United States Environmental Protection 1=1 m m Agency EPA/690/R-16/007F Final 09-27-2016 Provisional Peer-Reviewed Toxicity Values for o-Phenylenediamine (CASRN 95-54-5) Superfund Health Risk Technical Support Center National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGERS Elizabeth Owens, PhD National Center for Environmental Assessment, Cincinnati, OH Letitia Wong, PhD Oak Ridge Institute for Science and Education Research Participation Program National Center for Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212 PRIMARY INTERNAL REVIEWER Suryanarayana Vulimiri, PhD National Center for Environmental Assessment, Research Triangle Park, NC 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 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). li o-Phenyl enedi ami amine ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS AND ACRONYMS iv BACKGROUND 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVS 1 INTRODUCTION 2 REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 5 HUMAN STUDIES 11 Oral Exposures 11 Inhalation Exposures 11 ANIMAL STUDIES 11 Oral Exposures 11 Inhalation Exposures 19 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 19 Genotoxicity 30 Acute Toxicity 30 Other Routes 31 Metabolism/Toxicokinetic Studies 32 Mode-of-Action/Mechanistic Studies 32 Neurotoxicity 32 DERIVATION 01 PROVISIONAL VALUES 34 DERIVATION OF ORAL REFERENCE DOSES 34 Derivation of a Subchronic Provisional Reference Dose 34 Derivation of a Chronic Provisional Reference Dose 35 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 41 CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 41 MODE-OF-ACTION DISCI SSION 45 Key Events 45 Strength, Consistency, Specificity of Association 46 Analogy 46 Dose-Response Concordance 46 Temporal Relationships 46 Biological Plausibility and Coherence 46 Early-Life Susceptibility 47 Conclusions 47 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 47 Derivation of a Provisional Oral Slope Factor 47 Derivation of a Provisional Inhalation Unit Risk 50 APPENDIX A. SCREENING PROVISIONAL VALUES 51 APPENDIX B. DATA TABLES 52 APPENDIX C. BENCHMARK DOSE MODELING RESULTS 65 APPENDIX D. REFERENCES 89 in o-Phenyl enedi ami amine ------- COMMONLY USED ABBREVIATIONS AND ACRONYMS a2u-g alpha 2u-globulin MN micronuclei ACGIH American Conference of Governmental MNPCE micronucleated polychromatic Industrial Hygienists erythrocyte AIC Akaike's information criterion MOA mode of action ALD approximate lethal dosage MTD maximum tolerated dose ALT alanine aminotransferase NAG N-acetyl-P-D-glucosaminidase AST aspartate aminotransferase NCEA National Center for Environmental atm atmosphere Assessment ATSDR Agency for Toxic Substances and NCI National Cancer Institute Disease Registry NOAEL no-observed-adverse-effect level BMD benchmark dose NTP National Toxicology Program BMDL benchmark dose lower confidence limit NZW New Zealand White (rabbit breed) BMDS Benchmark Dose Software OCT ornithine carbamoyl transferase BMR benchmark response ORD Office of Research and Development BUN blood urea nitrogen PBPK physiologically based pharmacokinetic BW body weight PCNA proliferating cell nuclear antigen CA chromosomal aberration PND postnatal day CAS Chemical Abstracts Service POD point of departure CASRN Chemical Abstracts Service Registry PODadj duration-adjusted POD Number QSAR quantitative structure-activity CBI covalent binding index relationship CHO Chinese hamster ovary (cell line cells) RBC red blood cell CL confidence limit RDS replicative DNA synthesis CNS central nervous system RfC inhalation reference concentration CPN chronic progressive nephropathy RfD oral reference dose CYP450 cytochrome P450 RGDR regional gas dose ratio DAF dosimetric adjustment factor RNA ribonucleic acid DEN diethylnitrosamine SAR structure activity relationship DMSO dimethylsulfoxide SCE sister chromatid exchange DNA deoxyribonucleic acid SD standard deviation EPA Environmental Protection Agency SDH sorbitol dehydrogenase FDA Food and Drug Administration SE standard error FEVi forced expiratory volume of 1 second SGOT glutamic oxaloacetic transaminase, also GD gestation day known as AST GDH glutamate dehydrogenase SGPT glutamic pyruvic transaminase, also GGT y-glutamyl transferase known as ALT GSH glutathione SSD systemic scleroderma GST glutathione-S-transferase TCA trichloroacetic acid Hb/g-A animal blood-gas partition coefficient TCE trichloroethylene Hb/g-H human blood-gas partition coefficient TWA time-weighted average HEC human equivalent concentration UF uncertainty factor HED human equivalent dose UFa interspecies uncertainty factor i.p. intraperitoneal UFh intraspecies uncertainty factor IRIS Integrated Risk Information System UFS subchronic-to-chronic uncertainty factor IVF in vitro fertilization UFd database uncertainty factor LC50 median lethal concentration U.S. United States of America LD50 median lethal dose WBC white blood cell LOAEL lowest-observed-adverse-effect level iv o-Phenyl enedi ami amine ------- FINAL 09-27-2016 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR 0-PHENYLENEDIAMINE (CASRN 95-54-5) BACKGROUND A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant scientific literature using established Agency guidance on human health toxicity value derivations. All PPRTV assessments receive internal review by a standing panel of National Center for Environmental 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 use 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. This document has been reviewed in accordance with U.S. EPA policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. QUESTIONS REGARDING PPRTVS Questions regarding the content 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-Phenyl enedi ami amine ------- FINAL 09-27-2016 INTRODUCTION o-Phenylenediamine, CASRN 95-54-5, is primarily used as a chemical intermediate, especially in the production of benzimidazole-derived agricultural fungicides, such as benomyl, and substituted benzimidazoles used as a veterinary anthelmintic (HSDB, 2013). o-Phenylenediamine is also used in the manufacture of dyes, although its use in hair dyes was banned by the European Union in September of 2007 (HSI)B, 2013). o-Phenylenediamine is also used as a fluorescence indicator, a photographic developing agent, and a laboratory reagent (HSDB. 2013). o-Phenylenediamine is solid at room temperature. As a diamine with pKa values of <2 and 4.47, o-phenylenediamine is expected to exist partially as a cation in the environment. As a result, it is not expected to volatilize from moist soil or water surfaces (HSDB, 2013). In addition, the estimated Henry's law constant for the neutral form of o-phenylenediamine indicates a low propensity to volatilize from water surfaces. Furthermore, the moderate vapor pressure of o-phenylenediamine's neutral form indicates that evaporation from dry soil is not expected. However, a moderate vapor pressure suggests that o-phenylenediamine, if released to the air, would remain in the vapor phase (HSDB, 2013). o-Phenylenediamine"s ability to leach from soil to groundwater is dependent on local conditions. In areas with high amounts of organic matter, leaching of o-phenylenediamine may be inhibited due to the high reactivity of the aromatic amine groups (HSDB, 2013). In other areas, o-phenylenediamine deposited on soil is likely to leach to groundwater or undergo runoff after a rain event, due to its high water solubility and relatively low soil adsorption coefficient. The empirical formula for o-phenylenediamine is C6H8N2 (see Figure 1). Synonyms include 2-aminoaniline, 1,2-benzenediamine, o-benzenediamine, o-diaminobenzene, 1,2-diaminobenzene, and 1,2-phenylenediamine. A table of physicochemical properties for o-phenylenediamine is provided below (see Table 1). NH '2 NH. '2 Figure 1. o-Phenylenediamine Structure 2 o-Phenyl enedi ami amine ------- FINAL 09-27-2016 Table 1. Physicochemical Properties of o-Phenylenediamine (CASRN 95-54-5) Property (unit) Value Physical state White crystals3 Boiling point (°C) 257a Melting point (°C) 102. la Density (g/cm3) 0.6b Vapor pressure (mm Hg at 25 °C) 2.06 x 10 3 a pH (unitless) 9.7 (saturated aqueous solution at 24°C)b pKa (at 25°C) pKal = <2; pKa2 = 4.47a Solubility in water (g/L at 20°C) 31b Octanol-water partition constant (log Kow) 0.15a; 0.12b Henry's law constant (atm-m3/mol at 25°C) 7.20 x 10 9 (estimated)0 Soil adsorption coefficient (Koc) (mL/g) 34.5 (estimated)0 Relative vapor density (air =1) 3.7d Molecular weight (g/mol) 108.14a aHSDB (2013). bECHA (2015). CU.S. EPA (2012b). dSigma-Aldrich (2015). A summary of available toxicity values for o-phenylenediamine from U.S. EPA and other agencies/organizations is provided in Table 2. 3 o-Phenyl enedi ami amine ------- FINAL 09-27-2016 Table 2. Summary of Available Toxicity Values for o-Phenylenediamine (CASRN 95-54-5) Source (parameter)ab Value (applicability) Notes Reference Noncancer IRIS NV NA U.S. EPA (2016) HEAST NV NA U.S. EPA (201 la) DWSHA NV NA U.S. EPA (2012a) ATSDR NV NA ATSDR (2016) IPCS NV NA IPCS (2016): WHO (2016) Cal/EPA NV NA Cal/EPA (2014): Cal/EPA (2016a): Cal/EPA (2016b) OSHA NV NA OSHA (2011): OSHA (2006) NIOSH NV NA NIOSH (2016) ACGIH (TLV-TWA) 0.1 mg/m3 Based on the potential for "blood dyscrasia (e.g., reduction of RBCs), as well as eye and skin irritation" ACGIH (2015) Cancer IRIS NV NA U.S. EPA (2016) HEAST (OSF, WOE) OSF: 4.7 x 10 2 (mg/kg-d) WOE: B2 Based on liver tumors in rats treated orally with o-phenylenediamine dihydrochloride U.S. EPA (1985): U.S. EPA (2011b) DWSHA NV NA U.S. EPA (2012a) NTP NV NA NTP (2014) IARC NV NA IARC (2015) Cal/EPA (cancer potency value) 0.027 (mg/kg-d)"1 Based on liver tumors in male rats and female mice after oral exposure to o-phenylenediamine dihydrochloride Cal/EPA (2002) ACGIH (WOE) A3—confirmed animal carcinogen with unknown relevance to humans Based on the "production of hepatocellular carcinomas and hepatomas" in rat and mouse bioassays ACGIH (2015) aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic Substances and Disease Registry; Cal/EPA = California Environmental Protection Agency; DWSHA = Drinking Water Standards and Health Advisories; HEAST = Health Effects Assessment Summary Tables; IARC = International Agency for Research on Cancer; IPCS = International Programme on Chemical Safety; IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety and Health; NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration. Parameters: OSF = oral slope factor; TLV = threshold limit value; TWA = time-weighted average; WOE = weight-of-evidence. NA = not applicable; NV = not available; RBC = red blood cell. 4 o-Phenyl enedi ami amine ------- FINAL 09-27-2016 Literature searches were conducted in July 2013 and in March 2016 for studies published from 1900 that are relevant to the derivation of provisional toxicity values for o-phenylenediamine. Searches were conducted using U.S. EPA's Health and Environmental Research Online (HERO) database of scientific literature. The following databases were searched: PubMed, TOXLINE (including TSCATS1), and Web of Science. The following databases were searched outside of HERO for health-related values: ACGIH, ATSDR, Cal/EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA Office of Water (OW), U.S. EPA TSCATS2/TSCATS8e, NIOSH, NTP, and OSHA. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) Tables 3A and 3B provide an overview of the relevant noncancer and cancer databases for o-phenylenediamine, respectively, and include all potentially relevant repeat-dose, short-term-, subchronic-, and chronic-duration studies, as well as reproductive and developmental toxicity studies. Principal studies are identified in bold font. The phrases "statistical significance" and "significant," used throughout the document, indicate ap-value of < 0.05 unless otherwise noted. 5 o-Phenyl enedi ami amine ------- FINAL 09-27-2016 Table 3A. Summary of Potentially Relevant Noncancer Data for o-Phenylenediamine (CASRN 95-54-5) Category" Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetryb Critical Effects NOAELb BMDL/ BMCLb LOAELb Reference (comments) Notes0 Human 1. Oral (mg/kg-d) ND 2. Inhalation (mg/m3)b Exposure duration cannot be determined 12 workers, evaluation of medical records of operators and operating supervisors of a phenylenediamine manufacturing plant for >10 yr (unknown composition of phenylenediamines) NDr Skin irritation, no adverse effects on blood oxygen saturation or Hb levels ND NDr ND DuPont (1984) (Study to evaluate potential for methemoglobinemia, not a comprehensive evaluation) NPR Animal 1. Oral (mg/kg-d)b Short-term 7-14 M/0 F, ChR-CD® rat, palatability study of o-phenylenediamine administered in drinking water, 4 wk 0, 2,240 (Group Bl), 2,070 (Group B2) ppm ADD: 0, 167 (Group Bl), 132 (Group B2) Decreased body weight and water intake ND NDr ND Haskell Laboratories (1980) (Not a comprehensive evaluation of endpoints; limited to clinical signs, body weight, and water consumption) NPR 6 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 3A. Summary of Potentially Relevant Noncancer Data for o-Phenylenediamine (CASRN 95-54-5) Category" Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetryb Critical Effects NOAELb BMDL/ BMCLb LOAELb Reference (comments) Notes0 Subchronic 10 M/10 F, Crl:CD®BR rat, neurotoxicity study of o-phenylenediamine administered by gavage (suspended in aqueous methyl cellulose) for 90 consecutive d 0, 20, 40, 80 ADD: 0, 20, 40, 80 Increased incidence of slight palpebral closure (M and F); increased incidence of yellow staining of abdomen, perineum, inguen (groin), and/or underbody (F) 40 NDr 80 DuPont (1992a) (Not a comprehensive evaluation of endpoints; limited to mortality, clinical signs, body weight, food consumption, and neurotoxicity endpoints, including neurological histology) NPR Chronic 50 M/50 F, F344/DuCrj rat, o-phenylenediamine dihydrochloride administered in drinking water for 2 yr 0, 500,1,000, 2,000 (M); 0, 250, 500,1,000 (F) ppm as o-phenylenediamine dihydrochloride ADD: 0,13,25,51 (M); 0,11,20,35(F) as o-phenylenediamine Increased incidences of renal papillary mineralization and basophilic cell foci of the liver (M); increased incidences of urothelial hyperplasia of the renal pelvis and basophilic foci of the liver (F) ND 4.8 (papillary mineralization, M) 13 (M); 11(F) Matsumoto et al. (2012) PR, PS Chronic 50 M/50 F, Cij:BDFi mouse, o-phenylenediamine dihydrochloride administered in drinking water for 2 yr 0, 500, 1,000, 2,000 (M); 0, 1,000, 2,000, 4,000 (F) ppm as o-phenylenediamine dihydrochloride ADD: 0, 27, 56, 106 (M); 0, 63.3, 119, 234 (F) as o-phenylenediamine Increased ALP and relative liver weight (M and F); decreased terminal body weight and increased incidence of papillary hyperplasia of the gall bladder (M); increased incidences of eosinophilic change of the nasal cavity respiratory epithelium and hydronephrosis of the kidney (F) ND 23 (hydronephrosis, F) 27 (M); 63.3 (F) Matsumoto et al. (2012) (Note: Liver tumor incidences were significantly increased at all doses in both male and female mice) PR 7 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 3A. Summary of Potentially Relevant Noncancer Data for o-Phenylenediamine (CASRN 95-54-5) Number of Male/Female, Strain, Species, Study Type, BMDL/ Reference Category" Study Duration Dosimetryb Critical Effects NOAELb BMCLb LOAELb (comments) Notes0 2. Inhalation (mg/m3) ND 'Category (treatment/exposure duration: unless otherwise noted): Short-term = repeated exposure for >24 hours < 30 days (U.S. EPA. 20021: long-term (subchronic) = repeated exposure for >30 days <10% lifespan for humans (more than 30 days up to approximately 90 days in typically used laboratory animal species) (U.S. EPA. 20021: chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002). bDosimetry: Values are presented as an ADD (mg/kg-day) for oral noncancer effects and as an HEC (mg/m3) for inhalation noncancer effects. Where applicable, the dose of o-phenylenediamine was calculated from the dose of o-phenylenediamine dihydrochloride by multiplying by the ratio of the molecular weights of the two compounds (108.14 g/mol: 181.062 g/mol). °Notes: NPR = not peer reviewed; PR = peer reviewed; PS = principal study. ADD = adjusted daily dose; ALP = alkaline phosphatase; BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit; F = female(s); Hb = hemoglobin; HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; M = male(s); ND = no data; NDr = not determined; NOAEL = no-observed-adverse-effect level. 