbinates, degeneration, olfactory epithelium0 0/5 (0) 0/5 (0) 0/ 5 (0) 4/5* (80; 1.0) 5/5** (100; 1.8) 5/5** (100; 2.0) Female 0 94 (46)d 188 (91) 375 (181) 750 (363) 1500 (726) Sciatic nerve degeneration0 0/5 (0) 0/5 (0) 0/5 (0) 5/5** (100; 1.0) 5/5** (100; 2.0) 5/5** (100; 3.0) "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day ^ 24) x (days dosed ^ total days) x RGDR; body weights used to determine the RGDR were calculated as (initial body weight + final body weight) 2. 0 Values expressed as number observed with neoplasm/number examined for that neoplasm (% incidence; average severity of lesions 1 = minimal, 2 = mild, 3 = moderate, 4 = marked). dDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. * Significantly different from control (p < 0.05); Fischer exact test performed by the study authors. **Significantly different from control (p < 0.01); Fischer exact test performed by the study authors. 46 Nitromethane ------- FINAL 7-26-2013 Table B.4. Liver Weights of Male and Female B6C3Fi Mice After Inhalation Exposure to Nitromethane for 16 Days" Parameter Exposure Group, ppm (HEC, mg/m3)b Male 0 94 (46) 188 (91) 375 (181) 750 (363) 1500 (726) Sample size 5 5 5 5 5 5 Necropsy body weight0 (g) 27.4 ±0.6 29.0 ± 1.0 (106) 28.9 ±0.6 (105) 27.7 ±0.9 (101) 29.2 ±0.7 (107) 28.6 ±0.2 (104) Liver weight0 Absolute (g) 1.376 ± 0.044 1.538 ±0.083 (112) 1.552 ±0.045 (113) 1.526 ±0.078 (HI) 1.752 ±0.081 (127)** 1.680 ±0.053 (122)** Relative (mg/g) 50.15 ± 0.67 52.99 ±2.14 (106) 53.63 ±0.53 (107) 54.96 ± 1.53 (110)* 59.93 ± 1.38 (120)** 58.72 ± 1.61 (117)** Female 0 94 (46) 188 (91) 375 (181) 750 (363) 1500 (726) Sample size 5 5 5 5 5 5 Necropsy body weight0 (g) 23.3 ±0.2 23.7 ±0.3 (102) 23.6 ±0.3 (101) 23.8 ±0.4 (102) 24.7 ±0.4 (106)* 24.0 ±0.4 (103) Liver weight0 Absolute (g) 1.146 ± 0.020 1.256 ±0.035 (110)* 1.338 ±0.037 (117)** 1.364 ±0.047 (119)** 1.442 ±0.020 (126)** 1.410 ±0.044 (123)** Relative (mg/g) 49.24 ± 0.96 53.00 ± 1.40 (108)* 56.77 ± 1.15 (115)** 57.28 ± 1.56 (116)** 58.44 ± 1.18 (119)** 58.70 ±0.90 (119)** "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Weights expressed as mean ± SE (% of control). * Significantly different from control (p < 0.05); Williams' or Dunnett's test performed by the study authors. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by the study authors. 47 Nitromethane ------- FINAL 7-26-2013 Table B.5. Mean Body Weights of Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3)b Male 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 10 10 Weight0 (g) Initial 107 ±3 105 ± 2 (98) 113 ±2 (106) 109 ±3 (102) 106 ± 2 (99) 109 ±2 (102) Final 334 ±7 323 ± 7 (97) 345 ±4 (103) 336 ±5 (101) 327 ± 4 (98) 295 ± 10 (88)** Change 228 ±6 218 ±7 (96) 232 ±3 (102) 227 ± 4 (100) 221 ±5 (97) 185 ±9 (81)** Female 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 10 10 Weight0 (g) Initial 95 ± 1 96 ±2 (101) 97 ± 2 (102) 95 ±2 (100) 96 ±2 (101) 94 ± 2 (99) Final 185 ±5 197 ±3 (106) 197 ±3 (106) 198 ± 5 (107) 194 ±4 (105) 177 ± 4 (96) Change 90 ±3 101 ±2 (112) 100 ±2 (111) 103 ±4 (114)** 97 ±2 (108) 84 ± 3 (93) "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Weights expressed as mean ± SE (% of control). **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by study authors. 48 Nitromethane ------- FINAL 7-26-2013 Table B.6. Select Hematology for Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3)b 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Male Sample size 10 10 10 10 10 10 Hematocrit0 (%) Day 3 36.7 ±0.5 36.3 ±0.3 (99) 35.2 ±0.2 (96)* 33.1 ±0.3 (90)** 31.7 ±0.2 (86)** 32.3 ±0.2 (88)** Day 23 40.7 ± 0.3d 43.2 ±0.9 (106)e 40.4 ±0.4 (99)f 37.6 ±0.4 (92)* 34.0 ±0.4 (84)** 30.3 ±0.4 (74)** Week 13 46.3 ±0.1 46.6 ±0.4 (101) 46.1 ±0.4 (100) 44.6 ±0.3 (96)** 42.5 ±0.4 (92)** 39.2 ±0.4 (85)** Hemoglobin0 (g/dL) Day 3 13.9 ±0.2 13.5 ±0.1 (97) 13.3 ±0.1 (96)* 12.6 ±0.1 (91)** 12.2 ±0.1 (88)** 12.4 ±0.1 (89)** Day 23 15.3 ± 0.2d 16.1 ±0.3 (105)e 15.0 ±0.1 (98)f 14.3 ±0.1 (93)* 13.2 ±0.1 (86)** 11.9 ± 0.1 (78)** Week 13 15.3 ±0.1 15.4 ±0.1 (101) 15.2 ±0.1 (99) 14.8 ±0.1 (97)** 14.3 ±0.1 (93)** 13.4 ±0.2 (88)** Erythrocytes0 (106/|iL) Day 3 7.75 ± 0.10 7.58 ±0.08 (98) 7.38 ±0.08 (95)** 7.16 ±0.07 (92)** 6.97 ±0.04 (90)** 6.94 ±0.06 (90)** Day 23 8.74 ± 0.06d 9.37 ±0.18 (107)*e 9.00 ± 0.07 (103)f 9.36 ±0.09 (107)* 9.10 ±0.09 (104) 7.77 ±0.11 (89) Week 13 9.12 ± 0.03 9.43 ± 0.06 (103)** 9.53 ±0.08 (104)** 9.72 ±0.08 (107)** 10.10 ±0.09 (111)** 9.41 ±0.11 (103)** Nucleated erythrocytes (103/hL) Week 13 0.03 ± 0.02 0.04 ± 0.02 (133) 0.03 ± 0.03 (100) 0.11 ±0.03 (367) 0.08 ±0.03 (267) 0.27 ± 0.07 (900)** MCV° (fL) Day 3 47.2 ±0.3 47.9 ±0.3 (101) 47.7 ±0.4 (101) 46.2 ±0.1 (98)* 45.5 ±0.2 (96)** 46.3 ±0.2 (98)** Day 23 46.5 ± 0.3d 46.0 ±0.2 (99)e 45.0 ±0.4 40.2 ±0.3 (86)** 37.5 ±0.2 (81)** 39.1 ±0.2 (84)** Week 13 50.7 ±0.2 49.3 ±0.2 (97)** 48.4 ±0.2 (95)** 45.8 ±0.4 (90)** 42.0 ±0.6 (83)** 41.6 ±0.3 (82)** MCH° (pg) Day 3 17.9 ±0.1 17.8 ±0.1 (99) 18.1 ± 0.1 (101) 17.6 ±0.0 (98)* 17.5 ±0.1 (98)* 17.9 ±0.1 (100) Day 23 17.5 ± 0.02d 17.2 ±0.1 (98)e 16.7 ±0.1 (95)**f 15.3 ±0.1 (87)** 14.5 ±0.0 (83)** 15.3 ±0.1 (87)** Week 13 16.8 ±0.0 16.3 ±0.1 (97)** 16.0 ±0.0 (95)** 15.2 ±0.1 (90)** 14.1 ±0.2 (84)** 14.3 ±0.1 (85)** MCHC0 (g/dL) Day 3 37.8 ±0.2 37.3 ±0.1 (99) 37.8 ±0.1 (100) 38.1 ±0.1 (101)* 38.4 ±0.2 (103)* 38.6 ±0.1 (102)** Day 23 37.5 ± 0.2d 37.3 ±0.1 (99)e 37.3 ±0.2 (99)f 38.2 ±0.3 (102) 38.8 ±0.2 (103)* 39.3 ±0.2 (105)** Week 13 33.0 ±0.1 33.0 ±0.1 (100) 33.0 ±0.1 (100) 33.2 ±0.1 (101) 33.6 ±0.1 (102)** 34.3 ±0.3 (104)** 49 Nitromethane ------- FINAL 7-26-2013 Table B.6. Select Hematology for Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3)b 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Platelets0 (lo VjiL) Day 3 663.6 ± 15.5 741.9 ± 13.8 (112)** 708.2 ± 13.5 (107)** 732.7 ± 17.8 (110)** 781.2 ± 10.5 (118)** 870.6 ± 16.3 (131)** Day 23 643.8 ± 44. ld 663.4 ±8.8 (103)e 675.0 ± 16.0 (105)f 704.8 ± 19.2 (109) 878.4 ±22.5 (136)** 1325.2 ±24.0 (206)** Week 13 538.3 ± 7.5 527.8 ± 16.7 (98) 539.2 ±5.7 (100) 578.7 ±6.1 (108)** 625.0 ±9.2 (116)** 817.4 ±32.9 (152)** Methemoglobin0 (g/dL) Day 3 0.16 ± 0.02f 0.14 ±0.02 (88)f 0.19 ±0.02 (119)e 0.34 ±0.02 (213)** 0.21 ±0.03 (13l)*8 0.22 ±0.02 (138)*e Day 23 0.08 ± 0.01d 0.06 ±0.01 (75)e 0.08 ±0.01 (100)f 0.16 ±0.06 (200) 0.15 ±0.01 (188)* 0.28 ±0.02 (350)** Week 13 0.15 ± 0.01 0.17 ±0.02 (113) 0.17 ±0.01 (113)* 0.17 ±0.01 (113)* 0.21 ±0.01 (140)** 0.41 ±0.09 (273)** Female Sample size 10 10 10 10 10 10 Hematocrit0 (%) Day 3 38.9 ±0.6 38.7 ±0.3 (99) 38.1 ±0.4 (98) 36.7 ±0.3 (94)** 36.0 ±0.3 (93)** 36.6 ±0.4 (94)** Day 23 42.6 ±0.3 40.5 ±0.9 (95)** 41.1 ±0.5 (96)* 37.9 ±0.4 (89)** 35.2 ±0.3 (83)** 31.7 ±0.2 (74)**e Week 13 46.8 ±0.3 46.6 ±0.4 (100) 44.7 ±0.4 (96)** 44.4 ±0.5 (95)** 40.7 ±0.4 (87)** 37.8 ±0.4 (81)** Hemoglobin0 (g/dL) Day 3 14.9 ±0.2 14.9 ±0.1 (100) 14.6 ±0.2 (98) 14.0 ±0.1 (94)** 13.7 ±0.1 (92)** 14.1 ±0.2 (95)** Day 23 16.2 ±0.1 15.4 ±0.3 (95)** 15.6 ±0.2 (96)* 14.5 ±0.1 (90)** 13.5 ±0.1 (83)** 12.5 ±0.1 (77)**e Week 13 16.0 ±0.1 15.8 ±0.1 (99) 15.3 ±0.1 (96)** 15.3 ±0.1 (96)** 14.1 ±0.1 (88)** 13.4 ±0.2 (84)** Erythrocytes0 (106/|iL) Day 3 8.39 ± 0.15 8.42 ±0.07 (100) 8.34 ± 0.11 (99) 8.10 ±0.09 (97) 7.87 ±0.07 (94)** 8.14 ± 0.11 (97)* Day 23 9.03 ± 0.06 8.86 ±0.18 (98) 9.35 ±0.09 (104) 9.32 ±0.09 (103) 9.14 ±0.09 (101) 8.16 ±0.06 (90)e Week 13 8.71 ± 0.05 8.91 ±0.06 (102) 8.92 ±0.09 (102) 9.42 ±0.07 (108)** 9.24 ± 0.07 (106)** 8.51 ±0.10 (98) Nucleated erythrocytes0 Week 13 0.03 ± 0.01 0.07 ± 0.02 (233) 0.09 ±0.04 (300) 0.06 ± 0.04 (200) 0.22 ±0.09 (733) 0.30 ± 0.11 (1000)** MCV0 (fL) Day 3 46.5 ±0.2 45.8 ±0.2 (98)* 45.6 ±0.3 (98)* 45.3 ±0.2 (97)** 45.7 ±0.2 (98)** 45.0 ±0.2 (97)** Day 23 47.0 ±0.0 45.8 ±0.2 (97)** 43.9 ±0.2 (93)** 40.6 ±0.2 (86)** 38.4 ±0.2 (82)** 38.8 ±0.2 (83)**e Week 13 53.9 ±0.2 52.4 ±0.2 (97)** 50.1 ±0.3 (93)** 47.2 ±0.2 (88)** 44.2 ±0.5 (82)** 44.4 ±0.4 (82)** 50 Nitromethane ------- FINAL 7-26-2013 Table B.6. Select Hematology for Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3)b 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) mchc (pg) Day 3 17.7 ±0.1 17.7 ±0.1 (100) 17.5 ±0.1 (99) 17.3 ±0.1 (98)** 17.4 ±0.1 (98)** 17.3 ±0.1 (98)** Day 23 18.0 ±0.1 17.4 ±0.1 (97)** 16.7 ±0.1 (93)** 15.6 ±0.1 (87)** 14.8 ±0.1 (82)** 15.3 ±0.1 (85)**e Week 13 18.3 ±0.1 17.7 ±0.1 (97)** 17.1 ±0.1 (93)** 16.2 ±0.1 (89)** 15.3 ±0.1 (84)** 15.7 ±0.1 (86)** MCHC° (g/dL) Day 3 38.1 ± 0.1 38.4 ±0.1 (101) 38.4 ±0.1 (101) 38.1 ±0.1 (100) 38.1 ±0.1 (100) 38.6 ±0.1 (101)** Day 23 38.0 ±0.2 38.1 ±0.2 (100) 38.0 ±0.2 (100) 38.2 ±0.2 (101) 