United States Environmental Protection 1=1 m m Agency EPA/690/R-12/032F Final 12-03-2012 Provisional Peer-Reviewed Toxicity Values for oToluidine (CASRN 95-53-4) 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 MANAGER Chris Cubbison, PhD National Center for Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY ICF International 9300 Lee Highway Fairfax, VA 22031 PRIMARY INTERNAL REVIEWERS Audrey Galizia, Dr PH National Center for Environmental Assessment, Washington, DC Dan D. Petersen, PhD, DABT National Center for Environmental Assessment, Cincinnati, OH 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). l o-Toluidine ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS iii 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 Subchronic-duration Studies 10 Chronic-duration Studies 10 Developmental Studies 10 Reproductive Studies 10 Carcinogenicity Studies 10 ANIMAL STUDIES 11 Oral Exposures 11 Subchronic-duration Studies 12 Chronic-duration/Carcinogenic Studies 13 Developmental and Reproduction Studies 16 Inhalation Exposures 16 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 16 Short-term Studies 16 Other Studies 17 DERIVATION OI PROVISIONAL VALUES 22 DERIVATION OI ORAL REFERENCE DOSE 22 Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 22 Derivation of Chronic Provisional RfD (Chronic p-RfD) 25 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 25 CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR 25 MODE-OF-ACTION DISCI SSION 26 Summary of Mutagenicity and Genetic Toxicology Studies 26 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 26 Derivation of Provisional Oral Slope Factor (p-OSF) 26 Derivation of Provisional Inhalation Unit Risk (p-IUR) 29 APPENDIX A. PROVISIONAL SCREENING VALUES 30 APPENDIX B. DATA TABLES 32 APPENDIX C. BMD OUTPUTS 38 APPENDIX D. REFERENCES 43 li o-Toluidine ------- COMMONLY USED ABBREVIATIONS BMC benchmark concentration BMCL benchmark concentration lower bound 95% confidence interval BMD benchmark dose BMDL benchmark dose lower bound 95% confidence interval 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 NOAELrec 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 reference concentration (inhalation) p-RfD provisional reference dose (oral) RfC reference concentration (inhalation) RfD reference dose (oral) UF uncertainty factor UFa animal-to-human uncertainty factor UFC composite uncertainty factor UFd incomplete-to-complete database uncertainty factor UFh interhuman uncertainty factor UFl LOAEL-to-NOAEL uncertainty factor UFS subchronic-to-chronic uncertainty factor WOE weight of evidence 111 o-Toluidine ------- FINAL 12-3-2012 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR o-TOLUIDINE (95-53-4) 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 (http://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.gov/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 o-Toluidine ------- FINAL 12-3-2012 INTRODUCTION 0/7/70-Toluidine (o-toluidine; also referred to as 2-methylaniline or 2-aminotoluene) is a synthetic aromatic amine with amine and methyl groups in an ortho (1,2) configuration. o-Toluidine is used as an intermediate in the production of dyes, pigments, and rubbers (IARC, 2000). The empirical formula for o-toluidine is C7H9N (see Figure 1). A table of physicochemical properties is provided below (see Table 1). CH. Figure 1. o-Toluidine Structure Table 1. Selected Physicochemical Properties Table (o-Toluidine)a Property (unit) Value Boiling point (°C) 200.3 Melting point (°C) -16.3 Density (g/cirf) 0.9984 Vapor pressure (kPa at 20 °C) 0.013 Solubility in water (mg/L at 25°C) 15 Relative vapor density (air = 1) 3.72 Molecular weight (g/mol) 107.16 Flash point (°C) 85 Octanol/water partition coefficient (unitless) 1.32 aValues obtained from IARC (2000). No RfD, RfC, or cancer assessment for o-toluidine is included in the EPA's IRIS database (U.S. EPA, 2012a) or on the Drinking Water Standards and Health Advisories List (U.S. EPA, 2006). No RfD or RfC values have been reported in the Health Effects Assessment Summary Tables (HEAST; U.S. EPA, 2012b). The Chemical Assessments and Related Activities (CARA) list (U.S. EPA, 1994a) includes a listing for a Health and Environmental Effects Profile (HEEP) for o-toluidine. The toxicity of o-toluidine has not been reviewed by the 2 o-Toluidine ------- FINAL 12-3-2012 ATSDR (2008). The World Health Organization (WHO, 2011) has not produced an Environmental Health Criteria Document for o-toluidine but did produce a Concise International Chemical Assessment Document (WHO, 1998). CalEPA (2008) has not derived acute toxicity values for o-toluidine. The American Conference of Governmental Industrial Hygienists (ACGIH) has derived an 8-hour time-weighted average (TWA) exposure limit of 2 ppm (8.8 mg/m3) along with a skin notation for o-toluidine (ACGIH, 2008). The National Institute of Occupational Safety and Health (NIOSH, 2010) has not established a Recommended Exposure Limit (REL) but considers o-toluidine to be a potential occupational carcinogen that may be absorbed through skin. NIOSH set an immediately dangerous to life or health (IDLH) value for o-toluidine at 50 ppm (220 mg/m3). The Occupational Safety and Health Administration (OSHA, 2010) has derived a TWA Permissible Exposure Level (PEL) of 5 ppm (22 mg/m3) for o-toluidine. The HEAST (U.S. EPA, 201 lb) does not include a cancer assessment for o-toluidine, but HEEP (U.S. EPA, 1984) provides a potency factor of 0.24 (mg/kg-day) 1 for oral exposure. The International Agency for Research on Cancer (IARC, 2010) has classified o-toluidine as a Group 1 agent (Carcinogenic to Humans) based on sufficient evidence in humans and animals. o-Toluidine is included in the 12th Report on Carcinogens (NTP, 2011) and is listed as Reasonably Anticipated to be a Human Carcinogen. CalEPA (2008) has derived an inhalation slope factor of 1.8 x 10 1 per mg/kg-day, an inhalation unit risk of 5.1 x 10 5 per (J,g/m3, and an oral slope factor of 1.8 x 10 1 per mg/kg-day for o-toluidine. Literature searches were conducted on sources published from 1950s through November 2011 for studies relevant to the derivation of provisional toxicity values for o-toluidine, CASRN 95-53-4. 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 also searched for health- related information: ACGIH, ATSDR, CalEPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA OW, EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS. 3 o-Toluidine ------- FINAL 12-3-2012 REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) Table 2 provides an overview of the database for o-toluidine and includes all potentially relevant repeated subchronic- and chronic-duration studies. Entries for the principal studies are bolded. Unless otherwise qualified, the phrase "significant" means "statistical significance" with ap-wdXut of less than 0.05). 4 o-Toluidine ------- FINAL 12-3-2012 Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects NOAEL' BMDL/ BMCLa LOAELab Reference (Comments) Notes0 Human 1. Oral (mg/kg-d)a Subchronic ND Chronic ND Developmental ND Reproductive ND Carcinogenicity ND 2. Inhalation (mg/m3)a'd Subchronic ND Chronic ND Developmental ND Reproductive ND Carcinogenicity 1643 male and 106 female (total 1749), retrospective cohort, from <5-yr to over 20-yr exposure to o-toluidine with or without aniline Categories were "definitely exposed," "possibly exposed," and "probably not exposed" Bladder cancer standardized incidence ratio [SIR] (90% confidence interval [CI]) Definitely exposed: 6.48 (3.04-12.2) Duration of employment <5 yr = 0 5 to <10 yr= 8.8 (0.45-41.7) >10 yr = 27.2 (11.8-53.7) p < 0,001 for linear trend NA Not run NA Ward et al. (1991) 5 o-Toluidine ------- FINAL 12-3-2012 Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL' BMDL/ BMCLa LOAELab Reference (Comments) Notes0 Carcinogenicity Not reported but apparently the same cohort as listed above was used Not reported 19 additional cases of bladder cancer in the same cohort from 1973 to 1988 NA Not run NA Markowitz and Levin (2004) 1643 male and 106 female (total 1749), retrospective cohort, from <5 yr to over 20 yr; bladder cancer incidence was determined from December 31, 1988 to December 31, 1994 (re- analysis of Ward et al., 1991) Categories were "definitely exposed," "possibly exposed," and "probably not exposed" Bladder cancer SIR (95% CI) Definitely exposed: 5.84 (2.91-10.45)* Duration of employment <5 yr= 1.25 (0.03-6.97) 5-<10 yr= 3.67 (0.09-20.44) >10 yr = 11.09 (5.07-21.05)* *p < 0.001 NA Not run NA Carreon et al. (2010) 2160 males, cohort, at least 6 mo of employment from 1955-1984 Not reported Bladder cancer; for subgroup exposed to 0-toluidine, standardized mortality ratio [SMR] = 1589 Relative risk (95% CI) for years of employment: 1-4 yr = 6.73 (1.59-28.41)* >5 yr = 7.65 (1.03-56.87)* *p < 0.05 test for trendp = 0.002 NA Not run NA Sorahan et al. (2000) 6 o-Toluidine ------- FINAL 12-3-2012 Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL' BMDL/ BMCLa LOAELab Reference (Comments) Notes0 Carcinogenicity 2160 males, cohort, at least 6 mo of employment from 1955-1984 (update of study listed above) Not reported Bladder cancer; for subgroup exposed to 0-toluidine, SMR = 1116 (95% CI: 230-3261) Relative risk (95% CI) for years of employment: 1-4 yr= 4.68 (1.66-13.2)* >5 yr = 6.99 (1.69-28.9)* *p < 0.05 test for trendp < 0.001 NA Not run NA Sorahan (2008) Animal 1. Oral (mg/kg-day)a Subchronic 10 rat (sex and strain not specified), diet, duration not specified but at least 91 d Not specified; authors added o-toluidine (final concentration of 7.5 to 12 mg per d) in rice flour diet Three animals exhibited metaplasia and early epithelial proliferation in bladder mucous membrane ND Not run ND Ekman and Strombeck (1947) 20/0 F344/N rat, diet, 7 d/wk, 13 wk 0 or 301 (Adjusted)6'' Decreased body weight; increased relative weight of the liver, kidney, spleen, and testes; and increased incidence of lesions in the liver, kidneys, bladder, and spleen None Not run 301 NTP (1996a) PS, PR 20/0 F344/N rat, diet, 7 d/wk, 26 wk 0 or 285 (Adjusted)e,f Decreased body weight; increased relative weight of the liver, kidney, spleen, and testes; and increased incidence of lesions in the liver, kidneys, bladder, and spleen None Not run 285 NTP (1996b) 7 o-Toluidine ------- FINAL 12-3-2012 Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL' BMDL/ BMCLa LOAELab Reference (Comments) Notes0 Subchronic 20/0 F344/N rat, diet, 7 d/wk, 13 wk with 13 wk recovery 0 or 304 (Adjusted)e'f Decreased body weight after treatment and recovery; increased relative spleen weight; and increased incidence of lesions in the kidney, liver, and spleen after recovery None Not run 304 NTP (1996c) Chronic 50/50 F344 rat, diet, 7 d/wk, 101-104 wk 0, 231 and 525 in males; 0, 264, and 600 in females (Adjusted)6'8 Decreased survival None Not run None; 231 is a frank effect level (PEL) NCI (1979a) Developmental ND Reproductive ND Carcinogenicity 50/50 F344 rat, diet, 7 d/wk, 101-104 wk 0, 63.0, and 138.8 in males; 0, 63.7 and 140.4 in females6*1 Significant increase in several types of cancer, including skin (fibromas), spleen, and bladder cancer; BMDL based on subcutaneous fibroma in males NA 10.4 NA NCI (1979a) 25/0 CD rat, diet, 7 d/wk, 18 mo followed by 6 mo on the control diet 0, 72.1, and 144.1eh Significant increase in the incidence of dermal subcutaneous fibroma and fibrosarcoma NA 4.5 NA Weisburger et al. (1978a) PS, PR 30/0 F344 rat, diet, 7 d/wk, 72 wk followed by 21-wk recovery 0 or 44.2e'h Significant increases in fibromas of the skin, spleen, and mammary tissue, and in peritoneal tumors; nonsignificant increases in neoplasms of the bladder, liver, and mammary carcinomas; decreased survival (significance not reported) NA Not run NA Hecht et al. (1982) 8 o-Toluidine ------- FINAL 12-3-2012 Table 2. Summary of Potentially Relevant Data for o-Toluidine (CASRN 95-53-4) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL3 BMDL/ BMCLa LOAELab Reference (Comments) Notes0 Carcinogenicity 50/50 B6C3FJ mouse, diet, 7 d/wk, 102-103 wk 0, 23.0, and 73.8 in males; 0, 26.5, and 91.9 in females6'11 Significant increase in hepatocellular carcinomas, adenomas, and hemangiosarcomas; BMDL based on hepatocellular adenomas and carcinomas in females NA 22.2 NA NCI (1979b) 25/25 CD-I mouse, diet, 7 d/wk, 18 mo followed by 3 mo on the control diet 0,210.5, and 420.9 in males; 0, 211.8, and 423.7 in femalese,h Significant increase in vascular tumors NA 23 NA Weisburger et al. (1978b) 2. Inhalation (mg/m3)a Subchronic ND Chronic ND Developmental ND Reproductive ND Carcinogenicity ND ""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects and to a human equivalent dose (HED in mg/kg-d) for oral carcinogenic effects. All long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values from animal developmental studies are not adjusted to a continuous exposure. bNot reported by the study author, but determined from data. °PS = Principal study, PR = Peer reviewed, NPR = Not peer reviewed, NA = Not applicable, ND = No data. dThese studies were occupational studies where exposure is presumably via inhalation, but dermal and oral exposures are also possible. "Compound administered as toluidine hydrochloride. fAverage daily doses were provided in the study report in mg/kg-day presumably based on the concentrations in the diet (also provided in the study report), body weights, and food consumption (both of which were routinely measured). 