8 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 3B. Summary of Potentially Relevant Cancer Data for o-Phenylenediamine (CASRN 95-54-5) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects BMDLa Reference (comments) Notesb Human 1. Oral (mg/kg-d) ND 2. Inhalation (mg/m3) ND Animal 1. Oral (mg/kg-d)a Carcinogenicity 50 M/50 F, F344/DuCij rat, o-phenylenediamine dihydrochloride administered in drinking water for 2 yr 0, 500, 1,000, 2,000 (M); 0, 250, 500, 1,000 (F) ppm as o-phenylenediamine dihydrochloride HED:0, 3.2, 6.1, 13 (M); 0, 2.6, 4.8, 8.5 (F) as o-phenylenediamine Significantly increased incidences of hepatocellular adenomas and/or carcinomas in males and females (>6.1 and 4.8 mg/kg-d, respectively); significantly increased incidence of urinary bladder transitional cell papilloma or carcinoma in males (13 mg/kg-d); significant dose-related trend for thyroid follicular adenoma in males 2.5 (M) (combined tumors); 2.2 (F) (liver tumors) Matsumoto et al. (2012) PR Carcinogenicity 50 M/50 F, Crj:BDFi mouse, o-phenylenediamine dihydrochloride administered in drinking water for 2 yr 0, 500,1,000, 2,000 (M); 0,1,000, 2,000, 4,000 (F) ppm as o-phenylenediamine dihydrochloride HED: 0,3.8, 7.7,14.5 (M); 0, 8.70,16.4, 32.1 (F) as o-phenylenediamine Significantly increased incidences of hepatocellular adenomas and/or carcinomas (>3.8 and 8.70 mg/kg-d in males and females, respectively) and papillary adenomas of the gall bladder (at 14.5 mg/kg-d in males and 16.4 mg/kg-d in females) 0.84 (M) (combined tumors); 1.56 (F) (combined tumors) Matsumoto et al. (2012) PS, PR 9 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 3B. Summary of Potentially Relevant Cancer Data for o-Phenylenediamine (CASRN 95-54-5) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects BMDLa Reference (comments) Notesb Carcinogenicity 25 M/0 F, Charles River CD rat, o-phenylenediamine dihydrochloride administered in diet for 18 mo 0, 2,000, 4,000 ppm as o-phenylenediamine dihydrochloride HED: 0, 20.1, 40.1 as o-phenylenediamine Significantly increased incidence of liver tumors at 40.1 mg/kg-d NDr Weisburger et al. (1978) (Note: Animals were not exposed over a lifetime [only for 18 mo] and then were allowed to recover for 6 mo after the exposure period. All endpoints were only measured at study termination of 24 mo) PR Carcinogenicity 25 M/25 F, CD-I mouse, o-phenylenediamine dihydrochloride administered in diet for 18 mo 0, 4,000, 8,000 ppm for 5 mo; 0, 8,000, 16,000 ppm for 13 mo as o-phenylenediamine dihydrochloride HED: 0, 98.5, 200 (M); 0, 100, 204 (F) as o-phenylenediamine Significantly increased incidences of hepatomas in males at 98.5 mg/kg-d and females at >100 mg/kg-d NDr Weisburger et al. (1978) (Note: Animals were not exposed over a lifetime [only for 18 mo] and then were allowed to recover for 3 mo after the exposure period. All endpoints were only measured at study termination of 21 mo) PR 2. Inhalation (mg/m3) ND aDosimetry: The units for oral exposures are expressed as human equivalent dose (HED, mg/kg-day). HED = ADD (mg/kg-day) x default dosimetric adjustment factor (DAF) (U.S. EPA. 201101. Where applicable, the dose of o-phenylenediamine was calculated from the dose of o-phenylenediamine dihydrochloride by multiplying by the ratio of the molecular weights of the two compounds (108.14 g/mol: 181.062 g/mol). bNotes: PR = peer reviewed; PS = principal study. ADD = adjusted daily dose; BMDL = benchmark dose lower confidence limit; F = female(s); HED = human equivalent dose; M = male(s); ND = no data; NDr = not determined. 10 o-Phenyl enedi amine ------- FINAL 09-27-2016 HUMAN STUDIES Oral Exposures No studies examining possible associations between health effects in humans and oral exposure to o-phenylenedi amine have been identified. Inhalation Exposures DuPont (1984) The frequency of methemoglobinemia in employees of a phenylenediamine manufacturing plant was evaluated by reviewing employee medical records. The phenylenediamine isomer(s) that the workers were exposed to were not provided. Employees in the plant provided blood samples every 6 months or whenever exposure excursions above the company-established acceptable exposure level of 0.1 mg/m3 occurred. Records of all operators and operating supervisors working with phenylenediamines for >10 years were reviewed, and hemoglobin (Hb) and oxygen levels were examined for the period of potential phenylenediamine exposure. Neither Hb nor oxygen saturation levels among employees differed from reported normal levels. Hb levels among employees averaged 15.6 g/dL, compared with a normal range of 14-17.2 g/dL; oxygen saturation averaged 93.9%, compared with normal levels >92.0%. The study authors reported that the medical records showed 27 cases of skin irritation associated with phenylenediamine exposure between 1975 and 1982, but none of the affected workers exhibited blood oxygen saturation <90%. ANIMAL STUDIES Oral Exposures The effects of oral exposure of animals to o-phenylenedi amine were evaluated in one short-term-duration study (Haskell Laboratories. 1980). one subchronic-duration neurotoxicity study (DuPont. 1992a). and two chronic-duration carcinogenicity studies (Matsumoto et al. 2012; Weisburger et ai. 1978). Short-Term-Duration Studies Haskell Laboratories (1980) The palatability of o-phenylenedi amine (purity 99%) in drinking water was tested in an unpublished 4-week study conducted by Haskell Laboratories (1980). Two groups of seven male ChR-CD® rats received 4,000 ppm o-phenylenedi amine (purity 99%) in drinking water on Days 0-7, and then untreated water on Days 7-10. One group (Bl) then received 400 ppm on Days 10-14, 1,000 ppm on Days 14-17, 2,000 ppm on Days 17-24, and 4,000 ppm on Days 24-28; the other group (B2) received 1,000 ppm on Days 10-24 and 4,000 ppm on Days 24-28. These exposure concentrations corresponded to time-weighted average (TWA) concentrations of 2,240 ppm (Group Bl) and 2,070 ppm (Group B2); doses estimated for this review using TWA-measured body weight and water intake values were 167 and 132 mg/kg-day in Groups Bl and B2, respectively. A control group of 14 rats received tap water. Toxicological evaluations were limited to daily observations for clinical signs and twice-weekly body-weight and water consumption measurements. None of the animals were necropsied. Statistical analysis was not reported or performed. There were no deaths among rats of any group. Some rats reportedly exhibited brown facial discoloration, but no other clinical signs were noted. In both groups of treated rats, the initial 1-week exposure to 4,000 ppm o-phenylenedi amine in drinking water resulted in body-weight losses of >10% of their initial body weight after only 3 days and 18% after 7 days. 11 o-Phenyl enedi amine ------- FINAL 09-27-2016 Both groups resumed gaining weight when exposed to concentrations up to 2,000 ppm, but lost weight again during the final 3 days of exposure to 4,000 ppm. Terminal body weights in both groups of treated rats were much lower (-24%) than controls. Treated rats drank less water compared to controls over the 28-day study; TWA daily water intake values were 22 and 19 mL/day in Groups B1 and B2 (65 and 56% of controls, respectively), compared with 34 mL/day in controls. The observed body-weight decreases in this study may have been attributable to the markedly reduced water intake, possibly due to poor palatability of the treated water. Given the uncertainty in the role of water intake on body-weight decrements, and the lack of evaluations of other toxicological endpoints, effect levels cannot be determined from this study. Subchronic-Duration Studies DuPont (1992a) A sub chronic-duration neurotoxicity study of o-phenylenedi amine (purity >98%, suspended in aqueous methyl cellulose) administered by gavage to Crl:CD®BR rats was conducted by DuPont (1992a). The study was not published. Groups of 10 rats/sex/dose were given daily doses of 0, 20, 40, or 80 mg/kg-day for 90 consecutive days. The test material was prepared daily. Rats were examined daily for mortality, appearance, and behavior. Body weights were recorded twice weekly for 4 weeks and weekly for the remaining 8 weeks; food consumption was measured weekly. All rats received ophthalmological examinations before study commencement and before sacrifice. Neurotoxicity evaluations (including motor activity and functional observational battery [FOB] assessments, forelimb and hindlimb grip strength, and foot splay measurements) were performed prior to the first dose and again during Weeks 4, 8, and 13. At sacrifice at the end of exposure, the following tissues were removed from the control and high-dose rats for histology examination (sciatic nerve, forebrain, cerebrum, midbrain, cerebellum, pons, medulla oblongata, spinal cord, tibial nerve and Gasserian ganglia, dorsal root ganglia, dorsal and ventral root fibers, and gastrocnemius muscle). High performance liquid chromatography (HPLC) analysis showed that concentrations of o-phenylenedi amine in the test suspensions were within 85-100%) of the target levels. One male rat exposed to 40 mg/kg-day was removed from the study for poor health apparently unrelated to toxicity (severe mouth injury, black discharge from the eye, and irregular respiration). All remaining rats survived the study. Clinical signs of toxicity consisting of yellow staining of the perineum, inguen, abdomen, and/or underbody were observed at increased incidence in female rats exposed to the high dose (see Table B-l). But only the increased incidences of perineum and inguen staining were statistically significant compared with the controls. In contrast, clinical signs attributed to toxicity were not observed in male rats. Male rats in the high-dose group exhibited significant decreases in body-weight gain during the sixth week of treatment, and statistically nonsignificant decreases were also seen during Weeks 4, 9, and 12. In addition, a significant decrease (13% less than controls) in body-weight gain calculated over the entire exposure period was observed in the 40-mg/kg-day male rats (see Table B-l). In female rats, a significant decrease in body-weight gain occurred during Week 4 only. However, average body weights in all exposed groups of male and female rats did not differ significantly from controls throughout the exposure period. Likewise, food consumption rates were not affected by treatment, although there were sporadic reductions in feed efficiency (weight gain/food consumption) in the 40- and 80-mg/kg-day males and 80-mg/kg-day females, reflecting the reduced weight-gain values noted above. 12 o-Phenyl enedi amine ------- FINAL 09-27-2016 Among the FOB endpoints assessed, only the response to tail pinch showed any evidence of an effect of treatment. Depending on the time of assessment, two or three male rats in the 80-mg/kg-day group displayed enhanced response to tail pinch, versus zero or one in the control and lower dose groups; however, the incidence was not statistically significantly different from control at any time and there was no similar observation in females (see Table B-l). Forelimb and hindlimb grip strength and foot splay were not altered by exposure at any dose in males or females. An increase in relative foot splay in high-dose males during Week 4 was attributed to unusually low values obtained at baseline. Both male and female rats exposed to the highest dose exhibited statistically significantly increased incidences of slight palpebral closure (i.e., rats with their eyelids slightly closed) during Week 13 (see Table B-l). Motor activity measures were comparable among control and treated groups of rats. There were no exposure-related ocular effects in either male or female rats. Microscopic examination of nervous system tissues did not indicate any significant differences from controls for any lesion. The study authors identified the 40-mg/kg-day dose as a no-effect level in both male and female rats. At this dose, a significant decrease in body-weight gain (calculated over the study duration) was observed in male rats, but the absolute body weights did not differ at any time and no such decrease in overall body-weight gain was seen in high dose males. The high dose (80 mg/kg-day) is a lowest-observed-adverse-effect level (LOAEL) for increased incidence of slight palpebral closure (males and females). Similar clinical signs were seen in an acute oral neurotoxicity study conducted by the same laboratory, in which single gavage doses of 225-900 mg/kg were administered to rats fDuPont (1990). see discussion in the "Neurotoxicity" section], providing support for this effect level. At this dose, incidences of persistent or recurrent yellow staining of perineum, abdomen, inguen, and/or underbody in female rats were significantly increased, while these were not observed in the males. However, the biological significance of these observations is unclear because they could represent changes in grooming behavior, rather than to the direct toxic effect of o-phenylenediamine. The no-observed-adverse-effect level (NOAEL) is 40 mg/kg-day. Chronic-Duration/Carcinogenicity Studies Matsumoto et al. (2012) (Rat Study) A chronic-duration carcinogenicity study of o-phenylenediamine dihydrochloride (99.5% pure) was conducted in rats exposed via drinking water (Matsumoto et al.. 2012). Groups of 50 male and 50 female F344/DuCrj rats were exposed for 2 years to drinking water concentrations of 0, 250 (females only), 500, 1,000, or 2,000 ppm (males only). Exposure solutions were prepared twice weekly, and the concentrations determined analytically every 3 months during the study. The study authors calculated o-phenylenediamine dihydrochloride doses of 0, 22, 42, and 86 mg/kg-day in male rats and 0, 18, 33, and 58 mg/kg-day in female rats. Equivalent doses of o-phenylenediamine1 are 0, 13, 25, and 51 mg/kg-day in male rats and 0, 11, 20, and 35 mg/kg-day in female rats. The authors reported that the doses were chosen based on their 13-week unpublished study that showed that female rats were more sensitive than male rats. In that experiment, 2/10 female rats given 3,000 ppm o-phenylenediamine dihydrochloride died; however, timing of deaths and additional information were not reported. A complete report of this subchronic-duration study could not be located for further review. Calculated as the product of dose (mg/kg-day) o-phenylenediamine dihydrochloride and the ratio of molecular weights (108.14 g/molforo-phenylenediamine:181.062 g/mol for o-phenylenediamine dihydrochloride). 