38.4 ±0.2 (101) 39.5 ±0.2 (104)**e Week 13 34.1 ±0.2 33.9 ±0.2 (99) 34.2 ±0.2 (100) 34.4 ±0.2 (101) 34.7 ±0.2 (102) 35.4 ±0.2 (104)** Platelets0 (10:V|iL) Day 3 669.7 ± 26.8 586.2 ± 15.3 (88)* 609.0 ± 26.4 (91) 657.9 ± 17.9 (98) 668.0 ± 15.8 (100) 624.6 ± 17.6 (93) Day 23 649.2 ± 12.7 628.9 ± 14.4 (97) 637.2 ± 12.6 (98) 698.7 ± 14.0 (108) 811.9 ± 14.4 (125)** 1179.0 ± 18.5 (182)**e Week 13 534.2 ± 7.7 560.6 ± 10.8 (105) 528.4 ± 10.2 (99) 608.8 ± 11.7 (114)** 669.9 ± 10.8 (125)** 765.0 ±32.6 (143)** Methemoglobin0 (g/dL) Day 3 0.20 ± 0.03 0.27 ±0.10 (135) 0.17 ±0.04 (85) 0.10 ±0.02 (50)* 0.11 ±0.01 (55) 0.16 ±0.01 (80) Day 23 0.09 ± 0.01 0.10 ±0.01 (Hl)f 0.12 ±0.01 (133)* 0.12 ±0.01 (133)** 0.19 ±0.01 (211)** 0.35 ±0.01 (389)**e Week 13 0.20 ± 0.01 0.20 ±0.01 (100) 0.20 ±0.01 (100) 0.21 ±0.01 (105) 0.25 ±0.01 (125)** 0.40 ± 0.04 (200)** "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Results expressed as mean ± SE (% of control). dn = 6. en = 8. f« = 9. sn = 7. * Significantly different from control (p < 0.05); Williams' or Dunnett's test performed by study authors. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by study authors. 51 Nitromethane ------- FINAL 7-26-2013 Table B.7. Histopathological Findings for Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3) Male 0 94 (6.6)b 188 (13.9) 375 (27.1) 750 (53.0) 1500 (100.1) Nasal turbinates0 Degeneration, olfactory epithelium 0/10 (0) d 0/ 10 (0) 9/10** (90; 1.0) 10/10** (100; 1.0) 10/10** (100; 1.0) Hyaline droplets, respiratory epithelium 0/10 (0) d 0/10 (0) 0/10 (0) 1/10(10; 1.0) 8/10 (80; 1.0)** Hyperplasia, goblet cell 0/10 (0) d 0/10 (0) 0/10 (0) 1/10(10; 1.0) 10/10** (100; 2.0) Male 0 94 (43)e 188 (87) 375 (173) 750 (346) 1500 (691) Sciatic nerve degeneration0 0/10 (0) d 0/10 (0) 5/10* (50; 1.0) 10/10** (100; 1.2) 10/10** (100; 1.5) Spinal cord degeneration0 0/10 (0) d 0/10 (0) 9/10** (90; 1.0) 10/10** (100; 1.4) 10/10** (100; 2.0) Bone marrow hyperplasia0 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 9/10** (90; 1.1) 10/10** (100; 2.0) Female 0 94 (4.8)b 188 (9.7) 375 (19.2) 750 (38.1) 1500 (72.1) Nasal turbinates0 Degeneration, olfactory epithelium 0/10 (0) 0/10 (0) 1/10(10; 1.0) 10/10** (100; 1.0) 10/10** (100; 1.2) 10/10** (100; 1.8) Hyaline droplets, respiratory epithelium 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 4/10* (40; 1.0) 10/10** (100; 1.0) Hyperplasia, goblet cell 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 2/10 (20; 1.5) 10/10** (100; 1.7) Female 0 94 (43)e 188 (87) 375 (173) 750 (346) 1500 (691) Sciatic nerve degeneration0 0/10 (0) d 0/10 (0) 8/10** (80; 1.0) 10/10** (100; 1.1) 10/10** (100; 1.8) Spinal cord degeneration0 0/10 (0) d 0/10 (0) 2/10 (20; 1.0) 10/10** (100; 1.4) 10/10** (100; 1.9) Bone marrow hyperplasia0 0/10 (0) 0/10 (0) 1/10 (10; 2.0) 6/10** (60; 1.0) 7/10** (70; 1.1) 10/10** (100; 1.7) "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day ^ 24) x (days dosed ^ total days) x RGDR; body weights used to determine the RGDR were calculated as (initial body weight + final body weight) 2. °Values expressed as number observed with neoplasm/number examined for that neoplasm (% incidence; average severity of lesions 1 = minimal, 2 = mild, 3 = moderate, 4 = marked). dNot examined at this exposure concentration. "Doses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. * Significantly different from control (p < 0.05); Fischer exact test performed by study authors. **Significantly different from control (p < 0.01); Fischer exact test performed by study authors. 52 Nitromethane ------- FINAL 7-26-2013 Table B.8. Neurological Data for Male and Female F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameterb Exposure Group, ppm (HEC, mg/m3)c Male 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 10 10 Forelimb grip strength (kg) 0.617 ± 0.025 0.554 ±0.015 (90) 0.583 ±0.037 (94) 0.592 ±0.021 (96) 0.568 ±0.029 (92) 0.471 ±0.024 (76)** Hindlimb grip strength (kg) 0.433 ± 0.020 0.399 ±0.026 (92) 0.407 ± 0.020 (94) 0.378 ±0.023 (87) 0.382 ±0.017 (88) 0.213 ±0.020 (49)** Female 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 10 10 Forelimb grip strength (kg) 0.598 ± 0.020 0.633 ±0.021 (106) 0.700 ±0.011 (117)** 0.619 ±0.029 (104) 0.585 ±0.019 (98) 0.632 ±0.018 (106) Hindlimb grip strength (kg) 0.403 ± 0.018 0.425 ±0.011 (105) 0.435 ±0.014 (108) 0.419 ±0.013 (104) 0.333 ±0.019 (83)** 0.209 ±0.015 (52)** "Source: NTP (1997). bResults expressed as mean ± SE (% of control). Doses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by study authors. 