8Doses were converted from ppm (as presented in the study report) by using the following equation ppm x average daily food consumption x (1 body weight) x (days dosed ^ total days). Average food consumption was obtained from EPA (1988) and average body weights were obtained from the study report (NCI, 1979). hThese doses are HEDs that were calculated as follows: average daily dose in mg/kg-day x (average animal body weight average human body weight)0'25. 9 o-Toluidine ------- FINAL 12-3-2012 HUMAN STUDIES Oral Exposures No human oral exposure studies on o-toluidine were identified Inhalation Exposures The effects of inhalation exposure of humans to o-toluidine have been evaluated in five carcinogenicity studies (i.e., Ward et al., 1991; Markowitz and Levin, 2004; Carreon et al., 2010; Sorahan et al., 2000; Sorahan, 2008). Subchronic-duration Studies No subchronic-duration studies on o-toluidine were identified. Chronic-duration Studies No chronic-duration studies on o-toluidine were identified. Developmental Studies No developmental toxicity studies on o-toluidine were identified. Reproductive Studies No reproductive toxicity studies on o-toluidine were identified. Carcinogenicity Studies Human studies have been limited to those examining cancer outcomes, specifically bladder cancer, in subjects presumed to have occupational exposure to azo dyes. Although for the purpose of this document, the exposures are considered via inhalation because there are no specifics in the study reports. Occupational exposures to compounds, such as aromatic amines, might have an appreciable dermal component, likely due to the relatively low vapor pressure of these compounds (Baan et al., 2008). The majority of the studies examined exposures to mixtures of several chemicals, including o-toluidine, and, in some cases, other suspected or known carcinogens (i.e., Ward et al., 1991; Markowitz and Levin, 2004; Carreon et al., 2010; Sorahan et al., 2000; Sorahan, 2008). Ward et al. (1991) used a retrospective cohort design to investigate the incidence of bladder cancer in 1749 workers (1643 males, 106 females) at a New York chemical plant with exposures to o-toluidine and aniline. The workers were assigned to three groups: (1) "definitely exposed" (n = 708; those who worked in the o-toluidine department), (2) "possibly exposed" (n = 288; maintenance, janitors, yard workers, and shipping), (3) and "probably not exposed" (n = 753; all others). The observed (O) incidence rate of bladder cancer in the chemical plant was compared to that of the expected (E) general population of the State of New York (excluding New York City). Workers in the "definitely exposed" group had a standardized incidence ratio (SIR) for bladder cancer of 6.48 (90% confidence interval [CI]: 3.04-12.2; O/E = 7/1.08). SIRs for <5 years, 5 to <10 years, or 10 years or more of employment were 0 (O/E = 0/0.75), 8.8 (O/E = 1/0.11), and 27.2 (O/E = 6/0.22) respectively. Using <5 years as a reference group, the standard rate ratios for these categories were 1.0, 3.31, and 16.0, which were significant for trend (p < 0.001). Ward et al. (1996) reported urinary levels of o-toluidine in both exposed (98.7 ± 119.4 |ig/L) and unexposed (2.8 ±1.4 |ig/L) workers. They further looked at the 10 o-Toluidine ------- FINAL 12-3-2012 hemoglobin adducts showing higher levels of o-toluidine than aniline, thereby suggesting that exposure to o-toluidine exceeded that of aniline. Markowitz and Levin (2004) conducted a follow-up study of the Ward et al. (1991) cohort and found an additional 19 cases of bladder cancer based on data from attorneys who represented civil litigation and from a bladder cancer screening program sponsored by the chemical plant, which employed the workers. Their analysis indicated that further follow-up should be conducted to characterize the occupational cohort. Another reanalysis by Carreon et al. (2010) changed the group assignments of the workers based on a walk-through and observations of the workers in the chemical plant. Although the categories remained the same, the number of individuals in the categories changed, with more being placed in the definitely exposed category (i.e., definitely exposed, n = 962; possibly exposed, n= 187; and probably not exposed, n = 600). The bladder cancer incidence was determined for the period of December 31, 1988 to December 31, 1994. The calculated SIR for the updated "definitely exposed" cohort was reported as 5.84 (95% CI: 2.91-10.45), which was significantly higher than the "probably not exposed" group. Duration of exposure was also reevaluated with an SIR of 11.09 (95% CI: 5.07-21.05) for those employed for 10 years or more. These results support the initial Ward et al. (1991) study, which also found a significant excess risk of bladder cancer among the cohort of workers in the chemical plant. Sorahan et al. (2000) examined the cancer mortality and incidence in 2160 males working in a factory manufacturing chemicals for the rubber industry for at least 6 months from 1955 to 1984 in northern Wales. Exposure assessments were done by using job histories for 300 different jobs from 1930 to 1984. However, a specific exposure-versus-time matrix for o-toluidine was not possible, and the subjects were evaluated by duration of employment in the o-toluidine department. There were 1131 observed deaths between 1955 and 1996, which were quite close to the expected deaths of 1114.5. Of the workers in the o-toluidine department (n = 53), there were three deaths due to bladder cancer, compared to the expected rate of 0.2, the standard mortality ratio (SMR) was reported to be 1589 (no CI provided). Relative risks (RRs) of bladder cancer mortality due to working in the o-toluidine department for 1-4 years or >5 years, using untagged employment history, were both elevated, at 4.44 (95% CI: 0.76-25.79) and 5.48 (95% CI: 0.51-59.14), respectively. However, RRs of total bladder cancer incidence for those working in the o-toluidine department for 1-4 years or >5 years were 6.73 (95% CI: 1.59-28.41) and 7.65 (95% CI: 1.03-56.87), respectively, which were significantly increased and followed a significant trend. Sorahan (2008) provided an update, with bladder cancer incidence and deaths examined through December 2005. The SMR for workers in the o-toluidine department was 1116 (95%) CI: 230-3261). Relative risk of bladder cancer due to working in the o-toluidine department for 1-4 years or >5 years remained elevated and significant, consistent with the 2000 analysis at 4.68 (95% CI: 1.66-13.2) and 6.99 (95% CI: 1.69-28.9), respectively. ANIMAL STUDIES Oral Exposures In animal studies, o-toluidine is generally administered as o-toluidine HC1. The effects of oral exposure of animals to o-toluidine HC1 have been evaluated in subchronic-duration (Ekman 11 o-Toluidine ------- FINAL 12-3-2012 and Strombeck, 1947; NTP, 1996a,b,c), chronic-duration (NCI, 1979a,b), and cancer (NCI, 1979a,b; Weisburger et al., 1978a,b; Hecht et al., 1982) studies. The NTP rat studies examined 13-week (designated as NTP, 1996a), 26-week (designated as NTP, 1996b), and 13-week with a 13-week recovery period (designated as NTP, 1996c) exposures. The NCI (1979) and Weisburger et al. (1978) studies were conducted in rats and mice. Rat studies were designated as NCI (1979a) and Weisburger et al. (1978a), while mouse studies were designated NCI (1979b) and Weisburger et al. (1978b) as indicated in Table 2. When the complete study was discussed throughout the report, the letter designations were not employed. Two subacute (14-day) studies were also conducted and are reported under the section of "Other Data" below. Subchronic-duration Studies In a study by Ekman and Strombeck (1947), 10 rats (sex and strain not specified) were given a diet of rice flour with o-toluidine that was supplemented with a slice of carrot every other day (duration not specified). The methods of the study are not well reported, and the dose of the experiment was lowered soon after study initiation. The study authors reported the dose in the latter part of the study as approximately 7.5 to 12 mg of o-toluidine per day, with levels about twice as high during the beginning of the study. Study investigators reported the average lifespan of the animals to be 91 days. No other methods or details of the experiment were provided. Three of the rats were stated to have changes in the mucous membrane of the bladder, with metaplasia and early epithelial proliferation. NTP (1996) The 13-week component of the peer-reviewed rat study by NTP (1996a) is selected as the principal study for derivation of the screening subchronic p-RfD. NTP (1996a,b,c) conducted a study performed under Good Laboratory Practices (GLP) that compared the toxicity and carcinogenicity of o-nitrotoluene and o-toluidine HC1 administered in feed at one dose level. Sixty male F344/N rats (20/group) were administered 5000 ppm of o-toluidine HC1 (100% pure as tested by the study laboratory) in the diet and sacrificed after continuous exposure for 13 weeks (Group 1) or 26 weeks (Group 2). A third group was administered 5000-ppm o-toluidine HC1 for 13 weeks, followed with a 13-week recovery period before sacrifice. The study authors reported the daily doses received by these groups as 301 mg/kg-day for the 13-week continuous exposure, 285 mg/kg-day for the 26-week continuous exposure, and 304 mg/kg-day for the 13-week exposure with 13-week recovery group. Homogeneity and stability tests were conducted. Results demonstrated that 9% of the test compound was lost due to preparation, but the dose formulations were homogeneous and stable for 28 days when stored at -20°C to 5°C. Samples stored at room temperature lost 10% of the test material. There were a total of 40 unexposed control rats. However, 20 were used to examine the effects of altered gastrointestinal flora (which were used as part of the testing for o-nitrotoluene). Clinical signs, body weight, and food consumption were recorded throughout the study. At study termination, animals were necropsied, and the following organs were weighed: epididymis, right kidney, liver, spleen, and right testis. Histopathology was only routinely performed on gross lesions, epididymides, liver, kidneys, testes, spleen, and urinary bladder. Continuous data with a normal distribution were analyzed with a Dunnett's test. A Fischer's Exact test was used to evaluate histopathological lesions, and a Wilcoxon rank sum test was used for the placental glutathione »Y-transferase-positive data. 12 o-Toluidine ------- FINAL 12-3-2012 No mortalities were observed among treated or control animals (NTP, 1996a,b,c). Although statistical analyses were not performed, there was a 11% decrease in body-weight, relative to concurrent controls after 13 weeks of treatment. The lowered relative body-weight persisted after the 13-week recovery period (11% lower than the concurrent control). After 26 weeks of treatment, o-toluidine-treated animals had a body-weight 18% lower than the concurrent controls. This was accompanied