13 o-Phenyl enedi amine ------- FINAL 09-27-2016 The investigators performed daily observations of the animals for death and clinical signs of toxicity. Food and water intake and body weights were measured weekly for 14 weeks and monthly thereafter. At sacrifice, blood was collected for hematology and clinical chemistry, and urine samples were collected and analyzed. The measured hematological parameters included Hb, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets. Serum samples were analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), y-glutamyl transferase (GGT), and blood urea nitrogen (BUN). Urinalysis measured occult blood and pH. All animals (including those that died or were sacrificed moribund) received gross necropsy. Organ-weight measurements and histopathology examinations were performed, but the study authors did not provide details of the selected tissues. Survival was not significantly altered by treatment with o-phenylenediamine dihydrochloride (see Table B-2); the 2-year survival rates for all groups ranged between 72 and 88% and exhibited no dose-related reductions in males or females. The only clinical signs of toxicity were observed in the high-dose males, which exhibited hematuria (presence of blood in urine) after the first year on treatment (incidence and frequency not reported). Biological and/or statistically significant dose-related reductions in body weight (compared with controls) were evident in both sexes (see Table B-2). In males, terminal body weights were significantly reduced by 7, 14, and 30% (compared with controls) at 13-, 25-, and 51-mg/kg-day o-phenylenediamine, respectively. In females, terminal body weights were 6, 8, and 19% lower than controls at 11-, 20-, and 35-mg/kg-day o-phenyl en edi amine, respectively; the differences were significant at the two higher doses. All treated male rats exhibited significantly lower water intake than controls (10, 17, and 25% less in the low-, medium-, and high-dose groups), as did females in the mid- and high-dose groups (15 and 30% less than controls, respectively). Food intake was also reduced in high-dose males and females (compared with controls) throughout the study, and in all dosed males in the beginning and end of the study (data not shown). Among male rats, small (<4% difference from controls) reductions in MCV and MCH occurred, but the changes were not dose-related (see Table B-3). Among females, statistically significant, albeit modest (<3% difference from controls), reductions in Hb, MCV, MCH, and MCHC were seen at the high dose (see Table B-3). In addition, platelet count was statistically significantly increased (21% higher than controls) in high-dose females. Serum chemistry changes indicative of liver toxicity (see Table B-3) occurred in high-dose male and female rats, including marked, statistically significant, increases in AST and ALT (both sexes) and in ALP and GGT (females only). In males, AST and ALT were increased by >18- and >4-fold, respectively, compared with controls; in females, AST and ALT were both increased by >3-fold. Increases in ALP and GGT in high-dose females were 48% and 6-fold, respectively, compared with controls. Finally, both male and female rats exposed to the highest doses exhibited statistically significantly higher BUN than controls (36 and 9% higher than controls, respectively; see Table B-3). Statistically significant increases in relative liver weights were observed in both male and female rats at the high dose (16 and 75% higher than controls, respectively) and in males exposed to 25-mg/kg-day o-phenylenedi amine (8% higher) (see Table B-2). Absolute liver weight was increased in high-dose females (41% higher than controls), while male rats exhibited dose-related declines in absolute liver weight, likely related to the declines in body weight. Weights of organs other than liver were either not measured or not reported. 14 o-Phenyl enedi amine ------- FINAL 09-27-2016 In both male and female rats exposed to o-phenylenediamine dihydrochloride, non-neoplastic histopathology findings occurred in the liver, urinary bladder, and kidney. The incidences of these lesions are shown in Table B-4. All exposed male groups exhibited statistically significant increases in the incidences of basophilic cell foci of the liver, and males of the low- and mid-dose groups also exhibited increased incidences of clear cell foci. Exposed females at all doses exhibited statistically significantly increased incidences of basophilic cell foci, but not clear cell foci. Renal lesions occurring at statistically significantly increased incidences at all doses included papillary mineralization in males and renal pelvis urothelial hyperplasia in females (see Table B-4). Male rats exposed to >25-mg/kg-day o-phenylenediamine also exhibited urothelial hyperplasia of the renal pelvis, and high-dose males exhibited an increased incidence of papillary necrosis. In females, other renal lesions (papillary necrosis and mineralization) occurred at statistically significantly increased incidence at the high dose only. Only high-dose male rats exhibited a statistically significant increase in the incidence of bladder lesions (papillary and/or nodular hyperplasia of the transitional epithelium). The lowest dose in this study is a LOAEL for increased incidences of renal papillary mineralization and basophilic cell foci of the liver in males (13-mg/kg-day o-phenylenediamine) and increased incidences of urothelial hyperplasia of the renal pelvis and basophilic cell foci of the liver in females (11-mg/kg-day o-phenylenediamine). At this dose, male rats also exhibited a significantly lower terminal body weight than controls, although the decrease was <10% and was possibly attributed to concomitant decreases in food and water consumption rather than a direct adverse effect of o-phenylenediamine. A NOAEL is not identified. Tumors of the liver, including hepatocellular adenomas and carcinomas, were observed at significantly increased incidences in both male and female rats (see Table B-5). Statistically significant increases occurred at doses >25-mg/kg-day o-phenylenediamine in males and >20-mg/kg-day o-phenylenediamine in females. High-dose male rats also exhibited a significantly increased incidence of transitional cell papilloma and/or carcinoma of the urinary bladder; this tumor did not occur at increased incidence in females. Finally, a significant dose-related trend was seen in the incidence of follicular adenoma of the thyroid in male rats; pairwise comparisons with the control incidence were not significant at any dose. Treatment with o-phenylenediamine dihydrochloride did not alter the incidence of thyroid tumors in female rats. Matsumoto et al. (2012): Mouse Study A chronic-duration carcinogenicity study of o-phenylenediamine dihydrochloride (purity 99.5%) was conducted in mice exposed via drinking water (Matsumoto et al. 2012). Groups of 50 male and 50 female Crj :BDFi mice were exposed for 2 years to drinking water concentrations of 0, 500 (males only), 1,000, 2,000, or 4,000 (females only) ppm. Exposure solutions were prepared twice weekly, and the concentrations determined analytically every 3 months during the study. The study authors calculated o-phenylenediamine dihydrochloride doses of 0, 46, 94, and 177 mg/kg-day in male mice and 0, 106, 200, and 391 mg/kg-day in female mice. Equivalent doses of o-phenylenediamine2 are 0, 27, 56, and 106 mg/kg-day in male mice and 0, 63.3, 119, and 234 mg/kg-day in female mice. The study authors reported that the doses were chosen based on the authors' 13-week unpublished study that showed that male Calculated as the product of dose (mg/kg-day) o-phenylenediamine dihydrochloride and the ratio of molecular weights (108.14 g/molforo-phenylenediamine:181.062 g/mol for o-phenylenediamine dihydrochloride). 15 o-Phenyl enedi amine ------- FINAL 09-27-2016 mice were more sensitive than female mice. A complete report of this 13-week, subchronic-duration study could not be located for further review. As in the rat study above, daily observations for death and clinical signs of toxicity were performed. Food and water intake and body weights were measured weekly for 14 weeks and monthly thereafter. At sacrifice, blood was collected for hematology and clinical chemistry, and urine samples were collected and analyzed; red and white blood cells were measured in mice, while all other parameters were measured in both mice and rats. All of the mice (including those that died or were sacrificed moribund) received gross necropsy. Organ-weight measurements and histopathology examinations were performed, but the study authors did not provide details of the selected tissues. Survival of mice was not affected by treatment with o-phenylenediamine dihydrochloride (see Table B-6); 2-year survival rates ranged between 76 and 84% in males and between 48 and 68% in females. No exposure-related clinical signs of toxicity were noted at any dose. Terminal body weight was significantly decreased at all doses in male mice (16—36% lower than controls) and at >119 mg/kg-day in female mice (15-31% lower than controls). Intake of water was significantly reduced in all treated animals (7—35% lower than controls); food consumption rates were also significantly decreased in male and female mice at all doses (data not shown). Hematology analyses showed statistically significant reductions in red blood cells (RBCs) (males only), white blood cells (WBCs) (males only), Hb (males and females), and MCHC (males and females) in mice receiving the highest dose of o-phenylenediamine dihydrochloride (see Table B-7); the decreases (relative to controls) ranged between 1 and 5% for the erythrocyte parameters and between 9—55% for total leukocyte count. In addition, MCV and platelet counts were statistically significantly increased in both sexes at the high dose. The only hematology parameter with a statistically significant change at the middle dose was MCHC, which was decreased by 2% relative to controls in females receiving 119-mg/kg-day o-phenylenediamine. Statistically significant, marked increases in serum levels of liver enzymes occurred in both male and female mice. In males, ALT was statistically significantly increased (>2-fold) at doses >56 mg/kg-day, and ALP was significantly increased (by 77—172%) at all doses. Serum AST and GGT increased from controls at all doses in male mice and all doses except the lowest dose for GGT in female mice, but did not reach statistical significance. In female mice, serum ALP was significantly increased at all doses (from a 49% increase over controls at the low dose to a 3.5-fold increase at the high dose). Serum ALT was statistically significantly increased (>2-fold compared to controls) at 234-mg/kg-day o-phenylenediamine in females. In both male and female mice, statistically significantly increased BUN was noted at the middle and high doses; these changes ranged between 17—32% above controls. Relative liver weights were significantly increased in male mice at all doses (26-57%) and in females at the two higher doses (61—93%) compared with controls (see Table B-6). Absolute liver weights increased >10% compared to control in male mice exposed to 56 mg/kg-day and female mice >119 mg/kg-day, but not statistically significantly. Weights of organs other than the liver were either not measured or not reported. As was seen with the rats, histopathology lesions were noted in the livers and kidneys of mice; however, mice also developed microscopic lesions in the gall bladder, nasal cavity, and nasopharynx (see Table B-8). In male mice, statistically significant increases in the incidences of basophilic cell foci of the liver (>56-mg/kg-day o-phenylenediamine), acidophilic cell foci of the liver 16 o-Phenyl enedi amine ------- FINAL 09-27-2016 (27 and 56 mg/kg-day), papillary hyperplasia of the gall bladder (at all doses), eosinophilic changes in the olfactory epithelium (27 mg/kg-day), and eosinophilic changes in the respiratory epithelium of the nasal cavity (at 106 mg/kg-day) were noted. Females exhibited statistically significant increased incidences of clear cell (at 234 mg/kg-day), acidophilic cell of the liver (at 234 mg/kg-day), and basophilic cell foci of the liver (at 63.3 and 234 mg/kg-day); papillary hyperplasia of the gall bladder (at >119 mg/kg-day); hydronephrosis of the kidney (at all doses); inflammatory polyps of the renal pelvis (at 63.3 and 119 mg/kg-day; increase was not statistically significant at 234 mg/kg-day); eosinophilic change of the respiratory epithelium in the nasal cavity (at all doses); glandular respiratory metaplasia in the nasal cavity (at >119 mg/kg-day); and eosinophilic change of the nasal olfactory epithelium and the nasopharynx (at the highest dose). The lowest dose (27-mg/kg-day o-phenylenediamine in males and 63.3-mg/kg-day o-phenylenediamine in females) is a LOAEL based on increased serum ALP (both sexes), increased incidence of papillary hyperplasia of the gall bladder (males), and increased incidences of eosinophilic change of the nasal cavity respiratory epithelium and hydronephrosis of the kidney (females). At this dose, male mice also exhibited a significantly lower terminal body weight (>10%) compared to controls; however, this decrease may be the result of concomitant decreases in food and water intake. In addition, male and female mice exposed to the lowest dose exhibited an increase in relative liver weight (>10%); however, there is uncertainty whether the liver weight measurements were affected by significant increases in liver tumor incidences at all doses or the reduction in terminal body weight. A NOAEL is not identified. Exposure to o-phenylenediamine dihydrochloride for 2 years resulted in increased incidences of hepatocellular adenoma and/or carcinoma in male and female mice at all doses; the data are shown in Table B-9. In addition, significant increases in the incidence of papillary adenoma of the gall bladder were noted in high-dose male and mid-dose female mice (incidences were 0/46, 2/50, 4/49, and 5/47 in control, low-, mid-, and high-dose males and 0/50, 1/50, 5/50, and 3/50 in the respective female groups) (see Table B-9). This finding is remarkable because spontaneous papillary adenoma of the gall bladder is a rare tumor in mice. The researchers reported that papillary adenomas of the gall bladder occurred in only 9 of the 60,000 control and chemically treated B6C3Fi mice in the NTP database through 1998, and in none of the almost 2,600 historical control BDFi mice in the Japanese Bioassay Research Center. Weishurger etal. (19 78) (Rat Study) In a chronic-duration carcinogenicity study of 21 aromatic amines, Sprague-Dawley (S-D)-derived Charles River CD rats (25 males/group) were exposed to o-phenylenediamine dihydrochloride (97-99%) pure; purity of individual test compounds not specified) at concentrations of 0, 2,000, or 4,000 ppm in the diet for 18 months (Weisburger et ai, 1978). Doses of 0, 140, or 279 mg/kg-day as o-phenylenediamine dihydrochloride3 are calculated for this review; these doses correspond to equivalent doses of 0, 83.6, or 167 mg/kg-day o-phenylenediamine. The rats were observed for up to 6 months after the end of the treatment period and monitored daily for mortality and clinical signs of toxicity. Body weights were consistently recorded. Complete necropsies were conducted on all animals that died after 3Based on chronic reference values for food consumption (0.036 kg/day) and body weight (0.523 kg) in male S-D rats (U.S. EPA. 19881. Reference values for CD rats are not available; in the absence of strain-specific information, data for S-D rats were used because CD rats are derived from the S-D strain. 17 o-Phenyl enedi amine ------- FINAL 09-27-2016 >6 months of treatment or at study termination. Histological examinations of grossly abnormal organs, tumor masses, lung, liver, kidneys, spleen, adrenal, heart, bladder, stomach, intestines, reproductive organs, and pituitaries were performed. The results reported in the study were limited to neoplastic changes; no data on mortality, clinical signs of toxicity, body weights, or non-neoplastic findings were provided. The authors indicated that their protocol called for doses to be decreased if a body-weight-gain difference of >10% from controls was observed or if animals died due to toxicity, but apparently neither mortality nor significant body-weight-gain differences were observed because the doses were unchanged over the treatment period. Male rats exposed to 167-mg/kg-day o-phenylenediamine exhibited a significant (p < 0.025) increase in hepatocellular carcinomas when compared with either simultaneous controls or controls pooled across the experiments with all 21 compounds (see Table B-10). No liver tumors occurred at the low dose, and no other tumor types were reported. Weisburger etal. (19 78) (Mouse Study) In a companion mouse study, HaM/ICR-derived albino CD-I mice (25/sex/group) were treated with concentrations of 0, 4,000, or 8,000 ppm in the diet for 5 months, after which the dietary concentrations were increased to 8,000 and 16,000 ppm, respectively, for the remaining 13 months on treatment. The study authors did not indicate why the exposure concentrations were increased, but presumably, toxicity was low at the initial concentrations. These concentrations yielded TWA concentrations of 6,900 and 14,000 ppm for the low and high doses, respectively, corresponding to doses of 0-, 1,180-, or 2,390-mg/kg-day and 0-, 1,200-, or 2,440-mg/kg-day o-phenylenediamine dihydrochloride for male and female mice, respectively.4 These doses correspond to equivalent doses of 0-, 704-, or 1,430-mg/kg-day and 0-, 717-, or 1,450-mg/kg-day o-phenylenediamine for male and female mice, respectively. Mice in all groups were given control diets for 3 months after the end of exposure. The same toxicological parameters that were evaluated in rats were also evaluated in mice, and the same tissues were subjected to histological examination, except that pituitaries were not examined. No data regarding mortality, clinical signs of toxicity, or body weights were reported; however, as noted above, the increase in exposure concentration after 5 months suggests that the mice tolerated the initial doses. As seen with rats, both male and female mice exhibited significant (p < 0.05) increases in hepatocellular carcinomas (see Table B-10). In male mice, the increase was statistically significant only at the low dose (704-mg/kg-day o-phenylenediamine), and not at the high dose. In female mice, the increase was statistically significant at both doses, but the incidence at the low dose was significant only when compared with the pooled control group. This carcinogenicity study is limited in that small sample sizes were used, only two doses were tested, exposure duration was less than lifetime, and data reporting was incomplete (growth and survival data were not reported). 