53 Nitromethane ------- FINAL 7-26-2013 Table B.9. Reproductive Tissue Data for Male F344 Rats After Inhalation Exposure to Nitromethane for 13 Weeks" Parameterb Exposure Group, ppm (HEC, mg/m3)0 Male 0 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 Necropsy body weight (g) 338 ±7 341 ±4 (101) 331 ±4 (98) 299 ±11 (88)** Left cauda weight (g) 0.207 ± 0.004 0.210 ±0.004 (101) 0.204 ± 0.006 (99) 0.177 ±0.009 (86)** Left epididymis weight (g) 0.467 ± 0.009 0.468 ±0.006 (100) 0.444 ± 0.009 (95) 0.412 ±0.013 (88)** Left testis weight (g) 1.39 ±0.03 1.36 ±0.01 (98) 1.34 ±0.02 (96) 1.29 ±0.02 (93)** Sperm motility (%) 94.57 ± 1.30 92.16 ± 1.90 (97) 87.11 ± 1.88 (92)** 76.43 ±2.78 (81)** Sperm concentration (106/g cauda epididymal tissue) 449 ± 45 483 ± 24 (108) 434 ± 35 (97) 380 ± 42 (85) "Source: NTP (1997). bResults expressed as mean ± SE (% of control). Doses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by study authors. 54 Nitromethane ------- FINAL 7-26-2013 Table B.10. Select Hematology Parameters for Male Sprague-Dawey Rats After Inhalation Exposure to Nitromethane11 Parameter Exposure Group, ppm (HEC, mg/m3)b 0 98 (51) 745 (388) Sample size0 10 10 10 Hematocrit (%o) 10 d 41.0 ±0.5 40.0 ±0.5 (98)* 39.0 ±0.9 (95)* 1 mo 44.0 ±0.3 43.0 ±0.4 (98) 42.0 ±0.4 (95)** 3 mo 44.0 ±0.7 44.0 ±0.7 (100) 41.0 ±0.3 (93)** 6 mo 43.0 ±0.5 43.0 ±0.7 (100) 40.0 ±0.8 (93)** Hemoglobind (%) 10 d 13.9 ±0.21 13.3 ±0.17 (96)* 12.9 ±0.25 (93)** 1 mo 14.6 ±0.13 14.9 ±0.16 (102) 13.7 ±0.17 (94)** 3 mo 14.8 ±0.23 14.6 ±0.26 (99) 13.0 ±0.22 (88)** 6 mo 14.0 ±0.23 14.6 ±0.23 (104) 12.3 ±0.22 (88)** aSource: Lewis et al. (1979) and Huntingdon Research Center (1989). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Sample size was generally 9-10 animals per measurement, but the sample size was not always indicated. Expressed as mean ± SE (% of control). * Significantly different from control (p < 0.05); Student's /-test performed by study authors. **Significantly different from control (p < 0.01); Student's /-test performed by study authors. 55 Nitromethane ------- FINAL 7-26-2013 Table B.ll. Liver and Kidney Weights of Male and Female B6C3Fi Mice After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3)b Male 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 10 10 Necropsy body weight0 (g) 36.1 ±0.5 35.9 ±0.5 (99) 35.5 ±0.8 (98) 36.3 ±0.6 (101) 35.2 ±0.4 (98) 34.7 ±0.5 (96) Right kidney weight0 Absolute (g) 0.294 ± 0.009 0.329 ±0.006 (112)** 0.322 ±0.005 (110)* 0.332 ±0.007 (113)** 0.339 ±0.007 (115)** 0.315 ±0.008 (107) Relative (mg/g) 8.15 ± 0.20 9.15 ± 0.11 (112)** 9.10 ± 0.15 (112)** 9.15 ±0.20 (112)** 9.63 ±0.20 (118)** 9.08 ±0.18 (111)** Liver weight0 Absolute (g) 1.633 ± 0.040 1.700 ±0.023 (104) 1.678 ±0.031 (103) 1.731 ±0.027 (106) 1.789 ±0.029 (110)* 1.724 ±0.053 (106) Relative (mg/g) 45.27 ± 0.89 47.32 ±0.38 (105) 47.39 ±0.78 (105) 47.70 ± 0.60 (105)* 50.79 ±0.72 (112)** 49.62 ±0.99 (110)** Female 0 94 (43) 188 (87) 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 10 10 Necropsy body weight0 (g) 31.1 ± 0.7 31.5 ±0.7 (101) 32.8 ±0.7 (105) 34.2 ±0.8 (110)** 31.5 ± 0.5 (101) 30.4 ±0.5 (98) Right kidney weight0 Absolute (g) 0.210 ± 0.007 0.221 ±0.005 (105) 0.228 ± 0.005 (109)* 0.232 ±0.005 (110)* 0.231 ±0.006 (110)* 0.230 ± 0.006 (110) Relative (mg/g) 6.75 ± 0.18 7.03 ±0.15 (104) 6.97 ±0.15 (103) 6.80 ±0.17 (101) 7.33 ±0.21 (109)* 7.57 ±0.15 (112)** Liver weight0 Absolute (g) 1.536 ± 0.033 1.590 ±0.030 (104) 1.604 ±0.044 (104) 1.639 ±0.037 (107) 1.563 ±0.041 (102) 1.575 ±0.050 (103) Relative (mg/g) 49.49 ± 0.99 50.64 ± 1.15 (102) 48.89 ±0.72 (99) 47.97 ±0.95 (97) 49.52 ±0.76 (100) 51.77 ± 1.25 (105) "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Weights expressed as mean ± SE (% of control). * Significantly different from control (p < 0.05); Williams' or Dunnett's test performed by study authors. **Significantly different from control (p < 0.01); Williams' or Dunnett's test performed by study authors. 