by lower food consumption. There was a significant increase in the relative weight of the right kidney, liver, spleen, and right testis, but not in the epididymides, after both 13 (see Table B. 1) and 26 weeks (see Table B.2) of treatment. After 13 weeks of treatment followed by 13 weeks of recovery, only the relative weight of the spleen remained significantly elevated (155% of the control weight) (see Table B.2). However, the spleen weight, after the recovery period, was not as elevated as after 13 weeks (350% of the controls) or 26 weeks (433% of the controls) of continuous exposure. In the liver, accumulation of hemosiderin pigment of the Kupffer cell cytoplasm was significantly increased in all groups (20/20 after 13 weeks and 26 weeks, 11/20 after a 13-week recovery period compared to 0/20 in the controls after 13 weeks and 0/10 after 26 weeks). Placental isozyme of glutathione ^'-transferase (PGST)-positive foci (number and size) were also found to be significantly increased at 26 weeks (not measured in the other two treatment groups). All treated animals experienced a significant increase in hemosiderin pigment accumulation in the tubule epithelium of the kidney. There was a significant increase in transitional cell hyperplasia in the urinary bladder after 13 (10/20) and 26 (17/20) weeks of treatment but not after the 13-week recovery period (0/20). At necropsy, the spleens were stated to be grossly enlarged, with granular plaques on the capsular surface, with some plaques containing fluid-filled cysts at 13 weeks. At 13 and 26 weeks, all the treated animals had the following histopathological lesions in the spleen: congestion, hematopoietic cell proliferation, hemosiderin pigmentation, and fibrosis capsules. These were significantly increased compared with the incidence in the controls, and with the exception of hematopoietic cell proliferation, were still significantly increased after a 13-week recovery period. There was thrombosis in the spleen in a few animals after 13 (3/20) and 26 (2/20) weeks of treatment that were not observed in either the controls or the 13-week recovery animals. The only end points that seemed to progress in severity from 13 weeks to 26 weeks were the increase in spleen weight and the increase in the severity of fibrosis capsules in the spleen. After a 13-week recovery period, effects were still observed in the spleen, with relative weight still increased and numerous lesions still observed. The LOAEL is 5000 ppm (285 to 304 mg/kg-day), the only dose tested, based on the numerous effects observed among the three groups. Chronic-duration/Carcinogenic Studies NCI (1979a) NCI (1979a) sponsored a chronic-duration/carcinogenic feeding study of o-toluidine HC1 (purity >99% as measured by the study laboratory) to male and female F344 rats (20 controls/sex, 50/dose/sex), obtained from the NCI Frederick Cancer Research Center animal farm. The rats received concentrations of 0, 3000, or 6000 ppm in the diet for a period of 101 to 104 weeks (equivalent to 231 and 525 mg/kg-day in males and 264 and 600 mg/kg-day in females based on average body weight from the study and daily food consumption [U.S. EPA, 1988]). The human equivalent doses (HEDs) for the carcinogenicity study are equivalent to 63.0 and 138.8 mg/kg-day in males and 63.7 and 140.4 mg/kg-day in females. Doses were selected based on a 7-week study that was conducted to determine a maximum tolerated dose. Diets were freshly prepared every 1 to 1.5 weeks and stored at 5°C, but there is no indication that the diets 13 o-Toluidine ------- FINAL 12-3-2012 were tested for concentration, stability, or homogeneity. Animals were checked twice daily for morbidity and mortality. Clinical observations, palpations, and body-weight measurements were conducted once per month. Animals were necropsied and evaluated for gross signs of toxicity, and tissues were examined microscopically. The statistical tests conducted were numerous and typical of those conducted in NCI studies. Animals that died from other than natural causes were statistically censored at the time of death; animals dying from natural causes were not statistically censored. A time-adjusted analysis was applied when numerous early deaths resulted from causes that were not associated with the formation of tumors. There was a significant decrease in survival relative to controls, at both doses, in both male and female rats; there was approximately 90%, 27%, and 0% (males) and 80%, 55%, and 21% (females) survival at the end of the study for control, low, and high doses, respectively (estimated from Figure 2 of NCI, 1979a). The majority of mortalities in the high-dose group occurred late in the exposure, after Week 60. Decreases in mean body weights of surviving rats relative to concurrent controls were also noted beginning at around 40 weeks of treatment for the low-dose group and at 10 weeks of treatment for the high-dose group. Proliferative lesions in the spleen, abdomen, scrotum, urinary bladder epithelium, and renal pelvis of dosed rats were observed. There was an increase in the incidence of liver necrosis in high-dose females and both low- and high-dose male groups and bladder epithelial hyperplasia in both low- and high-dose groups in males and females (see Table B.3). o-Toluidine also induced subcutaneous fibromas and mammary fibroadenomas (see Table B.4). Statistical analyses conducted by the authors showed a significant, dose-related increase in subcutaneous fibroma in male (but not female) rats, with the incidence at both dose levels (28/50 low, 27/49 high) significantly higher than matched controls (0/20) in male rats. Sarcomas in multiple organs and fibrosarcomas in multiple organs were found to be significantly elevated in male rats as compared to controls. In female rats, sarcomas (fibrosarcomas, angiosarcomas, and osteosarcomas) of multiple organs were found to be significantly higher than controls. The study authors also reported that o-toluidine significantly induced urinary bladder transitional cell carcinomas in female rats, fibroadenomas/adenomas of the mammary gland in female rats, and mesotheliomas in multiple organs or tunica vaginalis in male rats. A NOAEL for noncancer effects was not reported by the study authors. No NOAEL can be derived from the data. There is no LOAEL because the lowest dose (231 mg/kg-day) is a frank effect level (FEL) based on decreased survival. Weisburger et al. (1978a) The peer-reviewed rat study by Weisburger et al. (1978a) is selected as the principal study for derivation of the cancer p-OSF. Weisburger et al. (1978a) administered o-toluidine HC1 (purity 97-99%) to groups of 25 male Sprague-Dawley-derived CD rats from Charles River in a 24-month study beginning at concentrations of 8000 or 16,000 ppm in the diet for 3 months, then reduced to 4000 or 8000 ppm in the diet for 15 additional months, after which they were fed a control diet for 6 additional months before scheduled sacrifice. Several chemicals were tested and reported in this report. For all compounds, the higher dose was selected to be the maximum tolerated dose, but if high mortality occurred or body weight became 10% lower than the concurrent control, the doses were reduced. Body weight was routinely measured to keep track of the differences. Each compound was run in conjunction with a control, which is considered the concurrent control. The pooled control combines the control groups for each compound (start times were stratified over an 8-month period). The time-weighted human equivalent doses are 72.1 and 144.1 mg/kg-day calculated based on the 3-month exposure to 8000 or 16,000 ppm, 14 o-Toluidine ------- FINAL 12-3-2012 the 15-month exposure to 4000 or 8000 ppm, and the 6 months on control diet (using average body weight [U.S. EPA, 1994b] and daily food consumption [U.S. EPA, 1988]), and the ratio of animal-to-human body-weight adjustment raised to the Vi power. Necropsies were performed only on animals that died or were sacrificed after 6 months of exposure. The following tissues were processed for histopathology: gross lesions and tumor masses, lung, liver, spleen, kidney, adrenals, intestines, stomach, heart, bladder, reproductive organs, and pituitary gland. A Fischer's Exact test was used to determine the differences between treatment groups and the control groups. There was a significant increase in subcutaneous fibromas and fibrosarcomas in both the low- (18/23) and high- (21/24) dose groups compared to the control group (concurrent: 0/16; pooled: 18/111) (see Table B.5). There was a nonsignificant increase in bladder tumors in both the low- (3/23) and high- (4/24) dose groups compared to the control (concurrent: 0/16; pooled: 5/111). Multiple tumors (this is assumed to be more than one tumor per rat, although the study authors did not specify) were significantly increased above the pooled controls in the high-dose group (8/24) but not the low-dose group (6/23) compared to the control groups (concurrent: 3/16; pooled: 14/111). Hecht et al. (1982) Hecht et al. (1982) administered o-toluidine HC1 (purity not reported) to 30 male F344 rats at a concentration of 0.028 mol/kg diet for 72 weeks. Study authors calculated a mean daily dose of o-toluidine HC1 to be 0.062 g/rat based on measured mean daily food consumption. This is equivalent to 163 mg/kg-day based on a standard average body weight of 0.380 kg in F344 rats during a chronic-duration study (U.S. EPA, 1994b). The human equivalent dose based on an animal-to-human body-weight ratio raised to the Vi power is 44.2 mg/kg-day. After 72 weeks of dosing, animals were placed on control diet. Animals were sacrificed when found moribund or after 93 weeks and were necropsied. It was stated that all major organs were processed for histopathology, but a detailed list was not provided. There was a decrease in survival of dosed rats beginning in the 18th month. Although the study authors stated that a chi-square test was used, it appears that results were compared to the o-nitrosotoluene group and not the control. Therefore, a Fisher's Exact test is used to analyze the data. There was a significant increase in the following tumors in treated rats compared to the concurrent controls: skin fibromas (25/30 versus 1/27), spleen fibromas (10/30 versus 0/27), mammary fibroadenomas (11/30 versus 0/27), and peritoneal tumors (14/30 versus 2/27). Smaller, nonsignificant, increases were observed for liver hepatomas (2/30 treated; 0/27 controls), bladder papilloma (3/30 treated; 0/27 controls), bladder carcinoma (1/30 treated; 0/27 controls), and mammary carcinomas (2/30 treated; 0/27 controls). The use of a single dose level precludes dose-response modeling. NCI (1979b) B6C3Fi mice (20 controls/sex, 50/dose/sex) were dosed in the diet at levels of 0, 1000, or 3000 ppm for a period of 102 to 103 weeks (NCI, 1979b). Study details were similar to those discussed above in the rat study (NCI, 1979a). HEDs are 23.0 and 73.8 mg/kg-day in males and 26.5 and 91.9 mg/kg-day in females calculated based on average body weight from the study and daily food consumption (U.S. EPA, 1988), and the ratio of animal-to-human body-weight adjustment raised to the Vi power. Animals were checked twice daily for morbidity and mortality. Clinical observations, palpations, and body-weight measurement were conducted once per month. Animals were necropsied and evaluated for gross signs of toxicity, and tissues were examined microscopically. There was a decrease in mean body weight in treated male and female mice, but, in contrast to the rats, there was no significant dose-related decrease in 15 o-Toluidine ------- FINAL 12-3-2012 survival. Statistical analyses conducted by the study authors indicate that incidence of hemangiosarcomas of all sites in male mice is significantly increased (p < 0.05 trend test; see Table B.6). In female mice, investigators reported a significant, dose-related increase in the number of combined hepatocellular carcinomas and adenomas, which was not observed in male mice. Weisburger et al. (1978b) Weisburger et al. (1978b) also administered o-toluidine HC1 (purity 97-99%) to groups of 25/dose/sex CD-I mice in a 24-month study, beginning at concentrations of 16,000 or 32,000 ppm in the diet for 3 