4Based on chronic reference values for food consumption (0.0064 and 0.0061 kg/day in males and females, respectively) and body weight (0.0373 and 0.0353 kg in males and females, respectively) in B6C3Fi mice (U.S. EPA. 1988). Reference values for CD-I mice are not available; in the absence of strain-specific information, U.S. EPA recommends using data for B6C3Fi mice. 18 o-Phenyl enedi amine ------- FINAL 09-27-2016 The lack of data on noncancer endpoints (other than the inference that toxicity was low based on the authors' decision to increase the doses) precludes the identification of noncancer effect levels for rats or mice. Inhalation Exposures No studies have been identified. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) Table 4A provides an overview of genotoxicity studies of o-phenylenediamine. Table 4B provides an overview of acute oral and inhalation lethality studies in rats. Acute dermal lethality and toxicity studies, skin and eye irritation studies, and a skin sensitization study are described briefly below in the "Other Routes" section. Dermal absorption of o-phenylenediamine was evaluated in a single in vitro study described below in the "Metabolism/Toxicokinetic Studies" stection; no other studies were pertinent to the toxicokinetics of o-phenylenedi amine. 19 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Genotoxicity studies in prokaryotic organisms Mutation Salmonella typhimurium strains TA98 and TA100 and their nitroreductase -deficient mutants, TA98NR and TA100NR 1, 10, 30, 300, 1,000, 3,000 ng/plate + (TA98, TA98NR) (TA100, TA100NR) Preincubation assay. o-Phenylenediamine was mutagenic to TA98NR at >10 |ig/plate with S9 added. Study authors reported that o-phenylenediamine was a potent mutagen in strain TA98 in the presence of S9, but data were not provided. Cytotoxic to TA98NR and TA100NR at 3,000 |ig/platc. Chung et al. (1996) Mutation S. typhimurium strains TA98 and TA100 0, 1, 10, 100, 1,000, 10,000 ng/plate + (TA98, TA100) Plate incorporation and preincubation assays. o-Phenylenediamine was mutagenic at >10 |ig/plate. Mutagenic in TA98 at lower concentrations than in TA100. Cytotoxicity observed at 10,000 |ig/platc (highest concentration tested). Gentile et al. (1987) Mutation S. typhimurium strain TA1538 0, 20, 40, 60, 80, 100 ng/plate NT + (TA1538) Plate incorporation assay. o-Phenylenediamine was mutagenic to TA1538 at >50 |ig/p'latc with S9 added (effective concentration estimated from data presented graphically). Ames et al. (1975) Mutation S. typhimurium strains C3076, D3052, G46, TA98, TA 100, TA1535, TA1537, and TA1538 0.1-100 ng/mL + (D3052, G46, TA98, TA100, TA1537, TA1538) (C3076, TA1535) Modified Ames gradient plate test. TA1538 and TA98 showed the greatest sensitivity to o-phenylenediamine, with positive results at >0.1 |ig/mL. Thompson et al. (1983) 20 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Mutation S. typhimurium strains TA100, TA1535, TA1537, and TA1538 1, 10, 50, 100, 250, 500, 750, 1,000 ng/plate + (TA1538) (TA100, TA1535, TA1537) Plate incorporation assay. Mutagenic in strain TA1538 only, at >250 |ig/plate. Zeieer et al. (1988) Mutation S. typhimurium strains TA98, TA100, and TA1537 0.01,0.1,0.5, 1, 2, 5 g/L ± (TA98) (TA100, TA1537) + (TA98) (TA100, TA1537) Plate incorporation assay. Mutagenic in TA98 at >0.01 g/L in the presence of S9. Mutagenic without S9 only at a concentration of 1 g/L, but not at higher concentrations. Vooed et al. (1980) Mutation S. typhimurium strains TA98 and TA100 68, 135,269, 539, 1,077 |ig/platc + (TA98) (TA100) Plate incorporation assay. Mutagenic in TA98 at >269 ng/plate. Assmann et al. (1997) Mutation S. typhimurium strains TA98, TA100, and TA1537 NR See comments See comments Preincubation assay. o-Phenylenediamine was reportedly positive, but dose, presence or absence of S9, and affected strain(s) were not specified. Isfaidate and Yoshikawa (1980) Mutation S. typhimurium strain TA98 3 ng/plate (without H202) + (with H2O2) Suspension assay. Mutagenicity tested before and after addition of H2O2. H2O2 oxidation products of o-phenylenediamine were mutagenic with S9. Watanabe et al. (1989) Mutation Escherichia coli strains WP2 and WP2uvrA- 0.1-100 ng/mL — — Modified Ames gradient plate test Thompson et al. (1983) 21 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Mutation Klebsiella pneumoniae 0.5 g/L - - Fluctuation test Vooed et al. (1980) Genotoxicity studies in nonmammalian eukaryotic organisms Mutation Saccharomyces cerevisiae D3 1-5% — — Toxicity observed at concentration between 1 and 5% (not further specified). Zeieer et al. (1988) Genotoxicity studies in mammalian cells in vitro Mutation Mouse lymphoma cells (L5178Y TK ±) 0.04,0.08, 0.12, 0.15, 1.0 mM + + Mutagenic at >0.08 mM Aseard et al. (2013) CAs CHO-K1 cells 187, 374, 748, 1,122 jig/mL + NA Increased percentage of aberrant cells at >187 |ig/mL. The TC50 (concentration cytotoxic to 50% of cells) was 374 ± 30 |ig/mL. S9 mix was not included because nitroreductase in the mix affects the direct-acting mutagenic activity of the test compound. Chung et al. (1996) CAs Chinese hamster lung fibroblasts NR + + CAs detected in 20% of metaphase cells at concentrations between 10 2 and 10 3 mg/mL (results shown graphically). Ishidate and Yoshikawa (1980) CAs Human lymphocytes 5, 10, 15 mM + NA A twofold increase in total aberration frequency/cell, including gaps, was observed at >5 mM o-phenylenediamine. Cebulska- Wasilewska et al. (1998) SCE Human lymphocytes 5, 10, 15 mM + NA A twofold increase in SCE/cell was observed at >10 mM o-phenylenediamine. Cebulska- Wasilewska et al. (1998) Unscheduled DNA synthesis Primaiy rat hepatocytes NR (see comments) NA + 50-500 nM/mL (effective concentration range) Thompson et al. (1983) DNA damage Mouse lymphoma cells (L5178Y TK ±) 0.04,0.08,0.12, 0.15, 1.0 mM + + General DNA damage (frank strand breaks and alkali-labile sites, comet assay) at 0.08 mM without S9 and 0.12 mM with S9. Oxidative DNA damage (modified comet assay with enzyme hOGGl) at 0.08 mM with and without S9. Aseard et al. (2013) 22 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References DNA damage Human lymphocytes 5, 10, 15 mM + NA Comet assay; increased tail moment at 15 mM (effective concentration defined as that yielding mean tail moment >control +2 SD). Cebulska- Wasilewska et al. (1998) Genotoxicity studies—in vivo Dominant lethal mutagenicity Male Charles River CD rats (20/group) treated with o-phenylenediamine in 0.2% aqueous solution by i.p. injection 3 times/wk for 8 wk, and then mated to untreated females; females sacrificed after 17 d and uteri examined 20 mg/kg No significant increase in postimplantation fetal loss. Burnett et al. (1977) Somatic mutation Mouse spot test; female C57BL/6JHan mice (unspecified number/group) mated with T-stock males and subsequently given i.p. injection of o-phenylenediamine in saline on GD 10; offspring examined for spots from Wk 2-4 after birth NR (see comments) Pre and postnatal mortality increased at 108 and 215 mg/kg; negative for spot test at "weighted" dose of 196 mg/kg (as reported by study authors). Gocke et al. (1983) Mouse bone marrow MN test (oral) NMRI mice (two/sex/group); 2 doses orally in 0.9% NaCl at 1 and 24 hr; sacrifice 6 hr after second exposure 108, 216, 324 mg/kg/dose + + Significant increase in frequency of MNPCEs at >108 mg/kg/dose. Wild et al. (1980) 23 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Bone marrow micronuclei NMRI mice (two/sex/group); 2 doses i.p. in 0.9% NaCl at 1 and 24 hr; sacrifice 6 hr after second exposure 27, 54, 108, 216, 324 mg/kg/ injection + + Significant increase in frequency of MNPCEs at >108 mg/kg/injection. Wild et al. f 1980) Bone marrow micronuclei Chinese hamster (two/sex/group); 2 doses i.p. in 0.9% NaCl at 1 and 24 hr; sacrifice 6 hr after second exposure 54, 108,216, 324 mg/kg/ injection + + Significant increase in frequency of MNPCEs at >216 mg/kg/injection. Wild et al. f 1980) Bone marrow micronuclei Male and female albino guinea pigs (three/group; unspecified strain); 2 doses i.p. in 0.9% NaCl at 1 and 24 hr; sacrifice 6 hr after second exposure 108, 216, 324 mg/kg/ injection + + Significant increase in frequency of MNPCEs at >108 mg/kg/injection. Wild et al. (1980) 24 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Inhibition of testicular DNA synthesis Male mice (three to four/group, unspecified strain), o-phenylenediamine administered in DMSO orally; 3H-thymidine incorporation measured in testicular DNA 200 mg/kg + + Significant inhibition of testicular DNA synthesis relative to controls. Seiler (1977) Genotoxicity studies in subcellular systems DNA damage XDNA 250 nM + NA Yielded DNA fragments >4 x 106 daltons in size. Yatnada et al. (1985) a+ = positive; - = negative. CA = chromosomal aberration; CHO = Chinese hamster ovary; DMSO = dimethylsulfoxide; DNA = deoxyribonucleic acid; GD = gestation day; i.p. = intraperitoneal; MN = micronuclei; MNPCE = micronucleated polychromatic erythrocyte; NA = not applicable; NR = not reported; NT = not tested; SCE = sister chromatid exchange; SD = standard deviation. 25 o-Phenyl enedi amine ------- Table 4B. Other Studies Test Materials and Methods Results Conclusions References Acute toxicity (oral/inhalation) Acute oral lethality rats Rats (strain and sex unspecified; 10/dose) were administered o-phenylenediamine via gavage at doses of 501, 562, 631, 708, 800, 891, 1,000, 1,122, or 1,259 mg/kg. Observation time following exposure, clinical signs, and necropsy findings not reported. Study reported in tabular form with few details. Mortality: 1/10, 0/10, 0/10, 2/10, 3/10, 4/10, 8/10, 8/10, and 9/10 deaths at 501, 562, 631, 708, 800, 891, 1,000, 1,122, and 1,259 mg/kg, respectively Oral LD5o = 900 mg/kg Rhone - Poulenc (1951); Woodard (1951) Acute oral lethality mice Mice (strain and sex unspecified; 10/dose) were administered o-phenylenediamine via gavage at doses of 794, 841, 891, 944, 1,000, 1,059, 1,122, or 1,259 mg/kg. Observation time following exposure, clinical signs, and necropsy findings not reported. Study reported in tabular form with few details. Mortality: 5/10, 0/10, 1/10, 2/10, 5/10, 4/10, 9/10, and 10/10 deaths at 794, 841, 891, 944, 1,000, 1,059, 1,122, and 1,259 mg/kg, respectively Oral LD5o = 1,000 mg/kg Rhone - Poulenc (1951); Woodard (1951) Acute oral lethality Individual male ChR-CD rats received o-phenylenediamine in peanut oil via single gavage doses of 450, 670, 1,000, 1,500, or 2,250 mg/kg. Animals were monitored for clinical signs of toxicity and mortality for 14 d after dosing. Study reported in tabular form with few details. The rat exposed to 2,250 mg/kg died 1 hr after dosing. There were no other deaths. Clinical signs in surviving rats included irregular breathing, pallor, dark red-brown urine, poor muscle tone, restlessness, lethargy, and initial weight loss. The study authors reported the ALD as 2,250 mg/kg. DuPont (1967a) Acute oral lethality Individual male ChR-CD rats received o-phenylenediamine in peanut oil or in acetone:peanut oil (1:10) via single gavage doses of 0, 300, 450, 670, 1,000, 1,500, 2,250, or 3,400 mg/kg. Clinical signs of toxicity and body weight were monitored and animals were observed for 14 d after dosing. At sacrifice, all animals were subjected to gross and histopathological examination. Study reported in tabular form with few details. Rats exposed to >1,500 mg/kg died within 2 d after dosing. Pathological changes in animals that died included fatty changes and congestion in the liver. Clinical signs of toxicity in surviving animals included weight loss and discolored urine at >450 mg/kg. Fatty changes and congestion of the liver were also seen at sacrifice of the rat exposed to 1,000 mg/kg. No pathology changes attributed to dosing were reported at doses <670 mg/kg. The study authors reported the ALD as 1,500 mg/kg. DuPont ( 1967b) Acute oral lethality Seven individual female Charles River rats were given single gavage doses of o-phenylenediamine in aqueous solution or methylcellulose suspension (10, 30, 100, 300, 1,000, 3,000, or 10,000 mg/kg). Clinical signs of toxicity and body weight were monitored, and animals were observed for 14 d following dosing. Gross necropsies were conducted on all animals. Study was reported in tabular form with few details. Mortality occurred at 3,000 and 10,000 mg/kg. Surviving rats exhibited hypoactivity and roughed fur (>100 mg/kg), ptosis (>300 mg/kg), and hyperpnea (1,000 mg/kg). Gross necropsy of decedents revealed hemorrhaging in the gastrointestinal tract and mottled kidneys. No gross pathological findings were noted in surviving animals. o-Phenylenediamine was lethal to rats at oral doses of >3,000 mg/kg. This conclusion should be viewed with caution due to issues with reliability of the performing laboratory.3 IBT Labs (1983) 26 o-Phenyl enedi amine ------- Table 4B. Other Studies Test Materials and Methods Results Conclusions References Acute inhalation lethality Male ChR-CD rats (six/group) were exposed to o-phenylenediamine aerosol for 4 hr at analytical TWA concentrations of 1.0, 1.3, 2.1, 3.1, 3.4, or 9.2 mg/L (1,000-9,200 mg/m3). Exposures were generated by heating the o-phenylenediamine and forming aerosols by blowing compressed nitrogen through a nebulizer immersed in the molten test material. The "pure" o-phenylenediamine concentrations were measured by ultraviolet spectroscopy in order to account for the known presence of oxidation products. The aerosol mass median diameters varied with concentration, ranging from 1.8-3.3 |im. The study was reported in tabular form and details of the toxicological assessments performed were not provided. The timing of sacrifice varied both among and within groups. Mortality occurred in all exposure groups: 1/6, 2/6, 1/6, 3/6, 3/6, and 6/6 deaths at 1,000, 1,300, 2,100, 3,100, 3,400, and 9,200 mg/m3, respectively. Irregular breathing was observed in all rats during exposure, and rats exposed to 1,300 and 2,100 mg/m3 did not respond to sound stimulation. Clinical signs of toxicity observed after the exposure period included hypersensitivity, aggression, and yellowed fur at 1,300 mg/m3; colored urine, tremors, weakness, and loss of balance or incoordination were seen at higher exposures. Hindquarter paralysis was seen in one surviving rat exposed to 2,100 mg/m3 and in two surviving rats in each of the 3,100- and 3,400-mg/m3 groups. Weight loss of-10% was noted in rats exposed to <2,100 mg/m3, while at >3,100 mg/m3, weight losses were -10-15%. Histopathological findings included evidence of respiratory tract irritation and decreased sperm formation (data and details not provided). Damage to testicular germinal epithelium was observed in animals sacrificed 7, 14, 18, and 46 d after exposure (affected groups and further details not provided). A 4-hr LC50 of 3,600 mg/m3 (95% CI 2,500-5,200 mg/m3) was estimated for o-phenylenediamine aerosol. Exposure to concentrations of >1,300 mg/m3 resulted in signs of neurotoxicity. DuPont (1969) 27 o-Phenyl enedi amine ------- Table 4B. Other Studies Test Materials and Methods Results Conclusions References Acute inhalation lethality Male and female S-D rats (10/sex) were exposed to a measured concentration of 2.46-mg/L (2,460 mg/m3) o-phenylenediamine aerosol (equivalent aerodynamic diameter of 7.2 |im ± 2.43) for 1 hr. The aerosol was generated by delivery of o-phenylenediamine powder mixed with dry filtered air into