56 Nitromethane ------- FINAL 7-26-2013 Table B.12. Reproductive Tissue Data for Male and Female B6C3Fi Mice After Inhalation Exposure to Nitromethane for 13 Weeks" Parameter Exposure Group, ppm (HEC, mg/m3)b Male 0 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 Sperm motility (%)c 93.50 ±0.46 85.09 ± 1.21 (91)** 86.47 ± 1.17(92)** 82.41 ± 1.30 (88)** Female 0 375 (173) 750 (346) 1500 (691) Sample size 10 10 10 10 Estrous cycle length (days) c'd 4.00 ± 0.00 4.33 ±0.14 (108)* 4.50 ±0.21 (113)* 4.71 ±0.26 (118)** "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Results expressed as mean ± SE (% of control). dEstrous cycle was longer than 12 days or was unclear for at least one mouse in all groups except the 750-ppm group. * Significantly different from control (p < 0.05); Shirley's test performed by study authors. **Significantly different from control (p < 0.01); Shirley's test performed by study authors. 57 Nitromethane ------- FINAL 7-26-2013 Table B.13. Histopathological Findings for Male and Female B6C3Fi Mice After Inhalation Exposure to Nitromethane for 13 Weeks3 Parameterb Exposure Group, ppm (HEC, mg/m3) Male 0 94 (7.1)c 188 (14.1) 375 (28.0) 750 (55.7) 1500 (112.7) Nasal turbinates Degeneration, olfactory epithelium 0/10 (0) 0/10 (0) 0/ 10 (0) 10/10** (100; 1.0) 10/10** (100; 1.3) 10/10** (100; 2.0) Hyaline droplets, respiratory epithelium 0/10 (0) 0/10 (0) 1/10(10; 1.0) 10/10** (100; 1.0) 10/10** (100; 1.0) 10/10** (100; 2.0) Male 0 94 (43)d 188 (87) 375 (173) 750 (346) 1500 (691) Spleen, extramedullary hematopoiesis 0/10 (0) 1/10(10; 1.0) 0/10 (0) 1/10 (10; 1.0) 2/10 (20; 1.0) 10/10** (100; 1.0) Female 0 94 (5.9)c 188 (12.2) 375 (24.9) 750 (48.0) 1500 (93.9) Nasal turbinates Degeneration, olfactory epithelium 0/10 (0) 0/10 (0) 7/10** (70; 1.0) 10/10** (100; 1.0) 10/10** (100; 2.0) 10/10** (100; 3.0) Hyaline droplets, respiratory epithelium 0/10 (0) 2/10 (20; 1.0) 9/10** (90; 1.0) 10/10** (100; 2.0) 10/10** (100; 2.0) 10/10** (100; 3.0) Female 0 94 (43)d 188 (87) 375 (173) 750 (346) 1500 (691) Spleen, extramedullary hematopoiesis 0/10 (0) 0/10 (0) 0/10 (0) 2/10 (20; 1.0) 3/10 (30; 1.0) 9/10** (90; 1.0) "Source: NTP (1997). bValues expressed as number observed with neoplasm/number examined for that neoplasm (% incidence; average severity of lesions 1 = minimal, 2 = mild, 3 = moderate, 4 = marked). Doses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day ^ 24) x (days dosed ^ total days) x RGDR; body weights used were for determining the RGDR were calculated as (initial body weight + final body weight) 2. dDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. **Significantly different from control (p < 0.01); Fischer exact test performed by study authors. 58 Nitromethane ------- FINAL 7-26-2013 Table B.14. Select Clinical Chemistry Parameters for Male New Zealand White Rabbits After Inhalation Exposure to Nitromethane" Exposure Group, ppm (HEC, mg/m3)b Parameter 0 98 (51) 745 (388) Sample size0 5 5 5 OCTd 1 mo 470 ± 147.9 788 ±65.7 (167)* 1790 ±310.0 (381)* (Su/mL) 3 mo 840 ± 277.2 1363 ± 170.0 (162) 1970 ± 159.4 (235)* 6 mo 2940 ± 1591.5 1650 ±352.1 (56) 1110 ±259.0 (38) Thyroxind 1 mo 3.4 ±0.42 2.8 ± 0.82 (82) 1.9 ±0.39 (56)* (Hg/dL) 3 mo 2.2 ±0.38 0.8 ±0.43 (36) 1.4 ±0.31 (64) 6 mo 2.1 ±0.28 1.1 ±0.33 (52)* 1.0 ±0.23 (48)* "Source: Lewis et al. (1979) and Huntingdon Research Center (1989). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Sample size per measurement was not specified, but five animals per treatment group were used. Expressed as mean ± SE (% of control). * Significantly different from control (p < 0.05); Mann-Whitney U test performed by the study authors. Table B.15. Thyroid Weights of Male Sprague-Dawley Rats After Inhalation Exposure to Nitromethane for 6 Months3 Parameter Exposure Group, ppm (HEC, mg/m3)b 0 98 (51) 745 (388) Thyroid weight0 Sample size 10 10 10 Absolute (mg) 22.5 ± 1.3 26.7 ± 1.7(119) 28.3 ±0.8 (126)** Relative (% x 1000) 3.9 ±0.2 4.4 ±0.2 (113) 5.3 ±0.2 (136)** aSource: Lewis et al. (1979). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Values expressed as mean ± SE (% of control). **Significantly different from control (p < 0.01); Student's t-test performed by the study authors. 