months, followed by 8000 or 16,000 ppm in the diet for 15 additional months, after which they were fed the control diet for 6 additional months before scheduled sacrifice. Methodology mirrored that in the rat experiment, except the pituitary was not routinely examined histologically. The time-weighted HEDs are 210.5 and 420.9 mg/kg-day in male mice and 211.8 and 423.7 mg/kg-day in female mice. Necropsies were performed only on animals that died after 6 months of exposure. Tissues were processed for histopathology. There was a significant increase in vascular tumors in both the low- and high-dose groups compared to the control in male and female groups (see Table B.7). Incidence of vascular tumors in the simultaneous control, pooled control, low-dose group, and high-dose group were 0/14, 5/99, 5/14, and 9/11, respectively, in male mice and 0/15, 9/102, 5/18, and 9/21, respectively, in female mice. Developmental and Reproduction Studies No studies could be located regarding the effects on development and reproduction resulting from oral exposure of animals to o-toluidine. Hiles and Abdo (1990) report on two dermal exposure studies that examined the developmental and reproductive effects of o-toluidine that indicate chemical-related effects. The original sources of Malysheva and Zaitseva (1982) and Malysheva et al. (1983) were unavailable for review at this time. The original study publications were published in a foreign language. The brief summary of the two studies stated that an unspecified number of animals (species not specified) were treated dermally (doses and purity not reported) for 4 months with o-toluidine. The summary also stated treatment resulted in changes in ovarian cycle, ovarian morphostructure, ability to reproduce in females, and stimulation of spermatogenesis in males. It is further stated that progeny were also affected, but no specific effects were reported. Inhalation Exposures There is no suitable information to provide in this regard. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) Short-term Studies Haskell Laboratory (1994) conducted a study sponsored by Dupont & Co. that examined urinary bladder toxicity in rats after a 14-day feeding exposure. o-Toluidine (purity 99.5%) was added to the diet via an ethanol vehicle and administered to F344 rats (10-15 rats/sex/dose) for 14 days at dose levels of 0, 500, 3000, or 6000 ppm. The mean daily intake was estimated by the study authors to be 40.4, 238, and 449 mg/kg-day in males and 43.5, 251, and 481 mg/kg-day in females. It was reported that the test substance was not stable in the diet (30% decrease in concentration after 7 days), and doses were not adjusted for test substance stability. Cage side observations and checks for moribund animals were made twice daily, body weights were 16 o-Toluidine ------- FINAL 12-3-2012 measured three times weekly, and urine was collected on Days 6-7 and Days 13-14. Average daily food consumption was measured weekly. At sacrifice, blood was collected and analyzed specifically for methemoglobin levels. Necropsies were performed at the end of the study period, and bladder and duodenum were removed and processed for analysis of cell proliferation (5 rats/sex/dose) and unscheduled DNA synthesis (5 rats/sex/dose). None of the animals died during treatment (Haskell Laboratory, 1994). Clinical signs of toxicity included wet and stained perineums in females of the high-dose group. There was a significant decrease in body weight in high-dose males and females that was likely due to the low weight gain during the first week of exposure. A significant decrease in food consumption was noted in the mid- and high-dose males and females during the first week. Significant increases in bladder epithelial cell proliferation were observed in mid- and high-dose females and high-dose males. Histopathology revealed mild urothelial hyperplasia in females, slight urothelial thickening in males, and unscheduled DNA synthesis (UDS) in both males and females in the high-dose groups. Furthermore, the investigators observed a significant and dose-related increase in methemoglobin levels in both sexes at all dose levels. The study authors established a NOAEL of 500 ppm (equivalent to 40.4 and 43.5 mg/kg-day in males and females, respectively) for urinary bladder toxicity (LOAEL is 238 and 251 mg/kg-day in male and females, respectively). The study authors could not establish a NOAEL for methemoglobinemia; therefore, the LOAEL is 40.4 and 43.5 mg/kg-day in males and females, respectively, based on increased levels of methemoglobin. Short et al. (1983) conducted a short-term toxicity study where they administered o-toludine (purity 99.3%) to male F344 rats (10/time point) via daily gavage at a dose level of 225 mg/kg-day for 5, 10, or 20 days. Body weight, organ weight, and histopathology were evaluated. Clinical signs reported by the study authors included transient cyanosis and rough hair coat. Of the 30 rats treated, 10 of the rats died before the end of the 20 days of treatment. Body weight was significantly lower after 5 and 10 days but was equal to the control by Day 20. Spleen weight was significantly increased at all time points, but liver and kidney weights were not affected. The effects on the spleen were confirmed with significant increases in spleen congestion, hemosiderosis, and hematopoiesis in the survivors at all three time points. Hypercellularity of bone marrow was significantly increased only at the 10-day time point. No changes in histopathology were noted in the liver. The LOAEL is 225 mg/kg-day and was the only dose tested. Other Studies Toxicokinetic data demonstrate that o-toluidine is readily absorbed via the gastrointestinal tract and excreted within 24 hours in the urine (Cheever et al., 1980; Son et al., 1980). A greater amount of o-toluidine is excreted unchanged (21%) compared to either of the other toluidine isomers (2.5%). o-Toluidine is not mutagenic in Ames assays (see Table 3). However, in a review or o-toluidine genotoxicity, Danford (1991) states that this is only the case in standard Ames assays, and positive mutagenicity results have been obtained at high concentrations using different types of metabolic activation systems. Additionally, Danford (1991) states that most yeast studies have also been negative, but o-toluidine has been found to cause deletion mutations in yeast at concentrations that reduced survival (Brennan and Schiestl, 1999). o-Toluidine was weakly positive for Trp+ revertants in modified yeast (Saccaromyces cerevisiae C658-K42) (Morita et al., 1989). There have been mixed results with cytogenic 17 o-Toluidine ------- FINAL 12-3-2012 assays. Danford (1991) concludes that it is a clastogen but only with prolonged exposure. Morita et al. (1997) summarized results from six independent micronuclei assays in male B6C3Fi mice receiving one to four intraperitoneal (ip) injections at doses up to 1000 mg/kg (the LD50 was stated to be 800 mg/kg). There was a small—but significant—increase in micronucleated polychromatic erythrocytes in the bone marrow only with the 800- or 1000-mg/kg dose. This was not considered biologically significant, due to the deaths in these animals, and the tests were considered negative. Chromosomal aberrations have been found in Chinese hamster ovary (CHO) cells in vitro (Gulati et al., 1985) but not after ip injection to B6C3Fi mice in vivo (McFee et al., 1989). o-Toluidine has been found to cause sister chromatid exchange (SCE) in both CHO cells in vitro (Gulati et al., 1985) and in B6C3Fi mice in vivo (McFee et al., 1989) and to cause transformations in BALB/c-3T3 and Syrian hamster embryo (SHE) transformation assays (Matthews et al., 1993; Kerckaert et al., 1998) (see Table 3). 18 o-Toluidine ------- FINAL 12-3-2012 Table 3. Other Studies Tests Materials and Methods Results Conclusions References Toxicokinetic Studied distribution of 14C following single dose of either 50- or 400 mg/kg subcutaneous dose of o-[methyl-14C]toluidine to male F344 rats (2/dose) after 24 and 48 h. Urine was the major excretory pathway at both dose levels, followed by fecal excretion and breath. Tissue concentration of radiolabel after 48 h was highest in liver with approximately 0.12% and 0.325% at the 50- and 400-mg/kg dose, respectively. NA Sonet al. (1980) Toxicokinetic Studied excretion and metabolism of 14C following single oral dose of 50 or 500 mg/kg of o-toluidine to male Sprague-Dawley rats; /Moluidinc and /w-toluidine were also tested. 92% of o-toluidine was excreted via the urine within 24 h of dosing. The amount of parent compound excreted was greater for o-toluidine (21%) than either of the other isomers (2.5%). Aminomethylphenols were the metabolite detected in the urine. NA Cheever et al. (1980) Genotoxicity Tested for reverse mutation in Salmonella typhimurium (Ames assay) TA1535, TA1537, TA1538, TA98, and TA100 with and without metabolic activation at a dose level of 1000 |ig or 9.33 (imole. Nonmutagenic in all strains tested. o-Toluidine was not considered to be mutagenic. Simmon (1979) Genotoxicity Tested for reverse mutation in Salmonella typhimurium (Ames assay) TA100 with metabolic activation and for DNA damage in Chinese hamster lung fibroblasts (V79) with metabolic activation incubated for 2 h with concentrations ranging from 0.3 to 10.0 mM (32-1070 mg). Nonmutagenic and did not cause DNA breakage. o-Toluidine was not considered to be mutagenic or cause DNA damage. Zimmer et al. (1980) Genotoxicity Tested for sperm head abnormalities using ip of 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, or 0.5 mg/kg-day for 5 days in male (CBA x BALB/c)Fi mice. Negative. o-Toluidine did not cause sperm head abnormalities. Topham (1980) 19 o-Toluidine ------- FINAL 12-3-2012 Table 3. Other Studies Tests Materials and Methods Results Conclusions References Genotoxicity Tested for reverse mutation in Salmonella typhimurium (Ames assay) TA1535, TA1537, TA1538, TA98, and TA100 with and without metabolic activation at doses of 0.32, 1.0, 3.2, or 10.0 ng/plate. Nonmutagenic in all test strains. o-Toluidine was not considered to be mutagenic. Baker and Bonin (1981) Genotoxicity Tested in vivo mutagenicity in the bone marrow micronucleus test (injected at 0 and 24 h, samples taken at 48, 72, and 96 h); dose level was at 50, 80, and 100% of LD50 in B6C3Fi mice (800 mg/kg). Nonclastogenic. o-Toluidine is not considered to be clastogenic. Salamone et al. (1981) Genotoxicity Tested for chromosomal aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells with concentrations up to 500 ng/ml in the absence of metabolic activation and 5000 ng/ml in the presence of metabolic activation. The highest dose was generally cytotoxic. However, in the one assay that was not toxic at 500 |ig/plate without metabolic activation, there was nearly twice as many SCEs as in the vehicle control. There was also an increase in chromosomal aberrations, with a concentration of 500 Hg/plate or greater depending on the assay conditions. o-Toluidine was considered positive for inducing SCE in the presence and absence of metabolic activation. o-Toluidine was considered positive for chromosomal aberration only when fixation was done at 6 h. Gulati et al. (1985) Genotoxicity Tested for chromosomal aberrations and sister chromatid exchanges (SCE) in vivo after a single ip injection at doses of 150, 300, or 600 (maximum tolerated dose) mg/kg to male B6C3Fi mice. There was not an increase in chromosomal aberrations or micronuclei, but there was an increase in the frequency of SCE in two separate trials. o-Toluidine was positive for inducing SCE but not chromosomal aberrations or micronuclei in vivo. McFee et al. (1989) Genotoxicity Tested for Trp+ reversion in Saccaromyces cerevisiae C658-K42 (strain with increased permeability), with concentrations ranging from 0.5 to 3.0 mg/mL. At 3.0 mg/mL there was an increase in Trp+ revertants (6.9 ± 1.9) in the presence of metabolic activation compared to the control (2.4 ± 1.3). It was concluded that o-toluidine was weakly positive. Morita et al. (1989) Genotoxicity BALB/c-3T3 cell transformation assay. 