the exposure chamber; concentrations were measured by collecting material on a filter and weighing it. Animals were observed for clinical signs of toxicity during exposure and daily thereafter for 14 d. Body weights were recorded prior to exposure, and after 7 and 14 d. Gross necropsies were performed on all animals. No deaths occurred. Clinical signs of toxicity were more pronounced or at higher incidence in males than in females. All exposed males exhibited decreased activity on D 2 and hyperactivity on D 2-5. Increased aggression was noted in both males and females. Other clinical signs included labored breathing, salivation, and body tremors. Male rats (6/10) developed alopecia in the second wk. At 10 d postexposure, 7/10 male rats and 3/10 females exhibited loss of muscle coordination in the hind limbs; one female became paraplegic. Mean body-weight gain for both sexes was normal for the duration of the study. Gross findings at necropsy included gelatinous areas on the lung surfaces in approximately 1/3 of the males and females, a "slight incidence" of enlarged cervical lymph nodes in both sexes, and enlarged adrenal glands in 4/10 females. Exposure to 2,460-mg/m3 o-phenylenediamine aerosol for 1 hr was not lethal to rats, but resulted in clinical signs of neurotoxicity as well as grossly visible lesions of the lung and enlarged lymph nodes and adrenal glands. Sherwin Williams (1992) 28 o-Phenyl enedi amine ------- Table 4B. Other Studies Test Materials and Methods Results Conclusions References Acute inhalation toxicity Male and female BLU:(LE)BR rats (five/sex/group) were exposed to 1,340-mg/m3 (nominal concentration) o-phenylenediamine vapor for 4 hr. Clinical signs of toxicity and body weight were monitored for 14 d following exposure. Gross necropsy was performed on all animals. The study was reported in tabular form with few details. All animals survived exposure and no clinical signs of toxicity were noted. Body-weight gain was within normal limits for all animals. There were no gross pathological findings at necropsy. A 4-hr exposure to 1,340-mg/m3 o-phenylenediamine vapor was not lethal to 10 rats. This conclusion should be viewed with caution due to issues with reliability of the performing laboratory.3 IBT Labs (1975c) aA total of 618/867 nonacute toxicity studies conducted by Industrial Bio-Test Laboratories (including subacute-duration, subchronic-duration, carcinogenicity, reproductive toxicity, genotoxicity, and neurotoxicity studies) were found to be invalid during a post hoc audit program conducted by U.S. EPA and the Canadian Health and Welfare Department (OECD. 20071. Discrepancies and deficiencies were also identified in acute studies, but the focus of the investigation was on repeated exposure studies that formed the basis of regulatory decisions. The laboratory closed in 1978. OECD (2007) outlined specific criteria for using data generated by Industrial Bio-Test Laboratories, and recommended rejecting a study when either a regulatory or internal audit revealed problems impacting the reliability of the findings, or when the findings of unaudited studies are inconsistent with data collected later by reputable laboratories. OECD (2007) recommended that studies that have not been audited should be used with caution and only as weak evidence if supported by later data from reputable laboratories. No information was available on internal or external auditing of this study. ALD = approximate lethal dosage; CI = confidence interval; LC50 = median lethal concentration; LD5o = median lethal dose; S-D = Sprague-Dawley; TWA = time-weighted average. 29 o-Phenyl enedi amine ------- FINAL 09-27-2016 Genotoxicity o-Phenylenedi amine has been tested in a number of in vitro and in vivo genotoxicity tests (see Table 4A for details), with predominantly positive results. o-Phenylenediamine induced mutations in Salmonella typhimurium in the presence of metabolic activation (Assmann et aL 1997; Chung et aL 1996; Zeiger et al.. 1988; Gentile et al.. 1987; Thompson et al.. 1983; Ishidate and Yoshikawa, 1980; Voogd et al, 1980; Ames et al, 1975); results without activation were uniformly negative. Mutagenicity was greatly enhanced in S. typhimurium with the addition of hydrogen peroxide treatment (applied to mimic conditions of o-phenylenediamine exposure in hair dye) (Watanabe et al. 1989). Mutagenicity assays in Escherichia coli (Thompson et al.. 1983). Klebsiella pneumoniae (Voogd et al.. 1980). and Saccharomyces cerevisiae (Zeiger et al. 1988) were negative both with and without metabolic activation. In mammalian cells, o-phenylenediamine induced mutations and deoxyribonucleic acid (DNA) damage in mouse lymphoma cells (L5 178Y TK±) (Asgard et al. 2013). Increased frequencies of chromosomal aberrations (CAs) were reported in Chinese hamster cells (Chung et al.. 1996) and Chinese hamster lung fibroblasts (Ishidate and Yoshikawa. 1980) treated with o-phenylenediamine. Furthermore, unscheduled DNA synthesis was seen in primary rat hepatocytes incubated with o-phenylenediamine (Thompson et al.. 1983). o-Phenylenediamine also induced CAs, sister chromatid exchanges (SCEs), and DNA damage in human lymphocytes (Cebulska-Wasilewska et al.. 1998). Incubation of XDNA with o-phenylenediamine resulted in increased DNA damage (Yamada et al.. 1985). In in vivo animal tests, o-phenylenediamine did not induce dominant lethal mutagenicity (to assess mutation in the germinal cells) in male rats following intraperitoneal (i.p.) administration (Burnett et al.. 1977) or somatic mutations of fetal cells in a spot test in female mice treated intraperitoneally (Gocke et al.. 1983). o-Phenylenediamine induced increased frequencies of bone marrow micronuclei in mice following oral and i.p. administration, and in hamsters and guinea pigs following i.p. administration (Wild et al.. 1980). Treatment of male mice with o-phenylenediamine resulted in significant inhibition of testicular DNA synthesis compared with control mice; the study authors reported that a positive response (e.g., inhibition) in this test was seen with many chemical carcinogens and mutagens, but not with compounds that are not carcinogenic or mutagenic (Seiler. 1977). Acute Toxicity The acute oral and inhalation toxicity studies of o-phenylenediamine are detailed in Table 4B. Oral median lethal dose (LD50) values of 900 and 1,000 mg/kg in rats and mice, respectively, were estimated by Rhone-Poulenc (1951) and Woodard (1951). In two studies using individual male ChR-CD rats receiving single gavage doses of o-phenylenediamine, DuPont (1967a. 1967b) estimated the approximate lethal dose of o-phenylenediamine to be 1,500 mg/kg (DuPont. 1967b) or 2,250 mg/kg (DuPont. 1967a). Industrial Bio-Test Laboratories (IB T Labs. 1983) assessed the lethality of single gavage doses of o-phenylenediamine in individual female Charles River rats; the authors reported deaths at doses of >3,000-mg/kg o-phenylenediamine. The latter study should be viewed with caution, as studies by the 30 o-Phenyl enedi amine ------- FINAL 09-27-2016 performing laboratory were found to have a number of deficiencies in an audit conducted by the U.S. EPA and the Canadian Health and Welfare Department.5 No deaths occurred in an acute inhalation study in which male and female S-D rats were exposed for 1 hour to o-phenylenediamine aerosol at a concentration of 2,460 mg/m3; however, signs of neurotoxicity, including loss of muscle coordination and limb paralysis were observed (Sherwin Williams. 1992). Exposure of male ChR-CD rats to o-phenylenediamine aerosol for 4 hours at analytically determined TWA concentrations of 1,000-9,200 mg/m3 resulted in deaths at all exposure levels; the 4-hour median lethal concentration (LCso) in rats was estimated to be 3,600 mg/m3 (I)uPont. 1969). All exposure levels >1,300 mg/m3 resulted in signs of neurotoxicity, with hindlimb paralysis seen at concentrations >2,100 mg/m3 (DuPont. 1969). There were no deaths, and no evidence of toxicity (based on clinical observations, body-weight measurement, and gross necropsy) when male and female BLU:(LE)BR rats were exposed for 4 hours to o-phenylenediamine vapor at a nominal concentration of 1,340 mg/m3 in a study performed by Industrial Bio-Test Laboratories (IBTLabs, 1975c); however, results from this laboratory must be viewed with caution.5 Other Routes Studies by Haskell Laboratories (1970b) and DuPont (1968). in which o-phenylenediamine was applied to intact skin for 24 hours under occlusion and animals were observed for 2 weeks, indicated that the dermal approximate lethal dose (ALD) of o-phenylenediamine was -1,500 mg/kg in male albino rabbits. No mortality occurred within the 14-day observation period when o-phenylenediamine was applied as a slurry in water to the abraded skin of female New Zealand white (NZW) rabbits at doses as high as 3,000 mg/kg (IB I' Labs. 1975a. b); however, results from this laboratory may not be reliable.5 At a dermal dose of 200 mg/kg (administered to intact skin under occlusion), o-phenylenediamine did not affect the 48-hour survival of six male albino rabbits; weight loss was noted on the day after treatment (details not provided) (Haskell Laboratories. 1982). Haskell Laboratories (1981) reported that o-phenylenediamine was slightly irritating, but not corrosive, to the skin of albino rabbits (sex not specified) following application of 0.5 g to clipped skin under occlusive wrapping for 4 hours. Solid o-phenylenediamine (-0.04-0.09 g) applied as a paste in hydrophilic ointment to the intact or abraded skin of male guinea pigs produced mild skin irritation (Haskell Laboratories. 1970a). Application of o-phenylenediamine in acetone: dioxane containing 13% guinea pig fat at exposure concentrations between 5-25% resulted in increased incidence and severity of erythema (compared with controls) in male guinea pigs (Haskell Laboratories. 1967). Mild to moderate sensitization was noted in male guinea pigs following the first challenge test after treatment with nine applications of o-phenylenediamine at 5 A total of 618/867 nonacute toxicity studies conducted by Industrial Bio-Test Laboratories (including subacute, subchronic-duration, carcinogenicity, reproductive toxicity, genotoxicity, and neurotoxicity studies) were found to be invalid during a post hoc audit program conducted by U.S. EPA and the Canadian Health and Welfare Department (OECD. 20071. Discrepancies and deficiencies were also identified in acute studies, but the focus of the investigation was on repeated exposure studies that formed the basis of regulatory decisions. The laboratory closed in 1978. OECD (2007) outlined specific criteria for using data generated by Industrial Bio-Test Laboratories, and recommended rejecting a study when either a regulatory or internal audit revealed problems impacting the reliability of the findings, or when the findings of unaudited studies are inconsistent with data collected later by reputable laboratories. OECD (2007) recommended that studies that have not been audited should be used with caution and only as weak evidence if supported by later data from reputable laboratories. No information was available on internal or external auditing of this study. 31 o-Phenyl enedi amine ------- FINAL 09-27-2016 concentrations between 1-25% (Haskell Laboratories. 1967). The second challenge test in this study produced fewer sensitized animals, and there was no evidence of cross-sensitization with ^-phenyl enediamine. o-Phenylenediamine was mildly to moderately irritating to the eyes of rabbits when applied as a solid or dissolved in propylene glycol (see Footnote 5) (IBTLabs. 1975c; Haskell Laboratories. 1970a). Haskell Laboratories (1970a) also reported corneal haziness and slight iris congestion after exposure to the solid. In general, ocular effects were resolved within 7 days. Metabolism/Toxicokinetic Studies As a solid chemical or in solution, o-phenylenediamine is readily oxidized; however, little is known on the metabolism of o-phenylenedi amine. When used as a chromogenic laboratory substrate, o-phenylenediamine is oxidized to 2,3-diaminophenazine (Tarcha et al. 1987). which also has been identified following treatment of o-phenylenediamine with hydrogen peroxide (Watanabe et al.. 1989). Bronaugh and Congdon (1984) measured the percutaneous absorption of o-phenylenediamine in excised human abdominal skin under alkaline conditions designed to mimic the pH of hair dye. A permeability constant of 4.5 x 10 4 cm/hour was estimated. Concentration-dependent binding of o-phenylenediamine to the stratum corneum was observed, with Km (bound/free) values ranging from 6.86-45 at concentrations from 77.2-0.45 M x 105. Mode-of-Action/Mechanistic Studies Methemoglobin formation was measured in five male rats given o-phenylenediamine intraperitoneally at a dose of 100 |imol/kg and sacrificed 5 hours later (Watanabe et al.. 1976). The methemoglobin level in treated rats was 10.8 ± 3.5%; the level in control rats was not reported. Serum liver enzyme levels (AST and ALT) were not significantly altered by treatment. In vitro incubation of o-phenylenediamine (0.5 |imol) with Hb (0.1 |imol) for 5 hours resulted in a significant increase in the percent methemoglobin (5.9 vs. 4.2% in untreated,/* < 0.01). The importance of this finding is uncertain, given the small increase in vitro and the fact that cyanosis has not been reported in animals exposed to o-phenylenediamine at high oral and inhalation doses. Neurotoxicity DuPont (1990) conducted an acute oral neurotoxicity study of o-phenylenediamine suspended in aqueous methyl cellulose, administered by gavage to groups of 12 male and 12 female Crl:CD®BR rats. After pre-exposure baseline motor activity and FOB assessments, single doses of 0, 225, 450, or 900 mg/kg were administered. Body weights and clinical signs were recorded before dosing, on the day of dosing, and on 1,4, and 7 days after dosing; clinical signs were also recorded 1 hour after dosing. Food consumption measurements were taken on the day of dosing as well as 1, 3, 4, and 7 days after dosing. Neurotoxicity assessments consisted of motor activity, FOB, forelimb and hindlimb grip strength, and foot splay assessments conducted at 1 and 24 hours after dosing and repeated 4 days after dosing. Six rats (two males and four females) exposed to 900-mg/kg o-phenylenediamine died between Days 1 and 4; the cause(s) of death were not reported (DuPont. 1990). Clinical signs of toxicity occurred from dosing through Day 4 postdosing, and were increased in a dose-related fashion. These signs included bright yellow urine, abnormal respiration (>450 mg/kg), and 32 o-Phenyl enedi amine ------- FINAL 09-27-2016 staining on the body (900 mg/kg). Significant, dose-related body-weight losses occurred in both sexes of rat at all doses of o-phenylenedi amine during the first day after dosing and when body-weight change was assessed from Days 0 (day of dosing) to 4; all rats resumed gaining weight after Day 4. Mean body weights of high-dose males and females, as well as females exposed to 450 mg/kg, were significantly lower than controls on Day 4. FOB assessment showed significant increases in palpebral closure (males and females) and altered posture (females) at all doses of o-phenylenedi amine; decreased arousal in both sexes at >450 mg/kg; and labored breathing (both sexes), salivation (males), increased ease of removal from home cage (males), and altered fur appearance (females) at the highest dose. Forelimb and hindlimb grip strength and foot splay were not altered by exposure to o-phenylenedi amine. Motor activity was significantly decreased at all doses when tested on the day of dosing. Dose-related decrements in activity persisted at later measurements, with gradual recovery toward normal levels of activity occurring at the lower doses. The study authors concluded that the effects of o-phenylenedi amine on motor activity and FOB assessment parameters reflected an overall systemic toxicity that led to general malaise and decreased arousal in the exposed animals, rather than a specific neurotoxic effect of o-phenylenedi amine. 