59 Nitromethane ------- FINAL 7-26-2013 Table B.16. Nonneoplastic Lesions in Male and Female B6C3Fi Mice After Inhalation Exposure to Nitromethane for 103 Weeks" Parameterb Exposure Group, ppm (HEC, mg/m3) Males 0 188 (22.2)c 375 (44.9) 750 (91.6) Nose Nasolacrimal duct, Inflammation 2/50 (4) (1.5) 3/49 (6) (1.3) 10/50 (20)* (1.9) 10/50 (20)* (1.9) Olfactory epithelium, atrophy, focal 3/50 (6) (1.0) 8/49 (16)* (1.1) 0/50 (0) 0/50 (0) Olfactory epithelium, degeneration 0/50 (0) 10/49 (20)** (1.1) 50/50 (100)** (2.5) 50/50 (100)** (3.1) Olfactory epithelium, metaplasia 0/50 (0) 1/49 (2) (2.0) 41/50 (82)** (1.8) 49/50 (98)** (2.0) Respiratory epithelium, degeneration, hyaline 5/50 (10) (1.0) 5/49 (10) (1.2) 50/50 (100)** (1.9) 50/50 (100)** (2.0) Females 0 188 (87)d 375 (173) 750 (346) Liver Eosinophilic focus 4/50 (8) 7/49 (14) 11/49 (22)* 15/50 (30)* Females 0 188 (21.9)c 375 (43.2) 750 (87.9) Nose Nasolacrimal duct, Inflammation 1/50 (2) (2.0) 0/49 (0) 3/50 (6) (1.7) 3/50 (6) (2.0) Olfactory epithelium, atrophy, focal 2/50 (4) (1.0) 6/49 (12) (1.0) 0/50 (0) 0/50 (0) Olfactory epithelium, degeneration 0/50 (0) 22/49 (45)** (1.1) 50/50 (100)** (2.7) 50/50 (100)** (3.2) Olfactory epithelium, metaplasia 0/50 (0) 2/49 (4) (1.0) 46/50 (92)** (1.9) 48/50 (96)** (2.2) Respiratory epithelium, degeneration, hyaline 16/50 (32) (1.1) 39/49 (80)** (1.5) 50/50 (100)** (2.0) 50/50 (100)** (2.5) "Source: NTP (1997). bValues expressed as number observed with neoplasm/number examined for that neoplasm (% incidence) (severity when available). Doses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x RGDR; average body weights from the study were used to determine the RGDR. dDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. * Significantly different from control (p < 0.05); Logistic regression test performed by the study authors. **Significantly different from control (p < 0.01); Logistic regression test performed by the study authors. 60 Nitromethane ------- FINAL 7-26-2013 Table B.17. Thyroid Weights of Male New Zealand White Rabbits After Inhalation Exposure to Nitromethane for 6 Months3 Exposure Group, ppm (HEC, mg/m3)b Parameter 0 98 (51) 745 (388) Thyroid Sample size 5 5 5 weight0 Absolute (mg) 235 ±33 265 ±31 (113) 325 ±31 (138) Relative (% x 1000) 6.0 ±0.6 7.0 ± 1.0(117) 8.0 ±0.7 (133) "Source: Lewis et al. (1979d). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Values expressed as mean ± SE (% of control). Table B.18. Mammary Tumors in Female F344 Rats After Inhalation Exposure to Nitromethane for 103 Weeks3 Parameter0 Exposure Group, ppm (HEC, mg/m3)b 0 94 (43) 188 (87) 375 (173) Fibroadenoma 19/50 (38) 21/50 (42) 33/50 (66)** 36/50 (72)** Fibroadenoma or adenoma 20/50 (40) 21/50 (42) 33/50 (66)** 36/50 (72)** Carcinoma 2/50 (4) 7/50 (14) 1/50 (2) 11/50 (22)** Adenoma or carcinoma 4/50 (8) 7/50 (14) 1/50 (2) 13/50 (26)* Fibroadenoma, adenoma, or carcinoma 21/50 (42) 25/50 (50) 34/50 (68)** 41/50 (82)** "Source: NTP (1997). bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. °Values expressed as number observed with neoplasm/number examined for that neoplasm (% incidence). * Significantly different from control (p < 0.05); Fischer exact test performed by the study authors. **Significantly different from control (p < 0.01); Fischer exact test performed by the study authors. 61 Nitromethane ------- FINAL 7-26-2013 Table B.19. Neoplastic Lesions in Male and Female B6C3Fi Mice After Inhalation Exposure to Nitromethane for 103 Weeks3 Parameterb Exposure Group, ppm (HEC, mg/m3) Males 0 188 (87)c 375 (173) 750 (346) Harderian gland Adenoma 9/50 (18) 10/50 (20) 19/50 (38)* 32/50 (64)** Carcinoma 1/50 (2) 1/50 (2) 6/50 (12) 5/50 (10) Adenoma or carcinoma 10/50 (20) 11/50 (22) 25/50 (50)** 37/50 (74)** Males 0 188 (359)d 375 (727) 750 (1484) Lung Alveolar/bronchiolar adenoma 11/50 (22) 10/50 (20) 9/50 (18) 12/50 (24) Alveolar/bronchiolar carcinoma 2/50 (4) 3/50 (6) 3/50 (6) 11/50(22)** Alveolar/bronchiolar adenoma or carcinoma 13/50 (26) 13/50 (26) 12/50 (24) 20/50 (40) Females 0 188 (87)b 375 (173) 750 (346) Harderian gland Adenoma 5/50 (10) 7/50 (14) 16/50 (32)** 19/50 (38)** Carcinoma 1/50 (2) 2/50 (4) 4/50 (8) 3/50 (6) Adenoma or carcinoma 6/50 (12) 9/50 (18) 20/50 (40)** 21/50 (42)** Liver Hepatocellular adenoma 14/50 (28) 25/49 (51)* 17/49 (35) 35/50 (70)** Hepatocellular adenoma, multiple 3/50 (6) 13/49 (27)** 4/49 (8) 13/50 (26)** Hepatocellular carcinoma 10/50 (20) 