50% cytotoxicity occurred with a concentration of 4.33 mM. Statistical sensitivities for the three trials were stated to be 87/110, 106/110, and 59/110. o-Toluidine was considered active in the BALB/c-3T3 cell transformation assay. Matthews et al. (1993) 20 o-Toluidine ------- FINAL 12-3-2012 Table 3. Other Studies Tests Materials and Methods Results Conclusions References Genotoxicity Tested in vivo mutagenicity in the bone marrow micronucleus test with 1 to 4 ip injections up to 1000 mg/kg (LD50 = 800 mg/kg) in male B6C3Fi mice, results were measure 24 h after last dose. There was a slight increase in micronucleated polychromatic erythrocytes in the bone marrow of dying animals, which was not considered biologically significant. o-Toluidine was not considered to be clastogenic. Morita et al. (1997) Genotoxicity Tested for genotoxicity using the 24-h Syrian hamster embryo (SHE) transformation assay at a pH of 6.7 with concentrations up to 1200 iig/mL (higher concentrations were not soluble). There was a significant increase in transformations with concentrations ranging from 750 to 1200 ng/mL, but there was only 38% cytotoxicity at the highest dose tested. o-Toluidine was positive in the SHE transformation assay. Kerckaert et al. (1998) Genotoxicity Tested for deletion (DEL) recombination in yeast, with concentrations ranging from 3.0 to 6.0 mg/mL. An increase in revertants (more than 3-fold) was observed, with concentrations that caused decreased survival (5.0 and 6.0 mg/mL, survival 3.90 and 0.49% compared to 93% in the control). Survival was increased and revertants decreased when the antioxidant A-acetyl cysteine was added to the culture. o-Toluidine caused deletion mutations in yeast. Brennan and Schiestl (1999) 21 o-Toluidine ------- FINAL 12-3-2012 DERIVATION OF PROVISIONAL VALUES Tables 5 and 6 below present a summary of noncancer and cancer reference values, respectively. IRIS data are indicated in the table if available. DERIVATION OF ORAL REFERENCE DOSE Derivation of Subchronic Provisional RfD (Subchronic p-RfD) There are two subchronic-duration studies available (see Table 4). Ekman and Strombeck (1947) did not provide sufficient details. There is no information provided on the doses used, and there does not appear to be a control group. While the NTP (1996) study was a peer-reviewed GLP study, only one dose was tested. The NTP (1996) study was conducted to compare the effects of o-toluidine HC1 to o-nitrotoluene, to demonstrate the progression of toxic effects over time (13 weeks on the diet compared to 26 weeks on the diet), and, also, to illustrate the reversibility of effects (13 weeks on the diet followed by 13 weeks on control diet); therefore, doses were selected to ensure a response. A potential POD, blood methemoglobin, is suggested by a short-term study (Haskell Laboratory, 1994) that observed a dose-dependent increase in methemoglobin levels. However, other studies with toluidine isomers suggest that the methemoglobin response likely decreases with longer durations of exposure, while other organ effects likely related to blood effects such as methemoglobinemia become apparent (Malik-Brys and Senczuk, 1995). For example, some of the splenic and hepatic effects noted in the NTP (1996a) 13-week study are consistent with persistent chronic blood effects. The NTP (1996b) study demonstrates that the splenic effects progress with time on the diet. Therefore, the methemoglobin levels observed in the 14-day study (Haskell Laboratory, 1994) are supportive of some of the effects seen in the NTP (1996) study but are not useful for deriving the subchronic p-RfD. Table 4 below describes studies that are relevant for deriving the oral provisional RfDs. The NTP (1996a) 13-week study provides the best data to derive a subchronic p-RfD. The NTP (1996b) study, after 26 weeks, reported similar effects to those seen at 13 weeks in the NTP (1996a) study at a slightly lower dose level, and supports the selection of a POD from the NTP (1996a) 13-week study. Although both the NTP (1996a) and NTP (1996b) studies could serve as the principal study for derivation of a subchronic p-RfD, the 13-week study (NTP, 1996a) is selected because the exposure duration is considered to be subchronic for rodent studies. Because there is not enough dose-response information available, the data are not amenable to BMD modeling for derivation of the subchronic p-RfD. Further, the NTP (1996a) study provides only a LOAEL as the POD, thus requiring four full UFs (UFA, UFH, UFD, UFL) resulting in a composite UF (UFC) of 10,000. Consequently, the high uncertainty combined with a very limited database precludes derivation of a subchronic p-RfD. However, the NTP (1996a) study does provide sufficient information to derive a screening subchronic p-RfD in Appendix A. A chronic p-RfD is not derived because no acceptable chronic-duration toxicity studies are available and extrapolation from the subchronic study, NTP (1996a) would entail applying a full suite of 10-fold UFs (UFA, UFD, UFH, UFL, UFS) resulting in a composite UFC of 100,000. Additionally, the low survival seen in the chronic-duration NCI (1979a) rat study (approximately 27% at the low dose of 231 mg/kg-day in males and 55% at 264 mg/kg-day in females) suggests that the subchronic-duration LOAEL of 301 mg/kg-day from the principal study (NTP, 1996a) should not be extrapolated to a chronic-duration LOAEL. 22 o-Toluidine ------- FINAL 12-3-2012 Table 4. Summary of Relevant Oral Systemic Toxicity Studies for o-Toluidine References Species, # /Sex (M/F) Exposure (ppm unless otherwise noted) Frequency/ Duration NOAELadj" (mg/kg-d) LOAELadj" (mg/kg-d) Critical End point Ekman and Strombeck (1947) Rat, 10 (sex not specified) Not specified; authors added o-toluidine (7.5 to 12 mg/d) in rice flour diet At least 91 d ND ND Three animals exhibited metaplasia and early epithelial proliferation in bladder mucous membrane NTP (1996a) Rat, 20 M 0,5000 13 wk in diet None0 301d Decreased body weight; increased relative weight of the liver, kidney, spleen, and testes; and increased incidence of lesions in the liver, kidneys, bladder, and spleen NTP (1996b) Rat, 20 M 0, 5000 26 wk in diet None 285d Decreased body weight; increased relative weight of the liver, kidney, spleen, and testes; and increased incidence of lesions in the liver, kidneys, bladder, and spleen NCI (1979a) Rat, 50M/50F 0, 3000, 6000 101-104 wk in diet None None. 23 ld is anFEL Decreased survival "NOAEL/jy = NOAEL (ppm or mg/kg food) x food consumption (kg/day) ^ body weight (kg). bLOAELADJ = LOAEL (ppm or mg/kg food) x food consumption (kg/day) ^ body weight (kg). °No NOAEL was identified. The NOAEL is considered equal to a LOAEL 10 for screening purposes. dThese values were provided in the study report. ND = Not determined. 23 o-Toluidine ------- FINAL 12-3-2012 Table 5. Summary of Noncancer Reference Values for o-Toluidine Toxicity Type (Units) Species/Sex Critical Effect p-Reference Value3 POD Method POD UFC Principal Study Screening subchronic p-RfD F344 rat/M Increased spleen weight and incidence of spleen lesions 2 x 1(T2 LOAEL 301 10,000 NTP (1996a) Chronic p-RfD (mg/kg-day) None Subchronic p-RfC (mg/m3) None Chronic p-RfC (mg/m3) None "The screening subchronic p-RfD adjusts for the fact that o-toluidine HC1 was administered instead of o-toluidine using the following calculation: p-RfD o-toluidine = p-RfD o-toluidine HC1 x (molecular weight of o-toluidine ^ molecular weight of o-toluidine HC1) or 3 x 10 2 mg/kg-day x (107.16 143.62) = 2 x 10 2 mg/kg-day. Table 6. Summary of Cancer Reference Values for o-Toluidine Toxicity Type Species/Sex Tumor Type Cancer Value" Principal Study p-OSF F344 rat/M Subcutaneous fibroma and fibrosarcoma 1.6 x 10 2 (mg/kg-day) 1 Weisburger et al. (1978a) p-IUR None aThe p-OSF adjusts for the fact that o-toluidine HC1 was administered instead of o-toluidine using the following calculation: p-OSF o-toluidine = p-OSF o-toluidine HC1 x (molecular weight of o-toluidine ^ molecular weight of o-toluidine HC1) or 2.2 x 10 2 (mg/kg-day) 1 x (107.16 143.62) = 1.6 x 10 2 (mg/kg-day) 24 o-Toluidine ------- FINAL 12-3-2012 Derivation of Chronic Provisional RfD (Chronic p-RfD) No chronic p-RfD values can be derived. The Weisburger et al. (1978a,b) study provides only cancer incidence and does not provide information on any possible noncancer effects or on mortality. The only chronic-duration study available that provides any nonneoplastic results (i.e., NCI, 1979a) also had decreased survival (i.e., 27% survival in males and 55% in females at 3000 ppm) in rats relative to controls (i.e., 90% and 80% survival in control males and females, respectively) at the lowest dose tested (see Table B.3). The decreased survival in the NCI (1979a) study occurred at doses (231 mg/kg-day in males and 264 mg/kg-day in females) that were equivalent to the dose that caused reduced survival in the NTP (1996) subchronic-duration rat studies (301 mg/kg-day), which precludes using the subchronic-duration study to extrapolate to a chronic p-RfD. In addition, using a subchronic-duration LOAEL to extrapolate to a chronic-duration NOAEL would entail applying a full suite of 10-fold UFs (UFA, UFD, UFh, UFl, UFs) resulting in a UFC of 100,000. Consequently, derivation of a chronic p-RfD is precluded. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS No subchronic or chronic p-RfC values can be derived. There are no animal inhalation studies, and the epidemiology studies in humans do not provide any concentrations for o-toluidine, nor can the studies make any definitive relationship between o-toluidine exposure and any toxic effect. CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR Table 7 identifies the cancer weight-of-evidence (WOE) descriptor for o-toluidine. Table 7. Cancer WOE Descriptor for o-Toluidine Possible WOE Descriptor Designation Route of Entry (Oral, Inhalation, or Both) Comments "Carcinogenic to Humans " N/A N/A Occupational studies, although consistently and positively associated with bladder cancer, cannot establish any definitive link with exposures to o-toluidine and are confounded with exposure to other known or suspected carcinogens. "Likely to Be Carcinogenic to Humans" Selected Oral Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), o-toluidine is "Likely to be Carcinogenic to Humans" based on evidence of carcinogenicity in rats and mice in the NCI (1979a,b) and Weisburger et al. (1978a,b) oral bioassays. "Suggestive Evidence of Carcinogenic Potential" N/A N/A "Inadequate Information to Assess Carcinogenic Potential" N/A N/A "Not Likely to Be Carcinogenic to Humans " N/A N/A No strong evidence of noncarcinogenicity in humans or animals is available. 