33 o-Phenyl enedi amine ------- FINAL 09-27-2016 DERIVATION OF PROVISIONAL VALUES Tables 5 and 6 present summaries of noncancer and cancer references values, respectively. Table 5. Summary of Noncancer Reference Values for o-Phenylenediamine (CASRN 95-54-5) Toxicity Type (units) Species/Sex Critical Effect p-Reference Value POD Method POD (HED) UFc Principal Study Subchronic p-RfD (mg/kg-d) NDr Chronic p-RfD (mg/kg-d) Rat/M Increased incidence of renal papillary mineralization 4 x 1(T3 BMDLio 1.2 300 Matsumoto et al. (2012) Subchronic p-RfC (mg/m3) NDr Chronic p-RfC (mg/m3) NDr BMDLio = 10% benchmark dose lower confidence limit; HED = human equivalent dose; M = male(s); NDr = not determined; POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose; UFC = composite uncertainty factor. Table 6. Summary of Cancer Reference Values for o-Phenylenediamine (CASRN 95-54-5) Toxicity Type (units) Species/Sex Tumor Type Cancer Value Principal Study p-OSF (mg/kg-d) 1 Mouse/M Combined tumors 1.2 x kt1 Matsumoto et al. (2012) p-IUR (mg/m3)-1 NDr M = male(s); NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor. DERIVATION OF ORAL REFERENCE DOSES The database of oral studies in experimental animals includes one short-term-duration study (Haskell Laboratories. 1980). one subchronic-duration neurotoxicity study (DuPont. 1992a). and two chronic-duration carcinogenicity studies (Nlatsumoto et ai. 2012; Wei simmer et at.. 1978). A chronic provisional reference dose (p-RfD) was derived based on the available studies. Derivation of a Subchronic Provisional Reference Dose Available data on oral exposure to o-phenyl en edi amine are not sufficient to derive a subchronic p-RfD that is different from the chronic p-RfD. A single, unpublished, subchronic-duration study of o-phenylenediamine in rats, conducted by DuPont (1992a). is available. The study was designed to examine the potential neurotoxic effects of oral exposure 34 o-Phenyl enedi amine ------- FINAL 09-27-2016 to o-phenylenediamine based on neurotoxicity seen in acute inhalation toxicity studies (Sherwin Williams. 1992; DuPont 1969). Although clinical signs of toxicity and neurobehavioral evaluations were thoroughly investigated, neither clinical chemistry nor hematology were assessed and necropsy investigations were limited to histopathology of nervous system tissues. Findings in the study did not suggest that neurotoxicity was a sensitive endpoint in rats after subchronic oral exposure. A chronic-duration study of rats exposed to o-phenylenediamine via drinking water (Nlatsumoto et aL 2012) at doses lower than the subchronic-duration neurotoxicity study (DuPont 1992a) identified the liver, kidney, and urinary bladder as target organs. Because the available subchroni c-durati on study (DuPont 1992a) provides no information on potential toxicity to organs identified as targets in the chronic-duration study (Nlatsumoto et aL 2012). it was not considered suitable to use in deriving a subchronic p-RfD. In the absence of relevant subchronic data, the chronic p-RfD of 4 x 10 3 mg/kg-day (described below) can be adopted as the subchronic p-RfD. Derivation of a Chronic Provisional Reference Dose The chronic-duration study in adult rats exposed to o-phenylenediamine in drinking water is considered the principal study for deriving the chronic p-RfD (Nlatsumoto et aL 2012). The critical effect from this study is increased incidence of renal papillary mineralization in male rats. The study conducted by Nlatsumoto et al. (2012) reported administration of o-phenylenediamine (as o-phenylenediamine dihydrochloride) in drinking water to F344/DuCrj rats and Cij:BDFi mice (50/sex/dose) for 2 years. This study followed GLP procedures, was published in a peer-reviewed journal, had adequate reporting and consideration for appropriate study design, methods, and conduct, including appropriate numbers of animals per group and three treatment doses in each of two species. The study includes comprehensive assessment of body weight, hematology, serum chemistry, urinalysis, liver weight, and gross and microscopic pathology of various organs. No other chronic-duration exposure study of o-phenylenediamine assessed noncancer endpoints. Thus, the study by Nlatsumoto et al. (2012) was considered suitable for derivation of a chronic p-RfD. Statistically significant effects were observed following exposure in all treatment groups of mice or rats; thus, NOAELs were not identified in the Nlatsumoto et al. (2012) study. The LOAELs in male and female rats were 13 and 11 mg/kg-day, respectively (see Table 3A). Endpoints that were significantly different from controls (either statistically significantly altered, or of such magnitude of change to be considered biologically significant) at the lowest dose in male or female rats and exhibited a dose-related change included increased incidence of renal papillary mineralization in males, increased incidence of renal pelvis urothelial hyperplasia in females, and increased incidences of basophilic cell foci of the liver in males and females (see Table B-4). The LOAELs in male and female mice were 27 and 63.3 mg/kg-day, respectively (see Table 3 A). Endpoints that were significantly different from controls (either statistically significantly altered, or of such magnitude of change to be considered biologically significant [e.g., >10% change]) at the lowest dose in male or female mice and exhibited a dose-related change included decreased terminal body weight in males, increased relative liver weight and serum ALP in males and females, increased incidence of papillary hyperplasia of the gall bladder in males, increased incidences of eosinophilic change of the nasal cavity respiratory epithelium in females, and hydronephrosis in females (see Tables B-6 to B-8). 35 o-Phenyl enedi amine ------- FINAL 09-27-2016 Several endpoints that were altered in rats and mice following exposure to the lowest dose of o-phenylenediamine were not considered as potential noncancer points of departure (PODs). Basophilic cell foci in the liver of male and female rats was not considered due to uncertain toxicological significance. These common lesions were not accompanied by other endpoints indicative of liver toxicity, such as changes in serum chemistry enzymes (i.e., ALT or AST) or further pathological lesions. In mice, terminal body weight (males) and relative liver weight (males and females) were not considered for potential POD selection because the changes were not considered to be directly related to the toxicity of o-phenylenediamine or biologically significant (i.e., the change was <10%). Terminal (2-year) body-weight changes were likely a result of decreased food and water consumption. Multiple factors confound the interpretation of increased 2-year relative liver weight in treated male and female mice. Significant increases in proliferative liver lesions were observed at all treatment doses, potentially causing the increased organ weight. In addition, as absolute liver weight was not significantly increased at the lowest dose, reduced terminal body weight may skew interpretation of increases in the organ-to-body weight ratio (i.e., relative weight) at this dose level. Measurements of serum ALP were highly variable, lacked a dose response, and were not accompanied by other non-neoplastic endpoints characteristic of gall bladder toxicity. Thus, increased serum ALP was not judged to be biologically relevant to the toxicity of o-phenylenedi amine. Eosinophilic change of the nasal cavity respiratory epithelium (female mice) was also not considered because the effect may have resulted from accidental sipping of drinking water containing o-phenylenedi amine via the nose. Thus, the biological significance of the pathological effect observed in the nasal cavity following exposure via the oral route is unclear. Finally, papillary hyperplasia in the gall bladder of male mice was not considered because it may be considered a preneoplastic lesion. Increased incidences of rare papillary adenomas of the gall bladder were reported in male and female mice following exposure to o-phenylenediamine (Nlatsumoto et aL 2012) (see Table B-9). These lesions were accompanied by increased incidences of papillary hyperplasia in the gall bladder which may act as a precursor to further carcinogenesis. The remaining data for effects occurring at the lowest doses tested were selected for benchmark dose (BMD) modeling and are provided in Table 7. 36 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 7. Selected Non-neoplastic Endpoints in Male and Female F344/DuCrj Rats and CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3 Male Rats Dose, mg/kg-d (as o-phenylenediamine) 0 13 25 51 Kidney—Papillary mineralization 7/50b 18/50° 16/50 26/50 Female Rats Dose, mg/kg-d (as o-phenylenediamine) 0 11 20 35 Kidney—Urothelial hyperplasia: pelvis 2/50 12/50 10/50 17/50 Female Mice Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Kidney—Hydronephrosis 2/50 12/50 13/50 11/50 aMatsimioto et at (2012). bNumber of animals affected/number of animals examined. °A11 treatment groups were significantly different from control atp< 0.05 by Fisher's exact test performed by the study authors. dMean ± standard deviation. All dichotomous models in the U.S. EPA's Benchmark Dose Software (BMDS, Version 2.5) were fit to the data of each potential dichotomous endpoint occurring at the lowest doses tested from the study conducted by Matsumoto et al. (2012) (see Table 7). Appendix C presents the details of the modeling procedure and results for all endpoints. The results are summarized in Table 8. 37 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 8. Potential Chronic PODs in Male and Female Rats and Mice Exposed Orally to o-Phenylenediamine (CASRN 95-54-5) for 2 Years3 Endpoint NOAEL mg/kg-d LOAEL mg/kg-d Animal PODb mg/kg-d POD (HED)C mg/kg-d Male Rats Kidney—Papillary mineralization"1 ND 13 BMDLio = 4.8 BMDLio (HED) = 1.2 Female Rats Kidney—Urothelial hyperplasia: pelvis ND 11 BMDLio — 5.6 BMDLio (HED) = 1.3 Female Mice Kidney—Hydronephrosis ND 63.3 BMDLio = 23.1 BMDLio (HED) = 3.23 "Matsimioto et al. (2012). bBMD modeling results are described in detail in Appendix C. cPOD (HED) = Animal POD (mg/kg-day) x DAF of 0.24 for rats or 0.14 for mice (U.S. EPA. 2011^. dChosen as the critical effect for derivation of the chronic p-RfD. BMD = benchmark dose; BMDLio = 10% benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; ND = no data; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose. 38 o-Phenyl enedi amine ------- FINAL 09-27-2016 In the EPA's Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lc). the Agency endorses a hierarchy of approaches to derive human equivalent oral exposures from data derived from laboratory animal species, with the preferred approach being physiologically based toxicokinetic modeling. Other approaches may include using some chemical-specific information, without a complete physiologically based toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of human equivalent oral exposures, EPA endorses body-weight scaling to the 3/4 power (i.e., BW3/4) as a default to extrapolate toxicologically equivalent doses of orally administrated agents from all laboratory animals to humans for the purpose of deriving an RfD under certain exposure conditions. More specifically, the use of BW3 4 scaling for deriving an RfD is recommended when the observed effects are associated with the parent compound or a stable metabolite, but not for portal-of-entry effect endpoints. A validated human physiologically based toxicokinetic model for o-phenylenedi amine is not available for use in extrapolating doses from animals to humans. Furthermore, the selected lesions of the kidney in rats or mice are not portal-of-entry effects. Therefore, scaling by BW3/4 is relevant for deriving human equivalent doses (HEDs) for these effects. Following U.S. EPA (2011c) guidance, all potential PODs are converted to HEDs by application of dosimetric adjustment factors (DAFs)6 as follows: DAF = (BWa1/4 - BWh1/4) where: DAF = dosimetric adjustment factor BWa = animal body weight BWh = human body weight Using a BWa of 0.25 kg for rats and 0.025 kg for mice, and a BWh of 70 kg for humans, the resulting default DAFs are 0.24 and 0.14 for rats and mice, respectively (U.S. EPA. 2011c). Each POD candidate is multiplied by the appropriate species-specific DAF to obtain a POD (HED) (see Table 8). The lowest POD (HED) following chronic exposure to o-phenylenedi amine is increased incidence of renal papillary mineralization in male rats (BMDLio (HED) =1.2 mg/kg-day). This POD is protective of other effects observed following o-phenylenedi amine exposure and increased renal papillary mineralization is consistently observed across sexes and coherent with other o-phenylenediamine-induced renal effects. Renal papillary mineralization not only exhibited a dose-response relationship in male rats, but was also increased in female rats at all doses with statistical significance at the highest dose. In addition, increased papillary mineralization was accompanied by other adverse o-phenylenediamine-induced events indicating kidney toxicity; male and female rats exhibited increased renal papillary necrosis, urothelial hyperplasia in the renal pelvis, and increased blood urea nitrogen. Based on the available data 6As described in detail in Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201101. rate-related processes scale across species in a manner related to both the direct (BW11) and allometric scaling (BW3'4) aspects such that BW3'4 ^ BW1'1 = BW converted to a DAF = BWa"4 - BWi,1'4. 39 o-Phenyl enedi amine ------- FINAL 09-27-2016 supporting effects on the kidney, the BMDLio (HED) for renal papillary mineralization in male rats (1.2 mg/kg-day) is selected as the POD for derivation of the chronic p-RfD. The chronic p-RfD for o-phenylenediamine is derived as follows: Chronic p-RfD = BMDLio (HED) UFc = 1.2 mg/kg-day -^300 = 4 x 10 3 mg/kg-day The composite uncertainty factor (UFc) for the chronic p-RfD for o-phenylenediamine is 300, as summarized in Table 9. Table 9. Uncertainty Factors for the Chronic p-RfD for o-Phenylenediamine (CASRN 95-54-5) UF Value Justification UFa 3 A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or toxicodynamic differences between rats and humans following o-phenylenediamine exposure. The toxicokinetic uncertainty has been accounted for by calculation of an HED through application of a DAF as outlined in the EPA's Recommended Use of Body WeightB/4 as the Default Method in Derivation of the Oral Reference Dose ('U.S. EPA. 201 lc). UFd 10 A UFd of 10 is applied to account for deficiencies and uncertainties in the database, specifically the lack of data on reproductive or developmental toxicity. UFh 10 A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in susceptibility, in the absence of information to assess toxicokinetic and toxicodynamic variability of o-phenylenediamine in humans. UFl 1 A UFl of 1 is applied because the POD is a BMDL, not a LOAEL. UFS 1 A UFS of 1 is applied because the POD comes from a chronic-duration study of rats. UFC 300 Composite Uncertainty Factor = UFA x UFD x UFH x UFL x UFS. BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; POD = point of departure; UF = uncertainty factor. The confidence in the chronic p-RfD for o-phenylenediamine is low as described in Table 10. 40 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 10. Confidence Descriptors for the Chronic p-RfD for o-Phenylenediamine (CASRN 95-54-5) Confidence Categories Designation Discussion Confidence in study M Confidence in the principal study is medium. Factors contributing to medium confidence in the principal study include: (1) appropriate numbers of animals in exposure and control groups for meaningful statistical analyses; (2) adequate numbers of exposure groups to establish dose-response relationships; and (3) assessment of a wide range of toxicological endpoints (body weight, hematology, serum chemistry, liver weight, and gross and microscopic pathology). The major factor restricting confidence in the principal study is the lack of a dose low enough to permit identification of a NOAEL. Confidence in database L Confidence in the database is low. The oral database for noncancer effects of o-phenylenediamine consists of a short-term-duration rat drinkine water palatabilitv studv assessing limited endpoints (Haskell Laboratories. 1980). a subchronic-duration neurotoxicity studv in rats (DuPont. 