14/49 (29) 8/49 (16) 12/50 (24) Hepatocellular adenoma or carcinoma 19/50 (38) 34/49 (69)** 22/49 (45) 40/50 (80)** Females 0 188 (355)d 375 (700) 750 (1423) Lung Alveolar/bronchiolar adenoma 3/50 (6) 3/50 (6) 2/49 (4) 9/50 (18) Alveolar/bronchiolar carcinoma 0/50 (0) 3/50 (6) 5/49 (10)* 3/50 (6) Alveolar/bronchiolar adenoma or carcinoma 3/50 (6) 6/50 (12) 6/49 (12) 12/50 (24)* "Source: NTP (1997). bValues expressed as number observed with neoplasm/number examined for that neoplasm (% incidence). Doses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x blood:gas partition coefficient. dDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x (hours per day 24) x (days dosed total days) x RGDRtb+pu; average body weights from the study were used to determine the RGDR. * Significantly different from control (p < 0.05); Logistic regression test performed by the study authors. **Significantly different from control (p < 0.01); Logistic regression test performed by the study authors. 62 Nitromethane ------- FINAL 7-26-2013 APPENDIX C. BMD OUTPUTS Multistage Model with 0.95 Confidence Level Multistage 1 0.8 0.6 0.4 0.2 0 BMDL BMD 0 5 10 15 20 25 dose 10:13 03/10 2011 Figure C.l. Multistage BMD Model for Respiratory Epithelium Hyaline Droplets Data (NTP, 1997) Text Output for Multistage BMD Model for Respiratory Epithelium Hyaline Droplets Data (NTP, 1997) Multistage Model. (Version: 3.2; Date: 05/26/2010) Input Data File: C:/l/NTP97_13wk_RespEpiHyalDrop_F_mouse_HiDosDrop_Multi3_l.(d) Gnuplot Plotting File: C:/l/NTP97_13wk_RespEpiHyalDrop_F_mouse_HiDosDrop_Multi3_l.pit Thu Mar 10 10:13:45 2011 [add_notes_here] The form of the probability function is: 63 Nitromethane ------- FINAL 7-26-2013 P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2-beta3* doseA3)] The parameter betas are restricted to be positive Dependent variable = DichEff Independent variable = Dose Total number of observations = 4 Total number of records with missing values = 0 Total number of parameters in model = 4 Total number of specified parameters = 0 Degree of polynomial = 3 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0 Beta(l) = 0 Beta(2) = 0 Beta(3) = 6.66264e+015 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Background -Beta(l) -Beta(2) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Beta(3) Beta (3) 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0 k k k Beta(1) 0 k k k Beta(2) 0 k k k Beta(3) 0.00121122 -k k k * - Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -8 .25485 -8 .27784 -27.6759 # Param's 4 1 1 Deviance Test d.f. 0.0459631 38.842 P-value 0.9974 <.0001 64 Nitromethane ------- FINAL 7-26-2013 0.0000 0.0000 0.000 0.000 10 0. 000 5.9362 0.2238 2 .238 2.000 10 -0.181 12.1994 0.8891 8.891 9.000 10 0.110 24.9367 1.0000 10.000 10.000 10 0. 000 Chi^2 =0.04 d.f. =3 P-value = 0.9975 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 4.43082 BMDL = 1.31366 BMDU = 5 .42422 Taken together, (1.31366, 5.42422) is a 90 % two-sided confidence interval for the BMD AIC: 18.5557 Goodness of Fit Scaled Dose Est. Prob. Expected Observed Size Residual 65 Nitromethane ------- FINAL 7-26-2013 Multistage Model with 0.95 Confidence Level Multistage 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 ESMDL BMD 0 10 20 30 40 50 60 70 80 90 dose 10:32 03/10 2011 Figure C.2. Multistage BMD Model for Respiratory Epithelium Degeneration Data (NTP, 1997) Text Output for Multistage BMD Model for Respiratory Epithelium Degeneration Data (NTP, 1997) Multistage Model. (Version: 3.2; Date: 05/26/2010) Input Data File: C:/l/NTP97_103wk_RespEpiDegHyal_F_mouse_Multi3_l.(d) Gnuplot Plotting File: C:/l/NTP97_103wk_RespEpiDegHyal_F_mouse_Multi3_l.pit Thu Mar 10 10:32:54 2011 [add_notes_here] The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2-beta3* doseA3)] The parameter betas are restricted to be positive 66 Nitromethane ------- FINAL 7-26-2013 Dependent variable = DichEff Independent variable = Dose Total number of observations = 4 Total number of records with missing values = 0 Total number of parameters in model = 4 Total number of specified parameters = 0 Degree of polynomial = 3 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 1 Beta(1) = 1.29371e+018 Beta(2) = 0 Beta(3) = 0 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Beta(l) -Beta(2) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Background Beta (3) Background 1 -0.47 Beta (3) -0.47 1 Parameter Estimates Interval Variable Limit Background Beta(1) Beta(2) Beta(3) Estimate 0.319859 0 0 0. 