25 o-Toluidine ------- FINAL 12-3-2012 MODE-OF-ACTION DISCUSSION The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) define mode of action (MO A) as the following: ".. .a sequence of key events and processes starting with the interaction of an agent with a cell, proceeding through operational and anatomical changes, and resulting in cancer formation.. .There are many examples of possible modes of carcinogenic action, such as mutagenicity, mitogenesis, inhibition of programmed cell death, cytotoxicity with reparative cell proliferation, and immune suppression" (p. 1-10). Summary of Mutagenicity and Genetic Toxicology Studies Data on the mutagenicity of o-toluidine are equivocal. o-Toluidine is not mutagenic in standard Ames assays but has been positive in modified assays including the use of different types of metabolic activation systems at high concentrations. It has also been generally negative in yeast studies, but it has been found to cause deletion mutations in yeast (Brennan and Schiestl, 1999) and was weakly positive for Trp+ revertants in modified yeast (Saccaromyces cerevisiae C658-K42) (Morita et al., 1989). There have also been mixed results with cytogenic assays. Danford (1991) concludes that it is a clastogen but only with prolonged exposure. Morita et al. (1997) summarize results from six independent micronuclei assays in male B6C3Fi mice receiving one to four ip injections at doses exceeding the LD50 (800 mg/kg). There was a small—but significant—increase in micronucleated polychromatic erythrocytes in the bone marrow only in dying animals. The study author did not consider this to be biologically significant, and the tests were considered negative. Chromosomal aberrations have been found in CHO cells in vitro (Gulati et al., 1985) but not after ip injection to B6C3Fi mice in vivo (Salamone et al., 1981). o-Toluidine has been found to cause SCE in both CHO cells in vitro (Gulati et al., 1985) and in B6C3Fi mice in vivo (McFee et al., 1989) and to cause transformations in BALB/c-3T3 and SHE transformation assays (Matthews et al., 1993; Kerckaert et al., 1998). Taken together, there are insufficient data to determine the MOA of o-toluidine. DERIVATION OF PROVISIONAL CANCER POTENCY VALUES There are insufficient data to determine the carcinogenic mode of action. Therefore, a linear approach is applied. Derivation of Provisional Oral Slope Factor (p-OSF) The study by Weisburger et al. (1978a) is selected as the principal study. The cancer end point is the incidence of subcutaneous fibromas and fibrosarcomas in male rats. This study is generally well conducted, and the data from this study are able to support a quantitative cancer dose-response assessment. There is no GLP statement, but the study otherwise meets the standards of study design, performance, and presentation of information to examine carcinogenicity. Details are provided in the "Review of Potentially Relevant Data" section. Among the available, acceptable studies, Weisburger et al. (1978a) represents the highest OSF from relevant studies in the database. A supporting study (i.e., NCI, 1979) is a peer-reviewed technical report from the National Cancer Institute (NCI). There were two acceptable carcinogenicity studies, each performed in rats and mice, showing significant increases in a number of different tumor end points. All relevant tumor end points in the Weisburger et al. (1978a,b) and NCI (1979a,b) studies were modeled using BMDS software version 2.1.2 (see Table C.l; U.S. EPA, 2010c). Individual tumor types were modeled 26 o-Toluidine ------- FINAL 12-3-2012 and all tumor types that could be combined were also modeled. All the remaining tumor types that were not significantly different from the control animal frequencies were not considered biologically relevant. Weisburger et al. (1978a) was selected as the principal study because the increase in subcutaneous fibromas and fibrosarcomas in male rats provided the highest OSF with adequate model fit. An increase in subcutaneous fibromas and/or fibrosarcomas was also found in the NCI study in rats (1979a) and mice (1979b) and in the Hecht et al. (1982) study in male rats. Although the Hecht (1982) study also found an increase in subcutaneous fibromas, with results at a lower dose (i.e., 44.2 compared to 72.1 mg/kg-day in the Weisburger et al. [1978a] study), the Hecht study only used one dose, and no dose-response can be established. However, the tumor findings of these three chronic-duration studies lend internal consistency to the entire o-toluidine database. Because the study administered o-toluidine HC1, the p-OSF is converted to reflect the molecular weight difference between o-toluidine and the salt form. In addition, because the study lowered the dose after 3 months, additional time-weighted adjustments were made for the two different concentrations. The following dosimetric adjustments were made for dietary treatment in adjusting doses for oral cancer analysis for the first 3 months (90 days) of treatment: (DOSEaDJ, HED)\¥eisburger et al., 1978a = (concentration) Weisburger et al., 1978a x food consumption per day x (1 -h body weight) x (days dosed ^ total days) x body-weight adjustment Body-weight adjustment = (BWa^BWh)1'4 BWa = 0.267 kg (average body weight for male rats in subchronic-duration study) (U.S. EPA, 1994b) BWh = 70 kg (human reference body weight) (U.S. EPA, 1997) Body-weight adjustment = (0.267 ^ 70)1/4 = 0.2485 (DOSEadj, HED)weisburger et ai., 1978a = 8000 mg/kg x (0.023 kg/day) x (1 -H 0.267 kg) X (90 days ^ 90 days) x 0.2485 = 689.08 mg/kg-day x 1 x 0.2485 (DOSEadj, HED)weisburger et ai., 1978a = 171.26 mg/kg-day The following dosimetric adjustments were made for dietary treatment in adjusting doses for oral cancer analysis for the last 15 months (455 days) of treatment: (DOSEadj, HED)weisburgeretai., 1978a = (concentration) Weisburger et al., 1978a x food consumption per day x (1 -h body weight) x (days dosed ^ total days) x body-weight adjustment Body-weight adjustment = (BWa^BWh)1'4 BWa = 0.523 kg (average body weight for male rats in chronic-duration study) (U.S. EPA, 1994b) BWh = 70 kg (human reference body) (U.S. EPA, 1997) 27 o-Toluidine ------- FINAL 12-3-2012 Body-weight adjustment = (0.523 /70)1/4 = 0.294 (DOSEadj, m:i))we,sburgcretaL 1978a = 4000 mg/kg x (0.036 kg/day) X (1 H- 0.523 kg) x (455 days ^ 455 days) x 0.294 = 275.328 mg/kg-day x 1 x 0.294 = 275.328 mg/kg-day x 0.294 (DOSEadj, HED)weisburger et al., 1978a = 80.95 mg/kg-day The time-weighted average HED including the 6 months on the control diet = [(DOSEadj, hed3 months x 90 days) + (DOSEadj, hed15 months x 455 days)] ^ study duration = [(171.26 x 90) + (80.95 x 455)] - (90 + 455 + 180) = 72.1 mg/kg-day; similar calculations for the high dose (16,000 ppm reduced to 8000 ppm) derived a value of 144.1 mg/kg-day. Table 8 presents BMD input data for incidence of subcutaneous fibromas and fibrosarcomas in male F344 rats exposed to o-toluidine in the diet for 18 months. Table 8. BMD Input for Incidence of Subcutaneous Fibromas and Fibrosarcomas in Male F344 Rats Exposed to o-Toluidine via the Diet3 (Dose)n (ppm) (DOSEadj,hed),, (mg/kg-d) Number of Subjects Responseb 0 0 16 0(0) 8000 reduced to 4000 72.1 23 18(78)c 16,000 reduced to 8000 144.1 24 21(87.5)c aWeisburger et al. (1978a). bNumber of rats with tumors, () = percentage of rats with tumors. Statistically significant in pairwise test versus control. Table 9 shows the modeling results. Adequate model fit is obtained for the subcutaneous fibroma and fibrosarcoma incidence data using the BMDS version 2.1.2 (U.S. EPA, 2010c) multistage cancer model. The BMD modeling results with 10% extra risk for subcutaneous fibroma and fibrosarcoma yield a BMDio of 6.08 mg/kg-day and a BMDLio of 4.50 mg/kg-day (see Table 13). 28 o-Toluidine ------- FINAL 12-3-2012 Table 9. Model Predictions for Subcutaneous Fibroma and Fibrosarcomas3 Model Goodness of Fit />-Valucb AIC for Fitted Model BMDl0 (mg/kg-day) BMDLl0 (mg/kg-day) Conclusions Multistage cancer 0.57 45.25 6.08 4.50 Lowest AIC Lowest BMDL aWeisburger et al. (1987). Values <0.10 fail to meet conventional goodness-of-fit criteria. AIC = Akaike's Information Criteria; BMD = benchmark dose; BMD10 = BMD at a response rate of 10% incidence, extra risk; BMDL = lower confidence limit (95%) on the benchmark dose. The curve and BMD output for the selected model, only, are provided in the BMD supplement to the document (Appendix C). The p-OSF0_toiuidine hci = 0.1^- BMDLio = 0.1 ^ 4.50 mg/kg-day = 2.2 x 10~2 (mg/kg-day)"1 Conversion of the p-OSF based on o-toluidine HCI salt (molecular weight of 143.62) to o-toluidine (molecular weight of 107.16) is as follows: p~OSFo-toiuidine = p-OSF0.toiuidineHci x (MW of o-toluidine ^ MW of o-toluidine HCI) = 2.2 x 10~2 (mg/kg-day)-1* (107.16 - 143.62) = 2.2 x 10~2 (mg/kg-day)_1x 0.746 = 1.6 x 10~2 (mg/kg-day)-1 Derivation of Provisional Inhalation Unit Risk (p-IUR) No human or animal studies examining the carcinogenicity of o-toluidine following inhalation exposure have been located. Therefore, derivation of an IUR is precluded. 29 o-Toluidine ------- FINAL 12-3-2012 APPENDIX A. PROVISIONAL SCREENING VALUES For reasons noted in the main PPRTV document, it is not possible to derive a provisional subchronic p-RfD for o-toluidine. However, information is available for this chemical which, although insufficient to support derivation of a provisional toxicity value, under current guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk Technical Support Center summarizes available information in an Appendix and develops a "screening value." Appendices receive the same level of internal and external scientific peer review as the PPRTV documents to ensure their appropriateness within the limitations detailed in the document. Users of screening toxicity values in an appendix to a PPRTV assessment should understand that there is considerably more uncertainty associated with the derivation of an appendix screening toxicity value than for a value presented in the body of the assessment. Questions or concerns about the appropriate use of screening values should be directed to the Superfund Health Risk Technical Support Center. DERIVATION OF SCREENING SUBCHRONIC ORAL REFERENCE DOSE The 13-week component of the NTP (1996a) report is selected as the principal study for the derivation of the screening subchronic p-RfD. The critical end point is spleen toxicity as measured by increased relative spleen weight accompanied by increased incidence of lesions in the spleen in male F344 rats. The effects progressed with longer duration and were not entirely reversible after a 13-week recovery period. This study is peer reviewed and performed according to GLP principles. Details are provided in the "Review of Potentially Relevant Data" section. BMD analysis is not possible with these data because there was only one dose level presented in addition to the control. Among the available, acceptable studies, NTP (1996a) represents the lowest POD for developing a subchronic p-RfD. The POD in this study is a LOAEL of 301 mg/kg-day. Dosimetric adjustments for daily exposure No dosimetric adjustments were made for the dose in the principal study for dietary treatment, as the study authors report the average daily dose of 301 mg/kg-day. The screening subchronic p-RfD for o-toluidine, based on the LOAEL of 301 mg/kg-day in male rats exposed for 13 weeks, is derived as follows: Screening Subchronic p-RfD0_t0iuidineHci = LOAELadj ^ UF = 301 mg/kg-day ^ 10,000 = 3 x 10~2 mg/kg-day This value is normalized to account for the actual amount of o-toluidine from the compound used, o-toluidine HC1: Screening Subchronic p-RfD o-toluidine P~RfDo-toluidineHCl ^ (MW of O-toluidine • MW of o-toluidine HC1) = 3 x 10~2 mg/kg-day x (107.16 - 143.62) = 3 x 10~2 mg/kg-day x 0.746 = 2 x 10~2 mg/kg-day 30 o-Toluidine ------- FINAL 12-3-2012 Table A.l summarizes the uncertainty factors for the screening subchronic p-RfD for o-toluidine. DERIVATION OF SCREENING CHRONIC ORAL REFERENCE DOSE A screening chronic p-RfD is not derived because the only available chronic-duration study that reported noncancer effects (i.e., NCI, 1979) also reported low survival at both tested doses. Further, deriving a screening chronic p-RfD based on the same subchronic study (NTP, 1996) that was used to derive the screening subchronic p-RfD would result in a UFC of 100,000 based on 10-fold values for each of the following UFs: UFA, UFD, UFH, UFL, UFS. Consequently, the derivation of a screening chronic p-RfD is precluded. See the section titled "Derivation of Chronic Provisional RfD (Chronic p-RfD)" for further discussion. Table A.l. Uncertainty Factors for Screening Subchronic p-RfD for o-Toluidine UF Value Justification Notes ufa 10 A UFa of 10 is applied for interspecies extrapolation to account for potential toxicokinetic and toxicodynamic differences between rats and humans. There are no data to determine whether humans are more or less sensitive than rats to the liver, spleen, kidney, and bladder toxicity of o-toluidine. ufd 10 A UFd of 10 is applied because there are no acceptable two-generation reproduction studies or developmental studies. 