1992a). and a chronic-duration studv in rats and mice exDOsed via drinking water (Matsumoto et al.. 2012). There are no reproductive or developmental toxicity studies in animals. The database deficiencies restrict confidence in the determination of the critical effect from oral exposure to o-phenylenediamine. Confidence in chronic p-RfDa L The overall confidence in the chronic p-RfD for o-phenylenediamine is low. aThe overall confidence cannot be greater than the lowest entry in the table (low). L = low; M = medium; NOAEL = no-observed-adverse-effect level; p-RfD = provisional reference dose. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS No studies of humans or animals exposed to o-phenylenediamine via inhalation have been identified in the available literature, precluding derivation of provisional inhalation reference concentrations (p-RfCs). CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR Table 11 provides the cancer weight-of-evidence (WOE) descriptor for o-phenylenediamine. 41 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 11. Cancer WOE Descriptor for o-Phenylenediamine (CASRN 95-54-5) Possible WOE Descriptor Designation Route of Entry (oral, inhalation, or both) Comments "Carcinogenic to Humans " NS NA There are no human data to support this. "Likely to Be Carcinogenic to Humans" Selected Oral There are no human data on the potential carcinogenicity of o-phenylenediamine by any exposure route. Chronic (>18 mo) exposure to o-phenylenediamine dihydrochloride in the drinking water or diet resulted in significantly increased incidences of liver tumors in male and female rats and in male and female mice in two studies (Matsumoto et aL. 2012; Weisbureer et aL. 1978). a significantly increased incidence of urinary bladder tumors and a significant dose-related trend for thyroid follicular adenoma in male rats (Matsumoto et aL. 2012). and significantly increased incidences of rare papillary adenomas of the gall bladder in male and female mice (Matsumoto et aL. 2012). The induction of tumors at multiple sites and in both sexes and species tested, as well as the induction of rare tumors in mice, support this cancer WOE for o-phenylenediamine. "Suggestive Evidence of Carcinogenic Potential" NS NA The available evidence is more than suggestive of carcinogenicity and is judged sufficient for a stronger conclusion. "Inadequate Information to Assess Carcinogenic Potential" NS NA Adequate data are available to assess carcinogenic potential. "Not Likely to Be Carcinogenic to Humans " NS NA Evidence of the carcinogenic potential of o-phenylenediamine is available in animals. NA = not applicable; NS = not selected; WOE = weight of evidence. Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a), exposure to o-phenylenediamine is "Likely to Be Carcinogenic to Humans" based on evidence of carcinogenicity in orally treated male and female rats and mice (see Table 11). There are no human data on the potential carcinogenicity of o-phenylenediamine by any exposure route. The carcinogenicity of o-phenylenediamine has been tested in two chronic-duration studies of rats and mice exposed orally (Matsumoto et aL 2012; Wei simmer et ai. 1978). Chronic (> 18 months) exposure to o-phenylenediamine dihydrochloride in the drinking water or diet resulted in significantly increased incidences of tumors in all studies, including increased liver tumors in male and female rats and mice in all studies (Matsumoto et ai. 2012; Weisburger et ai. 1978). increased incidence of urinary bladder tumors and thyroid follicular adenoma in male rats (Matsumoto et ai, 2012), and increased incidences of rare papillary adenomas of the gall bladder in male and female mice (Matsumoto et ai, 2012) 42 o-Phenyl enedi amine ------- FINAL 09-27-2016 (see Table 12). The papillary adenomas of the gall bladder observed in male and female mice following exposure to o-phenylenediamine are considered a rare tumor in mice. Matsumoto et ai (2012) reported that papillary adenomas of the gall bladder occurred in only 9 of the 60,000 control and chemically treated B6C3Fi mice in the NTP database through 1998, and in none of the almost 2,600 historical control BDFi mice in the Japanese Bioassay Research Center. 43 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table 12. Incidences of Selected Neoplastic Lesions in Male and Female F344/DuCrj Rats and CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3 Male Rats Dose, mg/kg-d (as o-phenylenediamine, ADD [HED])b 0 (control) 13 (3.2) 25 (6.1) 51 (13) Hepatocellular adenoma and/or carcinoma—liver 4/5 0"° (8) 3/50 (6) 16/50* (32) 22/50* (44) Transitional cell papilloma and/or carcinoma—urinary bladder 2/5 0# (4) 0/50 (0) 0/50 (0) 10/50* (20) Follicular adenoma—thyroid 0/5 0# (0) 1/50 (2) 0/50 (0) 4/50 (8) Female Rats Dose, mg/kg-d (as o-phenylenediamine)b 0 (control) 11 (2.6) 20 (4.8) 35 (8.5) Hepatocellular adenoma and/or carcinoma—liver l/50# (2) 3/50 (6) 19/50* (38) 44/50* (88) Male Mice Dose, mg/kg-d (as o-phenylenediamine)b 0 (control) 27 (3.8) 56 (7.7) 106 (14.5) Hepatocellular adenoma and/or carcinoma—liver 18/50# (36) 29/50* (58) 39/50* (78) 38/50* (76) Papillary adenoma—gall bladder 0/46# (0) 2/50 (4) 4/49 (8) 5/47* (10) Female Mice Dose, mg/kg-d (as o-phenylenediamine)b 0 (control) 63.3 (8.70) 119 (16.4) 234 (32.1) Hepatocellular adenoma and/or carcinoma—liver 6/5 0# (12) 23/50* (46) 31/50* (62) 41/50* (82) Papillary adenoma—gall bladder 0/50 (0) 1/50 (2) 5/50* (10) 3/50 (6) "Matsimioto et al. (2012). bDose expressed as average daily animal dose of o-phenylenediamine (mg/kg-day) and as an HED in parentheses. HF.D = Dose x (BW„ ^ BWh)1/4 where Dose = average daily animal dose of o-phenylenediamine, BWa = reference animal body weight, BVV'h = 70 kg, reference human body weight (U.S. EPA. 19881. Example calculation: HED = 13 x (0.25/70)1'4 = 3.2 mg/kg-day. °Number of animals affected/total number of animals (% of animals affected); % calculated by EPA. * Statistically different from controls, p < 0.05 by Fisher's exact test performed by study authors. "Statistically dose-related positive trend atp< 0.05 by Peto's test performed by study authors. ADD = adjusted daily dose; HED = human equivalent dose. 44 o-Phenyl enedi amine ------- FINAL 09-27-2016 As stated in the Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a). examples of supporting data to conclude that a chemical is "Likely to Be Carcinogenic to Humans " include (1) "an agent that has tested positive in animal experiments in more than one species, sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans"; (2) "a rare animal tumor response in a single experiment that is assumed to be relevant to humans." Based on these examples from the U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) and the data available in male and female rats and mice, exposure to o-phenylenediamine is "Likely to Be Carcinogenic to Humans. " MODE-OF-ACTION DISCUSSION The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) defines mode of action (MO A) "as 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." Examples of possible modes of carcinogenic action include mutagenic, mitogenic, anti-apoptotic (inhibition of programmed cell death), cytotoxic with reparative cell proliferation, and immunologic suppression. The available data support a determination that o-phenylenediamine may be carcinogenic by a mutagenic MOA. There are no data to suggest alternative MO As. Elements to consider in determining whether a carcinogen acts by a mutagenic MOA are provided in Section 5 of the 2005 Cancer Supplementary Guidance (U.S. EPA. 2005b). which states that "determinations of chemicals that are operating by a mutagenic MOA entails evaluation of test results for genotoxic endpoints, metabolic profiles, physiochemical properties, and structure-activity relationships." No data on the metabolism of o-phenylenedi amine were identified in the available literature. Key Events—The proposed MOA for o-phenylenedi amine carcinogenicity consists of the following key events: (1) metabolism to DNA-reactive metabolite(s), (2) covalent binding with DNA, (3) mutation of critical genes such as oncogenes, and (4) proliferation of initiated cells. o-Phenylenediamine has been tested in a number of in vitro and in vivo genotoxicity tests (see Table 4A), with predominantly positive results. o-Phenylenediamine induced mutations in S. typhimurium in the presence of metabolic activation (Assmann et ai. 1997; Chung et ai. 1996; Zeiger et al, 1988; Gentile et ai, 1987; Thompson et ai, 1983; Ishidate and Yoshikawa, 1980; Voogd et ai. 1980; Ames et ai, 1975). but not without metabolic activation. Mutagenicity assays in E. coli (Thompson et ai. 1983). K. pneumoniae (Voogd et ai. 1980). and S. cerevisiae (Zeiger et ai. 1988) were negative both with and without metabolic activation. In mammalian cells, o-phenylenediamine induced mutations and DNA damage in mouse lymphoma cells (Asgard et ai. 2013). increased frequencies of CAs in Chinese hamster ovary cells (Chung et ai. 1996) and Chinese hamster lung fibroblasts (Ishidate and Yoshikawa, 1980), induced unscheduled DNA synthesis in primary rat hepatocytes (Thompson et ai, 1983), and induced CAs, SCEs, and DNA damage in human lymphocytes (Cebul ska-Wasi lewska et ai, 1998). In in vivo animal tests, o-phenylenediamine did not induce dominant lethal mutagenicity (Burnett et ai, 1977) or somatic mutations in a spot test (Gocke et ai, 1983), but did induce increased frequencies of bone marrow micronuclei in mice, hamsters, and guinea pigs (Wild et ai, 1980). 45 o-Phenyl enedi amine ------- FINAL 09-27-2016 Strength, Consistency, Specificity of Association—There is consistent evidence from a variety of different in vitro and in vivo genotoxicity tests to suggest o-phenylenedi amine exposure induces mutations. These tests include mutagenicity and DNA damage assays in bacteria and eukaryotic cells. Less evidence is available to assess genotoxicity and mutagenicity in in vivo studies, although bone marrow micronuclei were consistently induced in several experimental species. The strength, consistency, or specificity of the association between o-phenylenedi amine exposure and subsequent key events (i.e., specific critical gene mutation and cell proliferation) cannot be evaluated due to the lack of data. Analogy—Data on the carcinogenicity of structurally related compounds are limited, and suggest that o-phenylenedi amine may differ toxicologically from the compounds most similar to it. m-Phenylenediamine was not carcinogenic in C3H or C57BL/6 mice exposed by skin application three times per week for 24 months at doses of 0.6 or 3 mg/week (Holland et al.. 1979). Chronic (>2 years) exposure of rats to/>-phenylenediamine (Imaida et al .. 1983) or rats and mice to/>-phenylenediamine dihydrochloride (NCI, 1979) in the diet did not significantly increase the incidence of any tumor type. There was a slight, statistically significant increase in the incidence of alveolar adenoma (18/88 vs. 12/86 in negative controls,/* = 0.04) in female offspring of mice exposed to />phenylenediamine via gavage during gestation [Holmberg et al. (1983) as translated in DuPont (1992bVI. Dose-Response Concordance—There are no data on genotoxic events in any tissue of animals exposed to o-phenylenedi amine at doses below those associated with tumor formation [>20 mg/kg-day in rats and >27 mg/kg-day in mice; Matsumoto et al. (2012)1. Significant increases in the incidences of non-neoplastic proliferative lesions (clear cell foci and basophilic foci) were seen at all doses in the livers of rats in the study by Matsumoto et al. (2012). with increased tumor incidences occurring at the mid and high doses. In mice, increased incidences of liver tumors (as well as foci of alterations) were seen at all doses (Matsumoto et al.. 2012). so there is no information on events occurring at lower doses. There were no significantly increased incidences of proliferative lesions in the urinary bladders of male rats at doses not associated with increased tumor formation (Matsumoto et al.. 2012). Male, but not female, mice exhibited significantly increased incidences of proliferative lesions in the gall bladder (papillary hyperplasia) at all doses, with an increased incidence of papillary adenomas in high-dose male mice. Temporal Relationships—No data are available with which to evaluate the temporal relationship between mutagenesis and tumor formation after o-phenylenedi amine exposure. Matsumoto et al. (2012) did not perform any interim sacrifices, and available short-term- and subchronic-duration studies (DuPont, 1992a; Haskell Laboratories, 1980) did not evaluate histopathology in any of the tissues in which tumor formation was seen in the chronic-duration study by Matsumoto et al. (2012). Biological Plausibility and Coherence—Supporting evidence for the association between mutagenesis and tumor formation comes from the observation that o-phenylenedi amine exposure produced increased incidences of tumors in a wide variety of tissues: liver tumors in male and female rats and in male and female mice (Matsumoto et al.. 2012; Weisburger et al.. 1978). urinary bladder tumors and thyroid follicular adenoma in male rats (Matsumoto et al.. 2012). and rare papillary adenomas of the gall bladder in male and female mice 46 o-Phenyl enedi amine ------- FINAL 09-27-2016 (Nlatsumoto et al.. 2012). Induction of tumors at multiple sites and in different species is characteristic of carcinogens acting via mutagenesis (U.S. EPA. 2005a). Available data do not suggest nongenotoxic MO As for the liver or urinary bladder. Nlatsumoto et al. (2012) noted in their chronic cancer bioassay in rats and mice that no pathology changes suggestive of nongenotoxic MO As (e.g., necrosis, hypertrophy, regenerative hyperplasia, or inflammation in the liver; or calculus formation in the urinary bladder) were observed in the liver or urinary bladder. Papillary hyperplasia was observed in the gall bladders of mice at lower doses than those inducing adenomas, suggesting the possibility that a nonmutagenic MOA could apply to these tumors. Early-Life Susceptibility—According to the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA. 2005b). those exposed to carcinogens with a mutagenic MOA are assumed to have increased early-life susceptibility. Data on o-phenylenediamine are not sufficient to develop separate risk estimates for childhood exposure. There are no data comparing the tumorigenicity of o-phenylenediamine after exposure during early life with tumorigenicity after exposure during adulthood. In the carcinogenicity bioassays of o-phenylenediamine, exposure of the animals began at 6 weeks of age (Nlatsumoto et al.. 2012) or between 6-8 weeks of age (Weisburger et al. 1978) and continued through adulthood. Conclusions—Available data on the tumorigenicity of o-phenylenediamine support a mutagenic MOA. Three lines of evidence provide support for the conclusion of a mutagenic MOA. First, o-phenylenediamine was capable of eliciting genotoxic effects, including mutations, in both bacteria and eukaryotic cells and in vivo tests. Second, administration of o-phenylenediamine to rats and mice resulted in the induction of tumors at multiple sites, a hallmark of a mutagenic MOA. Third, pathology changes suggestive of nongenotoxic MO As were not observed in liver or urinary bladder in chronic cancer bioassays in rats and mice (although potentially nongenotoxic preneoplastic lesions were seen in the gall bladder). Therefore, a mutagenic MOA for carcinogenicity is proposed for o-phenylenediamine and a linear approach would be appropriate to extrapolate from the POD in the derivation of the provisional oral slope factor (p-OSF) (U.S. EPA. 2005a). DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of a Provisional Oral Slope Factor As shown in Table 13, a provisional oral slope factor (p-OSF) for o-phenylenediamine was derived from the combined incidences of hepatocellular adenomas and/or carcinomas and papillary adenomas of the gall bladder in male mice reported by Nlatsumoto et al. (2012). As noted in Table 11, EPA concluded that exposure to o-phenylenediamine is "Likely to Be Carcinogenic to Humans. " The chronic-duration oral carcinogenicity study by Nlatsumoto et al. (2012) was selected as the primary study for derivation of the p-OSF for o-phenylenediamine. Of the two available publications, the study by Nlatsumoto et al. (2012) used lower doses for a longer duration (2 years), tested larger groups of animals, included male and female rats and mice, and was reported more completely than Weisburger et al. (1978) (18 months), which tested both species, but only male rats. In the study by Nlatsumoto et al. (2012). there was a significant increase in the incidence of hepatocellular adenomas and carcinomas in male and female rats and mice, urinary bladder tumors and thyroid follicular adenomas in male rats, and papillary 47 o-Phenyl enedi amine ------- FINAL 09-27-2016 adenomas of the gall bladder in male and female mice at several doses compared to controls (see Table 12). The incidences of the various tumor types were modeled using BMDS and the modeling results are presented in Table 13. Based on the BMD modeling results, the calculated cancer slope factors for the various tumor types in male and female mice or rats were calculated and also presented in Table 13. Because treatment with o-phenylenediamine produced multiple types of tumors in different tissues within a single study (i.e., within a single sex and species), the overall oral cancer slope factor for o-phenylenediamine exposure was derived based on the incidence data for combined tumors assuming that different tumor types are independent from each other. The overall tumor incidence was fit with the MSCombo multiple tumor model (see BMDS, Version 2.6; Appendix C for details), and the estimated 10% benchmark dose (BMDio), 10% benchmark dose lower confidence limit (BMDLio), and calculated cancer slope factors are presented in Table 13. This modeling provides an estimate of composite risk for developing any combination of tumors at any site within a single study. Modeling procedures and results are described in detail in Appendix C. Prior to dose-response modeling, doses administered to the animals in the studies by Matsumoto et al. (2012) were converted to HEDs according to the equation below: Dose (HED) = Dose x (BWa - BWh)14 where: Dose = average daily animal dose of o-phenylenediamine BWa = reference animal body weight7 BWh = 70 kg, reference human body weight (U.S. EPA. 1988) Using a BWa of 0.25 kg for rats and 0.025 kg for mice, and a BWh of 70 kg for humans, the resulting default DAFs are 0.24 and 0.14 for rats and mice, respectively (U.S. EPA. 2011c. 2005a). The animal doses, calculated HED values, and associated tumor incidences are provided in Table 12. 