000114246 Std. Err. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -56.1379 -56.1413 -105.132 Param's 4 2 1 Deviance Test d.f. 0. 00669167 97.989 P-value 0.9967 <.0001 AIC: 116.283 67 Nitromethane ------- FINAL 7-26-2013 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.3199 15.993 16.000 50 0. 002 21.9354 0.7963 39.020 39.000 49 -0.007 43.2380 0.9999 49.997 50.000 50 0. 058 87.9212 1.0000 50.000 50.000 50 0. 000 Chi^2 = 0.00 d.f. = 2 P-value = 0.9983 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 9.73372 BMDL = 1.60241 BMDU = 11.3184 Taken together, (1.60241, 11.3184) is a 90 % two-sided confidence interval for the BMD 68 Nitromethane ------- FINAL 7-26-2013 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation 0.9 0.8 0.7 0.6 0.5 0.4 0.3 BMDL BMD 0 20 40 60 80 100 120 140 160 180 dose 10:55 03/10 2011 Figure C.3. Multistage Cancer BMD Model for Mammary Fibroadenoma, Adenoma, or Carcinoma Data (NTP, 1997) Text Output for Multistage-Cancer BMD Model for Mammary Fibroadenoma, Adenoma, or Carcinoma Data (NTP, 1997) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/l/NTP97_103wk_MamFibroadenoORAdenoORCarc_F_rat_MultiCanc3_l.(d) Gnuplot Plotting File: C:/l/NTP97_103wk_MamFibroadenoORAdenoORCarc_F_rat_MultiCanc3_l.pit Thu Mar 10 10:55:56 2011 [add_notes_here] The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2-beta3* doseA3)] 69 Nitromethane ------- FINAL 7-26-2013 The parameter betas are restricted to be positive Dependent variable = DichEff Independent variable = Dose Total number of observations = 4 Total number of records with missing values = 0 Total number of parameters in model = 4 Total number of specified parameters = 0 Degree of polynomial = 3 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.4 00 60 6 Beta(1) = 0.00594339 Beta(2) = 6.26236e-006 Beta(3) = 0 the user, Background Beta (1) Beta (2) Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Beta(3) have been estimated at a boundary point, or have been specified by and do not appear in the correlation matrix ) Background Beta(l) Beta(2) 1 -0.67 0.49 -0.67 1 -0.94 0.49 -0.94 1 Parameter Estimates Interval Variable Limit Background Beta(1) Beta(2) Beta(3) Estimate 0.410209 0.004892 1.2 6923e-005 0 Std. Err. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -123.585 -123.826 -134.186 # Param's 4 3 1 Deviance Test d.f. 0.481622 21.2027 P-value 0.4877 <.0001 70 Nitromethane ------- FINAL 7-26-2013 AIC: 253.652 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.4102 20.510 21.000 50 0.141 43.3000 0.5340 26.701 25.000 50 -0.482 86.6000 0.6489 32.447 34.000 50 0. 460 172.8000 0.8266 41.331 41.000 50 -0.124 Chi^2 = 0.48 d.f. = 1 P-value = 0.4887 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 20.452 BMDL = 11.2764 BMDU = 67.5516 Taken together, (11.2764, 67.5516) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.00886811 71 Nitrom ethane ------- FINAL 7-26-2013 Table C.l. Multistage Cancer Model Predictions for Tumor Data for Nitromethane" Endpoint Sex Species IURd Goodness-of-Fit p-V alueb AICC Mammary fibroadenoma, adenoma, or carcinoma F Rat 0.00886811 0.4887 253.65 Mammary fibroadenoma or adenoma F Rat 0.00717979 0.1631 263.41 Mammary fibroadenoma F Rat 0.00705667 0.3518 263.95 Hepatocellular adenoma or carcinoma F Mouse 0.00338166 0.0008 261.71 Harderian gland adenoma or carcinoma M Mouse 0.00283946 0.1674 237.29 Hepatocellular adenoma F Mouse 0.00254843 0.0121 263.79 Harderian gland adenoma M Mouse 0.00214776 0.4731 235.45 Harderian gland adenoma or carcinoma F Mouse 0.00194971 0.2630 225.79 Harderian gland adenoma F Mouse 0.00164856 0.4568 207.67 Mammary carcinoma F Rat 0.00126694 0.0245 131.45 Harderian gland carcinoma M Mouse 0.00055152 0.2574 95.47 Hepatocellular carcinoma F Mouse 0.00050530 0.3112 213.71 Harderian gland carcinoma F Mouse 0.00038784 0.5942 82.16 aNTP (1997). bValues <0.10 fail to meet conventional goodness-of-fit criteria. °AIC = Akaike's Information Criteria. dAs calculated by BMDS. 72 Nitromethane ------- FINAL 7-26-2013 APPENDIX D. 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