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 10 A UFl of 10 is applied for using a POD based on a LOAEL. Only one dose was tested. UFS 1 A UFS of 1 is applied because a subchronic-duration study was utilized. UFC <3000 10,000 31 o-Toluidine ------- FINAL 12-3-2012 APPENDIX B. DATA TABLES Table B.l. Results in Male F344 Rats Exposed to Oral (in Feed) o-Toluidine for 13 Weeks3 Parameter Adjusted Daily Dose (mg/kg-d)b Control 301 Relative Body-weight (g) 331 293 (11%)C Food consumption (g/d) 14.9 13.5 (91%) Necropsy body weight (g) 345 ± 5d 298 ± 4 (86%)** Relative right kidney weight (mg/g) 3.27 ±0.03 3.50 ±0.05 (107%)** Relative liver weight (mg/g) 34.91 ±0.31 42.57 ± 0.36 (122%)** Relative spleen weight (mg/g) 2.12 ±0.03 7.43 ±0.16 (350%)** Relative right testis weight (mg/g) 4.61 ±0.03 5.07 ±0.07 (110%)** Hemosiderin pigmentation in the liver 0/10e 20/20** Kidney pigmentation 0/10 20/20** Transitional epithelium hyperplasia in the urinary bladder 0/10 10/20** Congestion in the spleen 0/10 20/20** Hematopoietic cell proliferation in the spleen 2/10 20/20** Hemosiderin pigmentation in the spleen 0/10 20/20** Thrombosis in the spleen 0/10 3/20 Fibrosis capsule in the spleen 0/10 20/20** aNTP (1996a). bAnimals were administered diets with 5000 ppm; adjusted daily doses were provided in the study report. °Number in parentheses is the percent of control. dMean ± standard error. eNumber of animals with lesions/number of animals. **p < 0.01 32 o-Toluidine ------- FINAL 12-3-2012 Table B.2. Results in Male F344 Rats Exposed to Oral (in Feed) o-Toluidine for 26 Weeks or for 13 Weeks with a 13-Week Recovery Period3 Parameter Adjusted Daily Dose (mg/kg-d)b Control 26 Wk (285) 13 Wk with 13-Wk Recovery (304) Body-weight (g) 382 314 (18%)° 342 (11%) Food consumption (g/d) 14.7 13.4 (91%) 13.9 (95%) Necropsy body weight (g) 389 ± 5d 316 ±3 (81%)** 351 ±9 (90%)** Relative right kidney weight (mg/g) 3.55 ±0.08 3.99 ±0.09 (112%)** 3.56 ±0.05 (100%) Relative liver weight (mg/g) 37.09 ±0.69 45.71 ±0.88 (123%)** 39.58 ± 1.13 (107%) Relative spleen weight (mg/g) 2.08 ±0.05 9.01 ±0.26 (433%)** 3.23 ±0.16 (155%)** Relative right testis weight (mg/g) 4.16 ±0.06 4.84 ±0.05 (116%)** 4.28 ±0.12 (103%) Hemosiderin pigmentation in the liver 0/10e 20/20 ** 11/20** Placental glutathione .S'-transfcrasc-positivc foci in the liver 17 ±2 145 ±61** Not conducted Kidney pigmentation 0/10 20/20** 20/20** Transitional epithelium hyperplasia in the urinary bladder 0/10 17/20** 0/20 Congestion in the spleen 0/10 20/20** 20/20** Hematopoietic cell proliferation in the spleen 3/10 20/20** 1/20 Hemosiderin pigmentation in the spleen 3/10 20/20** 18/20** Thrombosis in the spleen 0/10 2/20 0/20 Fibrosis capsule in the spleen 0/10 20/20** 20/20** Capsule, lymphatic, angiectasis in the spleen 0/10 6/20 15/20** aNTP (1996b,c). bAnimals were administered diets with 5000 ppm; adjusted daily doses were provided in the study report. °Number in parentheses is the percent of control. dMean ± standard error. "Number of animals with lesions/number of animals. **p < 0.01. 33 o-Toluidine ------- FINAL 12-3-2012 Table B.3. Incidence of Selected Noncancer Parameters in the F344 Rat Exposed to Oral (in Feed) o-Toluidine for 101 to 104 Weeks3 Parameter Exposure Group (ppm) (Average Daily Dose, mg/kg-d) Males 0 3000 (231) 6000 (525) Probability of survival13 90% 27% 0% Liver Necrosis 2/20 (10%)c 12/50 (24%) 17/49 (35%)* Bladder epithelial hyperplasia 0/20 (0%) 9/50 (18%)* 7/44 (16%) Females 0 3000 (264) 6000 (600) Probability of survival13 80% 55% 21% Liver Necrosis 0/20 (0%) 1/50 (2%) 15/49 (31%)* Bladder epithelial hyperplasia 0/20 (0%) 21/45 (47%)* 13/47 (28%)* aNCI (1979a). bActual mortality was not provided in the study report; results were estimated for the study termination from Kaplan and Meier curves provided in the study report. There were significant positive dose-response trends in mortality. °Number of animals with lesions/total number of animals (%). *p < 0.05 Fischer's Exact Test performed for this PPRTV document. 34 o-Toluidine ------- FINAL 12-3-2012 Table B.4. Incidence of Tumors in the F344 Rat Exposed to Oral (in Feed) o-Toluidine for 101 to 104 Weeks3 Parameter Exposure Dose (ppm) (Human Equivalent Dose, mg/kg-d) Males 0 3000 (63.0) 6000 (138.8) Subcutaneous fibroma 0/20 (0%)b 28/50 (56%)** 27/49 (55%)** Spleen sarcoma, NOS° 0/20 1/49 (2%) 3/42 (7%) Mesotheliomas of the tunica vaginalis or multiple organs 0/20 17/50 (34%)** 9/49 (18%)* Multiple organ osteosarcoma 0/20 (0%) 3/50 (6%) 5/49 (10%) Multiple organ fibrosarcoma 0/20 (0%) 8/50 (16%) 20/49 (41%)** Multiple organ sarcoma, NOS 0/20 (0%) 3/50 (6%) 11/49 (22%)* Multiple organ sarcoma, NOS, fibrosarcoma, angiosarcoma, or osteosarcoma 0/20 15/50 (30%)** 37/49 (76%)** Females 0 3000 (63.7) 6000 (140.4) Mammary fibroadenoma 6/20 (30%) 20/50 (40%) 35/49 (71%)* Bladder transitional-cell carcinoma 0/20 (0%) 9/45 (20%)* 22/47 (47%)** Multiple organ osteosarcoma 0/20 (0%) 0/50 (0%) 18/49 (37%)** Multiple organ sarcoma, NOS 0/20 (0%) 1/50 (2%) 2/49 (4%) Multiple organ sarcoma, NOS, fibrosarcoma, angiosarcoma, or osteosarcoma 0/20 3/50 (6%) 21/49 (43%)** Spleen sarcoma, NOS, angiosarcoma, or osteosarcoma 0/20 9/49 (18%)* 12/49 (24%)* Spleen fibroma 0/20 (0%) 4/49 (8%) 6/49 (12%) Spleen angiosarcoma 0/20 (0%) 7/49 (14%) 9/49 (18%)* Spleen sarcoma, NOS 0/20 (0%) 1/49 (2%) 3/49 (6%) aNCI (1979a). bNumber of animals with tumor/number of animals (%). °NOS = Not otherwise stated. *p < 0.05; **p < 0.01. 35 o-Toluidine ------- FINAL 12-3-2012 Table B.5. Incidence of Tumors in Male CD Rats Exposed to Oral (in Feed) o-Toluidine for 18 Months3 Parameter Human Equivalent Dose (mg/kg-d)b Concurrent Control Pooled Control 72.1 144.1 Subcutaneous fibroma and fibrosarcoma 0/16 (0%)c 18/111 (16%) 18/23 (78%)d 21/24 (88%)d Bladder tumors 0/16 (0%) 5/111 (5%) 3/23 (13%) 4/24 (17%) Multiple tumors 3/16 (19%) 14/111 (13%) 6/23 (26%) 8/24 (33%)e aWeisburger etal. (1978a). bAnimals were administered diets with 8000 or 16,000 ppm for 3 months followed by 4000 or 8,000 ppm for an additional 15 months, then placed on control diet for 6 months. °Number of animals with tumor/number of animals (%). Ap < 0.025 denotes significant difference from all controls. ep < 0.025 denotes significant difference from pooled controls only Table B.6. Incidence of Tumors in the B6C3Fi Mice Exposed to Oral (in Feed) o-Toluidine for 101 to 104 Weeks3 Parameter Exposure Group (ppm) (Human Equivalent Dose, mg/kg-d) Males 0 1000 (23.0) 3000 (73.8) Hepatocellular adenoma 1/20 (5%)b 3/50 (6%) 3/50 (6%) Hepatocellular carcinoma 4/19 (21%) 16/50 (32%) 11/50 (22%) Hepatocellular adenoma and carcinoma 5/19 (26%) 19/50 (38%) 14/50 (28%) Hemangiosarcoma (all sites) 1/20 (5%) 1/50 (2%) 10/50 (20%)° Females 0 1000 (26.5) 3000 (91.9) Hepatocellular adenoma 0/20 (0%) 2/49 (4%) 6/50 (12%) Hepatocellular carcinoma 0/20 (0%) 2/49 (4%) 7/50 (14%)° Hepatocellular adenoma and carcinoma 0/20 (0%) 4/49 (8%) 13/50 (26%)** Hemangiosarcoma (all sites) 1/20 (5%) 1/49 (2%) 2/50 (4%) aNCI (1979b). bNumber of animals with tumor/number of animals (%). Significant (p < 0.05) trend but incidence not significantly different from the control. **p < 0.01. 36 o-Toluidine ------- FINAL 12-3-2012 Table B.7. Incidence of Vascular Tumors in the CD-I Mice Exposed to Oral (in Feed) o-Toluidine for 18 Months3 Parameter Human Equivalent Dose (mg/kg-d)b Males Concurrent control Pooled control 210.5 420.9 Vascular tumors 0/14 (0%)c 5/99 (5%) 5/14 (36%)** 9/11 (82%)** Females Concurrent control Pooled control 211.8 423.7 Vascular tumors 0/15 (0%) 9/102 (9%) 5/18 (28%)* 9/21 (43%)** Males and females combined Concurrent control Pooled control 211.2 422.3 Vascular tumors 0/29 (0%) 14/201 (7%) 10/32 (31%) 18/32 (56%) aWeisburger et al. (1978b). bAnimals were administered diets with 16,000 or 32,000 ppm for 3 months followed by 8000 or 16,000 ppm for an additional 15 months, then were on control diet for an additional 3 months. °Number of animals with tumor/number of animals (%). *p < 0.05; **p < 0.025. 37 o-Toluidine ------- FINAL 12-3-2012 APPENDIX C. BMD OUTPUTS Table C.l. Multistage Cancer Model Predictions for Tumor Data for o-Toluidine Tumor Type/Site Gender/Species Goodness-of-Fit p-Valuea AICb for Fitted Model BMDL10C (mg/kg-d) Slope Factor Reference Subcutaneous fibroma and fibrosarcoma Male rat 0.57 45.25 4.50 0.0222316 Weisburger et al. (1978a) Vascular tumor data Female mouse 0.96 52.02 50.00 0.00199986 Weisburger et al. (1978b) Vascular tumor data Male mouse 0.54 31.99 23.00 0.00434693 Weisburger et al. (1978a) Bladder tumors Male rat 0.85 41.74 40.3 0.00248149 Weisburger et al. (1978a) Multiple tumors Male rat 0.64 71.32 34.25 0.00292001 Weisburger et al. (1978a) Subcutaneous fibroma Male rat 0.01 146.48 10.41 0.00960838 NCI (1979a) Multiple organ sarcomas, NOSd, fibrosarcoma, angiosarcoma, or osteosarcoma Male rat 1.00 119.64 13.77 0.00726365 NCI (1979a) Multiple organ sarcoma, NOS, fibrosarcoma, angiosarcoma, or osteosarcoma Female rat 0.59 92.80 47.03 0.00212622 NCI (1979a) Spleen sarcoma, NOS, angiosarcoma, or osteosarcoma Female rat 0.55 100.40 32.71 0.00305746 NCI (1979a) Multiple organ osteosarcoma Male rat 0.95 57.09 73.66 0.00135766 NCI (1979a) Multiple organ osteosarcoma Female rat 0.09 74.80 61.92 0.00161487 NCI (1979a) Multiple organ sarcoma, NOS Male rat 0.62 77.90 45.27 0.00220876 NCI (1979a) Multiple organ sarcoma, NOS Female rat 1.00 28.52 137.23 0.000728707 NCI (1979a) Multiple organ fibrosarcoma Male rat 0.76 112.81 22.89 0.00436794 NCI (1979a) Spleen fibromas Male rat 0.01 73.52 Failed failed NCI (1979a) Spleen fibromas Female rat 0.85 66.45 61.62 0.00162297 NCI (1979a) Spleen angiosarcomas Female rat 0.56 86.85 42.73 0.00234007 NCI (1979a) Spleen sarcomas Female rat 0.76 25.71 132.25 0.000756154 NCI (1979a) 38 o-Toluidine ------- FINAL 12-3-2012 Table C.l. Multistage Cancer Model Predictions for Tumor Data for o-Toluidine Tumor Type/Site Gender/Species Goodness-of-Fit p-Valuea AICb for Fitted Model BMDLioC (mg/kg-d) Slope Factor Reference Spleen sarcoma, NOS Male rat 0.81 24.93 122.54 0.000816071 NCI (1979a) Mammary fibroadenoma Female rat 0.86 154.40 14.99 0.00667223 NCI (1979a) Rat bladder transitional-cell carcinoma Female rat 0.82 112.41 19.10 0.0052354 NCI (1979a) Hepatocellular carcinoma Female mouse 1.00 59.21 39.22 0.00254972 NCI (1979b) Hemangio- sarcomas, all sites Male mouse 0.26 72.92 38.35 0.00260727 NCI (1979b) Hemangio- sarcomas, all sites Female mouse 0.21 31.41 100.95 0.000990631 NCI (1979b) Hepatocellular adenoma and carcinoma Female mouse 1.00 87.02 22.20 0.00450356 NCI (1979b) Hepatocellular adenoma Female mouse 0.99 55.43 43.29 0.00231009 NCI (1979b) "Values <0.10 fail to meet conventional goodness-of-fit criteria. bAIC = Akaike's Information Criteria. cBMDLio = lower confidence limit (95%) on the benchmark dose at 10% incidence, extra risk. dNOS = not otherwise stated. 39 o-Toluidine ------- FINAL 12-3-2012 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation 1 0.8 0.6 0.4 0.2 0 BMDL BMD 0 20 40 60 80 100 120 140 dose 14:12 01/13 2011 Figure C.l.° Multistage Cancer BMD Model for Subcutaneous Fibroma and Fibrosarcoma in Male F344 Rats Data (Weisburger et al., 1978) Text Output for 1° Multistage cancer BMD Model for Subcutaneous Fibroma and Fibrosarcoma in Male F344 Rats Data (Weisburger et al., 1978) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/18/Jan2011/Weisburger_1978_fibroma_fibrosarcoma_rat_m_MultiCancl_l.(d) Gnuplot Plotting File: C:/18/Jan2011/Weisburger_1978_fibroma_fibrosarcoma_rat_m_MultiCancl_l.pit Thu Jan 13 14:12:20 2011 Male rat subcutaneous fibroma and fibrosarcoma The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl) ] The parameter betas are restricted to be positive 40 o-Toluidine ------- FINAL 12-3-2012 Dependent variable = DichPerc Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 2 Total number of specified parameters = 0 Degree of polynomial = 1 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 = 0.14965 4 Beta(1) = 0.014428 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Background have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Beta(1) Beta (1) 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0 * * * Beta(1) 0.0173338 * * * * - Indicates that this value is not calculated. Model Full model Fitted model Reduced model Analysis of Deviance Table # Log(likelihood) -21.085 -21.6251 -41.8653 Param's 3 1 1 Deviance Test d.f. 1.08023 41.5606 P-value 0.5827 <.0001 AIC: 45 .2502 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.0000 0.000 0.000 16 0.000 72.0626 0.7132 16.405 18.000 23 0.736 41 o-Toluidine ------- FINAL 12-3-2012 144.1251 0.9178 22.026 21.000 24 -0.763 Chi^2 = 1.12 d.f. = 2 P-value = 0.5704 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 6.07834 BMDL = 4.4 9811 BMDU = 8 .32855 Taken together, (4.49811, 8.32855) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0222316 42 o-Toluidine ------- FINAL 12-3-2012 APPENDIX D. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). (2008) 2008 TLVs and BEIs: Threshold limit values for chemical substances and physical agents and biological exposure indices. ACGIH, Cincinnati, OH. 594529 ATSDR (Agency for Toxic Substances and Disease Registry). (2008) Toxicological profile information sheet. Available online at http://www.atsdr.cdc.gov/toxprofiles/index.asp. Accessed on 12/09/2011. 595415 Baan, R; Straif, K; Grosse, Y; et al. (2008) Carcinogenicity of some aromatic amines, organic dyes, and related exposures. Lancet Oncol 9(4):322-323. 673241 Baker, RSU; Bonin, AM. (1981) Study of 42 coded compounds with the Salmonella/mammalian microsome assay. In: de Serres, FJ; Ashby, J; eds. Progress in mutation research, vol 1. Evaluation of short term tests for carcinogens. Amsterdam: Elsevier, pp. 249-260. 018801 Brennan, RJ; Schiestl, RH. (1999) The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae. Mutat Res 430:37-45. 597183 CalEPA (California Environmental Protection Agency). (2008) Office of Environmental Health Hazard Assessment. Search Chronic RELs. Available online at http://www.oehha.ca.gov/air/allrels.html. Accessed on 12/09/2011. 595416 Carreon, T; Hein, MJ; Viet, SM; et al. (2010) Increased bladder cancer risk among workers exposed to o-toluidine and aniline: a reanalysis. Occup Environ Med 67:348-350. 595455 Cheever, KL; Richards, DE; Plotnick, HB. (1980) Metabolism of ortho-, meta-, and para-toluidine in the adult male rat. Toxicol ApplPharmacol 56(3):361-369. 064616 Danford, N. (1991) The genetic toxicology of ortho-toluidine. Mutat Res Rev Mutat Res 258(3):207-236. 597203 Ekman, B; Strombeck, JP. (1947) Demonstration of tumorigenic decomposition products of 2,3-azotoluene. Acta Physiol Scand47(l-2):43-50. 598199 Gulati, D; Sabharwal, P; Shelby, M. (1985) Tests for the induction of chromosomal aberrations and sister-chromatid exchanges in cultured Chinese hamster ovary (CHO) cells. In: de Serres, FJ; Ashby, J; eds. Progress in mutation research, vol 5. Evaluation of short-term tests for carcinogens. Amsterdam: Elsevier Science Publishers, pp. 413-426. 595458 Haskell Laboratory. (1994) Urinary bladder toxicity: 14-day feeding study with o-toluidine in rats with cover letter dated 06/21/94. Haskell Laboratory, Newark, DE. OTS0557449. 597218 Hecht, SS; El-Bayoumy, K; Rivenson, A; et al. (1982) Comparative carcinogenicity of o-toluidine hydrochloride and o-nitrosotoluene in F-344 rats. Cancer Lett 16:103-108. 064623 43 o-Toluidine ------- FINAL 12-3-2012 Hiles, RC; Abdo, KM. (1990) Ortho toluidine. In: Buhler DR; Reed DJ; eds. Ethel Browning's toxicity and metabolism of industrial solvents, 2nd ed. Vol II: nitrogen and phosphorus solvents. Amsterdam: Elsevier Publishing, pp. 202-207. 597930 IARC (International Agency for Research on Cancer). (2000) 0/t/?o-Toluidine. In: Some industrial chemicals. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 77. Lyon, France: IARC. Available online at http://monographs.iarc.fr/ENG/Monographs/vol77/mono77-l 1 .pdf. 597178 IARC (International Agency for Research on Cancer). (2010) 0/t/?o-Toluidine. Some aromatic amines, organic dyes, and related compounds. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 99. Lyon, France: IARC. Available online at http://monographs.iarc.fr/ENG/Monographs/vol99/mono99.pdf. 674890 Kerckaert, GA; LeBoeuf, RA; Isfort, RJ. (1998) Assessing the predictiveness of the Syrian hamster embryo cell transformation assay for determining the rodent carcinogenic potential of single ring aromatic/nitroaromatic amine compounds. Toxicol Sci 41:189-197. 597305 Malik-Brys, M and Senczuk, W. (1995) Toxicodynamic properties of toluidines in chronic poisoning Part III: the effect of the diet on the course of poisoning. Bromat Chem Toksykol 28(3):275-281. (translated from Polish). 673405 Malysheva, MV; Zaitseva, EP. (1982) Prolonged administration of o toluidine on the gonads of white female rats through the skin. MetodAspekty Izuch Bioi Deistiviya Khim 69-71. As cited in Hiles and Abdo, 1990. 597930 Malysheva, MV; Zaitseva, EP; Ivanov, IuV. (1983) Possibility of late effects of O-toluidine after its absorption through the skin. Gig Tri Prof Zabol 9:47-49. As cited in Hiles and Abdo, 1990. 597930 Markowitz, SB; Levin, K. (2004) Continued epidemic of bladder cancer in workers exposed to ortho-toluidine in a chemical factory. .1 Occup Environ Med 46(2): 154— 160. 597182 Matthews, EJ; Spalding, JW; Tennant, RW. (1993) Transformation of BALB/c-3T3 cells: V. Transformation responses of 168 chemicals compared with mutagenicity in Salmonella and carcinogenicity in rodent bioassays. Environ Health Per sped 101(Suppl 2):347-482. 201831 McFee, AF; Jauhar, PP; Lowe, KW; et al. (1989) Assays of three carcinogen/non-carcinogen chemical pairs for in vivo induction of chromosome aberrations, sister chromatid exchanges and micronuclei. EnvironMolMutagen 14(4):207-220. 597314 Morita, T; Asano, N; Awogi, T; et al. (1997) Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (groups 1, 2 A and 2B) the summary report of the 6th collaborative study by CSGMT/JEMS MMS. MutatRes 389:3-122. 194532 Morita, T; Iwamoto, Y; Shimizu, T; et al. (1989) Mutagenicity tests with a permeable mutant of yeast on carcinogens showing false-negative in Salmonella assay. Chem Pharm Bull (Tokyo) 37(2):407-409. 597317 44 o-Toluidine ------- FINAL 12-3-2012 NCI (National Cancer Institute). (1979a,b) Bioassay of o-toluidine hydrochloride for possible carcinogenicity. U.S. Department of Health, Education and Welfare, National Institutes of Health. Bethesda, MD; technical report series no. 153. NCI-CG-TR-153. Available online at http://ntp.niehs.nih.gov/ntp/htdocs/LT rpts/trl53.pdf. 064631 NIOSH (National Institute for Occupational Safety and Health). (2010) NIOSH pocket guide to chemical hazards. Index of chemical abstracts service registry numbers (CAS No.). Center for Disease Control and Prevention, U.S. Department of Health, Education and Welfare, Atlanta, GA. Available online at http://www.cdc.gov/niosh/npg/npgdcas.html. 070020 NTP (National Toxicology Program). (1996a,b,c) NTP technical report on comparative toxicity and carcinogenicity studies of o-nitrotoluene and o-toluidine hydrochloride administered in feed to male F344/N rats. United States Department of Health and Human Services, National Institutes of Health, Research Triangle Park, NC; toxicity report series no. 44. 597187 NTP (National Toxicology Program). (2011) 12th Report on carcinogens. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Available online at http://ntp- server.niehs.nih.gov/index.cfm?obiectid=32BA9724-FlF6-975E-7FCE50709CB4C932. 091126 OSHA (Occupational Safety and Health Administration). (2010) Air contaminants: occupational safety and health standards for shipyard employment, subpart Z, toxic and hazardous substances. U.S. Department of Labor, Washington, DC. OSHA Standard 1915.1000. Available online at http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=102 86. Accessed on 12/09/11. Salamone, MF; Heddle, JA; Katz, M. (1981) Mutagenic activity of 41 compounds in the in vivo micronucleus assay. In: de Serres, FJ; Ashby, J; eds. Progress in mutation research, vol 1. Evaluation of short-term tests for carcinogens. Amsterdam: Elsevier, pp. 686-697. 018902 Short, CR; King, C; Sistrunk, PW; et al. (1983) Subacute toxicity of several ring-substituted dialkylanilines in the rat. Fimdcim Appl Toxicol 3(4):285-292. 061463 Simmon, VF. (1979) In vitro mutagenicity assays of chemical carcinogens and related compounds with Salmonella typhimurium. J Natl Cancer Inst 62(4):893-899. 054655 Son, OS; Everett, DW; Fiala, ES. (1980) Metabolism of o-[methyl-14C]toluidine in the F344 rat. Xenobiotica 10(7-8):457-468. 094945 Sorahan, T. (2008) Bladder cancer risks in workers manufacturing chemicals for the rubber industry. OccupMed (Lond) 58(7):496-501. 548805 Sorahan, T; Hamilton, L; Jackson, J. (2000) A further cohort study of workers employed at a factory manufacturing chemicals for the rubber industry, with special reference to the chemicals 2-mercaptobenzothiazole (MBT), aniline, phenyl-beta-naphthylamine and o-toluidine. J Occup Environ Med 57(2): 106-115. 594246 45 o-Toluidine ------- FINAL 12-3-2012 Topham, JC. (1980) Do induced sperm-head abnormalities in mice specifically identify mammalian mutagens rather than carcinogens? Mutat Res 74(5):379-387. 010070 U.S. EPA (U.S. Environmental Protection Agency). (1984) Health and environmental effects profile for toluidines. U.S. Environmental Protection Agency, Cincinnati, OH; EPA/600/X-84/151. 064639 U.S. EPA (U.S. Environmental Protection Agency). (1988) Recommendations for and documentation of biological values for use in risk assessment. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH; EPA/600/6-87/008. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=34855. 064560 U.S. EPA (U.S. Environmental Protection Agency). (1994a) Chemical assessments and related activities (CARA). Office of Health and Environmental Assessment, Washington, DC; EPA/600/R-94/904. Available online at nepis.epa. gov/Exe/ZvPURL.cgi?Dockev=6000 lG8L.txt. 596444 U.S. EPA (U.S. Environmental Protection Agency). (1994b) Methods for derivation of inhalation reference concentrations (RfCs) and application of inhalation dosimetry. Office of Research and Development, Office of Health and Environmental Assessment, Washington, DC; EPA/600/8-90/066F. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=71993. 006488 U.S. EPA (U.S. Environmental Protection Agency). (1997) Exposure factors handbook (final report). National Center for Environmental Assessment, Washington, DC; EPA/600/P-95/002F a~c. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid= 12464. 594981 U.S. EPA (U.S. Environmental Protection Agency). (2005) Guidelines for carcinogen risk assessment. Risk Assessment Forum, Washington, DC; EPA/630/P-03/001F. Federal Register 70(66): 17765-17817. Available online at http://www.epa.gov/raf/publications/pdfs/CANCER GUIDELINES FINAL 3-25-05.PDF. 086237 U.S. EPA (U.S. Environmental Protection Agency). (2006) 2006 Edition of the drinking water standards and health advisories. U.S. Environmental Protection Agency, Washington, DC; EPA/822/R-06/013. Available online at http://www.epa.gov/waterscience/criteria/drinking/dwstandards.pdf. 091193 U.S. EPA (U.S. Environmental Protection Agency). (2012a) Integrated risk information system (IRIS). Office of research and Development, National Center for Environmental Assessment, Washington, DC. Available online at http J/www, epa. gov/iri s/. Accessed on 12/09/2011. 595423 U.S. EPA (U.S. Environmental Protection Agency). (2012b) Health effects assessment summary tables (HEAST). Prepared by the Office of Research and Development, National Center for Environmental Assessment, Cincinnati OH for the Office of Emergency and Remedial Response, Washington, DC. Available online at http://epa-heast.ornl.gov/. 595422 46 o-Toluidine ------- FINAL 12-3-2012 U.S. EPA (U.S. Environmental Protection Agency). (2010c) Benchmark dose software (BMDS) version 2.1.2. Available online at http://www.epa.gov/NCEA/bmds. Accessed on 05/16/2011. 200772 Ward, E; Carpenter, A; Markowitz, S; et al. (1991) Excess number of bladder cancers in workers exposed to ortho-toluidine and aniline. J Natl Cancer Inst 83(7):501-506. 597208 Ward, E; Sabbioni, G; DeBord, D; et al. (1996) Monitoring of aromatic amine exposures in workers at a chemical plant with a known bladder cancer excess. J Natl Cancer Inst 88(15): 1046—1053. 597209 Weisburger, EK; Russfield, AB; Homburger, F; et al. (1978a,b) Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. J Environ Pathol Toxicol 2(2):325-356. 064640 WHO (World Health Organization). (1998) o-Toluidine. Concise international chemical assessment document 7. Geneva: World Health Organization. Available online at http://www.who.int/ipcs/publications/cicad/en/cicad07.pdf. 085708 WHO (World Health Organization). (2011) Online catalogs for the Environmental Health Criteria Series. Available online at http://www.who.int/ipcs/publications/ehc/en/. Accessed on 12/09/2011. 595424 Zimmer, D; Mazurek, J; Petzold, G; et al. (1980) Bacterial mutagenicity and mammalian cell DNA damage by several substituted anilines. Mutat Res 11(4)317-326. 201823 47 o-Toluidine ------- |