7Time-weighted body weight was not reported by the study authors or calculable from reported study data. Default animal body weights (0.25 kg for rats and 0.025 kg for mice) were used in calculating HED values. o-Phenyl enedi amine 48 ------- FINAL 09-27-2016 Table 13. Goodness-of-Fit Statistics and BMDio and BMDLio Values for Tumors and Combined Tumors in Male and Female F344/DuCrj Rats and CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3'0 Multistage-Cancer Model Degree of Polynomial Goodness-of-Fit />-Valucb BMDio (HED) mg/kg-d BMDLio (HED) mg/kg-d Cancer Slope Factor (mg/kg-d) 1 Male Rats Hepatocellular adenoma and/or carcinoma—liver 2-degree (high dose dropped) 0.1 3.8 2.8 0.036 Transitional cell papilloma and/or carcinoma— urinary bladder 3-degree 0.08 11 9.1 0.011 Follicular adenoma— thyroid 1-degree 0.58 23 12 0.0083 Combined tumors 3.5 2.5 0.040 Female Rats Hepatocellular adenoma and/or carcinoma—liver 3-degree 0.42 3.1 2.2 0.045 Male Mice Hepatocellular adenoma and/or carcinoma—liver 1-degree 0.13 1.3 0.90 0.11 Papillary adenoma—gall bladder 1-degree 0.94 12 7.4 0.014 Combined tumors 1.1 0.84 0.12 Female Mice Hepatocellular adenoma and/or carcinoma—liver 1-degree 0.93 2.04 1.64 0.0610 Papillary adenoma—gall bladder 1-degree 0.33 32.1 19.4 0.00515 Combined tumors 1.92 1.56 0.0641 "Matsimioto et al. (2012). bValues >0.05 meet conventional goodness-of-fit criteria. °BMD modeling results are described in detail in Appendix C. BMDio (HED) = 10% benchmark dose human equivalent dose; BMDLio (HED) = 10% benchmark dose lower confidence limit human equivalent dose. 49 o-Phenyl enedi amine ------- FINAL 09-27-2016 The lowest BMDLio (HED) of 0.84 mg/kg-day, obtained from data on the combined tumors in male mice, was selected as the POD for calculation of the p-OSF. Because the Matsumoto et al. (2012) study was conducted for the full lifetime of the mice (2 years), no adjustment for less-than-lifetime observation was necessary. Because a mutagenic MOA has been implicated for o-phenylenediamine-induced tumors, a linear extrapolation to low dose was used to obtain the slope from the POD. The p-OSF of 1.2 x KT1 (mg/kg-day)-1 was derived as follows: p-OSF = BMR -h BMDLio (HED) = 0.1 ^ 0.84 mg/kg-day = 1.2 x 10 1 (mg/kg-day) 1 The p-OSF should not be used with exposure exceeding the POD (BMDLio [HED] = 0.84 mg/kg-day) because at doses higher than this value, the fitted dose-response model better characterizes the dose-response relationship. A WOE evaluation has concluded that o-phenylenediamine may be carcinogenic by a mutagenic MOA. According to the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA. 2005b) those exposed to carcinogens with a mutagenic MOA are assumed to have increased early-life susceptibility. Data on o-phenylenediamine are not sufficient to develop separate risk estimates for childhood exposure. The p-OSF of 1.2 x 10 1 (mg/kg-day) 1 calculated from data from adult exposure does not reflect presumed early-life susceptibility for this chemical, and age-dependent adjustment factors (ADAFs) should be applied to this parameter when assessing cancer risks. The current ADAFs and their age groupings are 10 for <2 years, 3 for 2 to <16 years, and 1 for 16 years and above (U.S. EPA. 2005b). The adjusted slope factors associated with these ADAFs are 1.2 (mg/kg-day) 1 for <2 years, 0.36 (mg/kg-day) 1 for 2 to <16 years, and 0.12 (mg/kg-day) 1 for 16 years and above. These slope factors are to be combined with age-specific exposure estimates when estimating cancer risks from early-life (<16 years of age) exposure to o-phenylenediamine. A cancer risk is derived for each age group and these are summed across age groups to obtain the total risk for the exposure period of interest. Derivation of a Provisional Inhalation Unit Risk No carcinogenicity studies of humans or animals exposed to o-phenylenedi amine via inhalation have been identified in the available literature, precluding derivation of inhalation cancer potency values. 50 o-Phenyl enedi amine ------- FINAL 09-27-2016 APPENDIX A. SCREENING PROVISIONAL VALUES No screening values for o-phenylenediamine are identified. 51 o-Phenyl enedi amine ------- FINAL 09-27-2016 APPENDIX B. DATA TABLES Table B-l. Selected Effects on Male and Female Crl:CD®BR Rats Exposed to o-Phenylenediamine (CASRN 95-54-5) via Gavage for 90 Days" Endpoint Exposure Group, mg/kg-d 0 20 40 80 Males Stained underbody 0/10b 0/10 1/10 1/10 Body weight on D 92 (g) 553.1 ±66.2 575.9 ± 25.8 (4%) 526.1 ±65.4 (-5%) 529.5 ± 47.9 (-4%) Body-weight gain D 1-92 (g) 340.3 ±66.5C 356.8 ±27.2 (5%) 296.3 ±47.5* (-13%) 312.8 ±49.3 (-8%) Slight palpebral closure in home cage, Wk 13 0/10 0/10 0/9 3/10 Slight palpebral closure in open field, Wk 13 0/10 0/10 0/9 4/10** Increased response to tail pinch, Wk 4 1/10 0/10 0/10 2/10 Increased response to tail pinch, Wk 6 1/10 1/10 0/10 3/10 Increased response to tail pinch, Wk 13 0/10 0/10 0/10 3/10 Females Stained abdomen 0/10 0/10 0/10 2/10 Stained inguen 0/10 0/10 1/10 7/10** Stained perineum 0/10 0/10 0/10 5/10** Stained underbody 0/10 0/10 0/10 2/10 Body weight on D 92 (g) 290.4 ±25.7 292.7 ±38.2 (0.8%) 288.0 ±20.5 (-0.8%) 270.9 ±28.3 ("7%) Body-weight gain D 1-92 (g) 132.8 ± 16.3 133.5 ±29.7 (0.5%) 125.1 ± 18.3 (-6%) 116.5 ± 19.8 (-12%) Slight palpebral closure in home cage, Wk 13 0/10 0/10 1/10 5/10** Slight palpebral closure in open field, Wk 13 0/10 0/10 0/10 2/10 Increased response to tail pinch, Wk 13 0/10 1/10 0/10 0/10 Slightly soiled fur 0/10 0/10 0/10 5/10** aDuPont (1992a). bNumber affected/number examined. °Mean ± standard deviation. * Significantly different from control (p < 0.05) based on Dunnett's test, as reported by the study authors. **Significantly different from control (p < 0.05) based on Fisher's exact test performed for this review. 52 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-2. Survival, Terminal Body Weights, and Liver Weights in Male and Female F344/DuCrj Rats Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Males Dose, mg/kg-d (as o-phenylenediamine) 0 13 25 51 Survival to termination13 41/50 36/50 42/50 42/50 Terminal body weight (g) 382 ± 33° 355 ±47* (-7%)d 330 ±22** (-14%) 269 ±29** (-30%) Absolute liver weight (g) 11.37 ±2.69 10.61 ± 1.29 ("7%) 10.65 ±2.41 (-6%) 9.27 ±3.41** (-18%) Relative liver weight (%) 2.99 ±0.75 3.01 ±0.34 (0.67%) 3.24 ±0.82** (8%) 3.46 ± 1.34** (16%) Females Dose, mg/kg-d (as o-phenylenediamine) 0 11 20 35 Survival to termination13 41/50 38/50 44/50 41/50 Terminal body weight (g) 253 ±23 237 ±30 (-6%) 234 ±24** (-8%) 204±19** (-19%) Absolute liver weight (g) 6.69 ±0.95 6.58 ± 1.10 (-2%) 6.81 ± 1.32 (2)% 9.41 ±3.63** (41%) Relative liver weight (%) 2.65 ±0.34 2.81 ±0.54 (6%) 2.93 ±0.63 (11%) 4.65 ± 1.85** (75%) "Matsumoto et al. (2012). bNumber of animals survived/number of animals examined. °Mean ± standard deviation. dPercent change from control. * Significantly different from control at p< 0.05 by Dunnett's test performed by the study authors. **Significantly different from control atp< 0.01 by Dunnett's test performed by the study authors. 53 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-3. Hematology and Serum Chemistry in Male and Female F344/DuCrj Rats Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Males Dose, mg/kg-d (as o-phenylenediamine) 0 13 25 51 Number of animals examined 40 36 42 42 Hematology Hb (g/dL) 13.2 ± 2.5b 13.2 ±3.4 (0%)c 13.6 ±2.4 (3%) 13.7 ±2.6 (4%) MCV (fL) 50.1 ±7.7 48.4 ± 2.3 (-3%) 48.8 ± 4.4* (-3%) 50.0 ±3.7 (-0.2%) MCH (pg) 16.8 ±2.0 16.2 ± 1.5* (-4%) 16.4 ± 1.3* (-2%) 16.8 ± 1.2 (0%) MCHC (g/dL) 33.5 ± 1.8 33.3 ±2.5 (-0.6%) 33.7 ± 1.6 (0.6%) 33.6 ± 1.4 (0.3%) Platelet (107|iL) 923 ± 238 869 ± 171 (-6%) 889 ± 226 (-4%) 822 ±238 (-11%) Seram chemistryb AST (IU/L) 97 ±49 76 ± 25 (-22%) 167 ± 270 (72%) 1,887 ± 10,973* (1,845%) ALT (IU/L) 45 ±23 41 ± 17 (-9%) 90 ± 168 (100%) 256 ± 1,059* (469%) ALP (IU/L) 228 ± 87 212 ± 68 (-7%) 202 ±49 (-11%) 231 ± 111 (1%) GGT (IU/L) 12 ±6 14 ± 6 (17%) 23 ± 36* (92%) 16 ± 12 (33%) BUN (mg/dL) 19.1 ±2.0 20.0 ± 2.8 (5%) 19.6 ±3.7 (3%) 26.0 ± 19.2** (36%) Urinalysis Positive occult bloodd 1/40 4/36 7/42 34/43## Urinary pHe 7.2 7.2 7.0 6.9# Females Dose, mg/kg-d (as o-phenylenediamine) 0 11 20 35 Number of animals examined 39 38 43 41 Hematology Hb (g/dL) 14.5 ± 1.8 14.1 ±3.2 (-3%) 13.9 ±2.8 (-4%) 14.0 ± 1.8* (-3%) MCV (fL) 53.2 ±3.4 55.5 ± 14.1 (4%) 54.4 ± 10.0 (2%) 52.1 ± 5.8** (-2%) MCH (pg) 18.4 ±0.8 18.8 ± 2.7 (2%) 18.5 ± 2.5 (0.5%) 17.9 ± 1.9** (-3%) MCHC (g/dL) 34.7 ± 1.0 34.3 ± 2.2 (-1%) 34.2 ± 1.9 (-1%) 34.4 ± 0.8** (-0.9%) Platelet (IOVjiL) 644±115 578 ± 154 (-10%) 661 ± 156 (3%) 777 ± 168** (21%) 54 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-3. Hematology and Serum Chemistry in Male and Female F344/DuCrj Rats Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Females Dose, mg/kg-d (as o-phenylenediamine) 0 11 20 35 Number of animals examined 39 38 43 41 Serum chemistry AST (IU/L) 127 ± 82 179 ±263 (41%) 179 ± 325 (41%) 596 ± 928** (369%) ALT (IU/L) 54 ±26 62 ± 54 (15%) 78 ± 218 (44%) 254 ± 322** (370%) ALP (IU/L) 139 ± 81 193 ±313 (39%) 141 ± 128 (1%) 206 ± 128** (48%) GGT (IU/L) 6 ± 5 7 ± 6 (17%) 9 ± 13 (50%) 42 ± 56** (600%) BUN (mg/dL) 17.2 ±5.3 17.1 ±2.7 (-0.6%) 18.8 ± 11.6 (9%) 18.7 ±3.2** (9%) Urinalysis Positive occult bloodd 1/41 4/39 4/44 16/41#" Urinary pHe 7.4 7.4 7.0# 6.7## "Matsimioto et al. (2012). bMean ± standard deviation. 'Percent change from control. dNumber of animals having positive occult blood/number of animals examined. "Number of animals in calculation of average pH was same as number of animals examined for positive occult blood. * Significantly different from control at p< 0.05 by Dunnett's test performed by the study authors. **Significantly different from control atp< 0.01 by Dunnett's test performed by the study authors. "Significantly different from control at p< 0.05 by x2 test with severity for urinalysis, performed by the study authors. ""Significantly different from control atp< 0.01 by x2 test with severity for urinalysis, performed by the study authors. ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; GGT = y-glutamyl transferase; Hb = hemoglobin; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume. 55 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-4. Incidences of Selected Non-neoplastic Lesions in Male and Female F344/DuCrj Rats Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3 Males Dose, mg/kg-d (as o-phenylenediamine) 0 13 25 51 Liver Clear cell foci 2/5 0b 9/50* 12/50** 3/50 Basophilic cell foci 19/50 31/50* 37/50** 38/50** Urinary bladder Simple hyperplasia: transitional epithelium 1/50 1/50 3/50 6/50 Papillary and/or nodular hyperplasia: transitional epithelium 0/50 2/50 1/50 7/50** Kidney Necrosis: papilla 0/50 0/50 0/50 15/50** Mineralization: papilla 7/50 18/50** 16/50* 26/50** Urothelial hyperplasia: pelvis 8/50 10/50 18/50* 22/50** Females Dose, mg/kg-d (as o-phenylenediamine) 0 11 20 35 Liver Clear cell foci 1/50 0/50 2/50 3/50 Basophilic cell foci 8/50 21/50** 39/50** 33/50** Urinary bladder Simple hyperplasia: transitional epithelium 0/50 1/50 0/50 2/50 Papillary and/or nodular hyperplasia: transitional epithelium 0/50 0/50 0/50 1/50 Kidney Necrosis: papilla 2/50 1/50 1/50 11/50** Mineralization: papilla 7/50 9/50 12/50 24/50** Urothelial hyperplasia: pelvis 2/50 12/50** 10/50* 17/50** aMatsimioto et al. (2012). bNumber of animals affected/number of animals examined. * Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors. **Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors. 56 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-5. Incidences of Selected Neoplastic Lesions in Male and Female F344/DuCrj Rats Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Males Dose, mg/kg-d (as o-phenylenediamine) 0 13 25 51 Liver Hepatocellular adenoma 3/50##b 2/50 12/50* 15/50** Hepatocellular carcinoma 1/50## 1/50 6/50 10/50** Hepatocellular adenoma and/or carcinoma 4/50## 3/50 16/50** 22/50** Urinary bladder Transitional cell papilloma 1/50## 0/50 0/50 6/50 Transitional cell carcinoma 1/50# 0/50 0/50 4/50 Transitional cell papilloma and/or carcinoma 2/50## 0/50 0/50 10/50* Thyroid Follicular adenoma 0/50## 1/50 0/50 4/50 Follicular adenocarcinoma 1/50 0/50 1/50 1/50 Females Dose, mg/kg-d (as o-phenylenediamine) 0 11 20 35 Liver Hepatocellular adenoma 1/50## 3/50 15/50** 36/50** Hepatocellular carcinoma 0/50## 0/50 4/50 18/50** Hepatocellular adenoma and/or carcinoma 1/50## 3/50 19/50** 44/50** Urinary bladder Transitional cell papilloma 1/50 0/50 1/50 1/50 Transitional cell carcinoma 0/50 0/50 0/50 0/50 Transitional cell papilloma and/or carcinoma 1/50 0/50 1/50 1/50 Thyroid Follicular adenoma 1/50 0/50 1/50 0/50 Follicular adenocarcinoma 0/50 0/50 1/50 0/50 "Matsiiiiioto et al. (2012). bNumber of animals affected/number of animals examined. * Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors. **Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors. #Significant dose-related positive trend atp< 0.05 by Peto's test performed by the study authors. ##Significant dose-related positive trend atp< 0.01 by Peto's test performed by the study authors. 57 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-6. Survival, Terminal Body Weights, and Liver Weights in Male and Female CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Males Dose, mg/kg-d (as o-phenylenediamine) 0 27 56 106 Survival to termination15 38/50 38/50 42/50 39/50 Terminal body weight (g) 48.1 ± 7.0° 40.3 ± 5.3* (-16%)d 35.1 ±4.1* (-27%) 31.0 ±2.6* (-36%) Absolute liver weight (g) 1.79 ±0.60 1.90 ±0.63 (6%) 2.09 ± 0.96 (17%) 1.87 ±0.50 (4%) Relative liver weight (%) 3.92 ±2.26 4.95 ±2.63* (26%) 6.16 ±3.22* (57%) 6.12 ± 1.98* (56%) Females Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Survival to termination13 24/50 29/50 28/50 34/50 Terminal body weight (g) 31.1 ± 3.4 28.4 ± 4.4 (-9%) 26.4 ± 5.6* (-15%) 21.5 ±2.1* (-31%) Absolute liver weight (g) 1.49 ±0.30 1.58 ±0.44 (6%) 1.99 ± 1.21 (34%) 2.02 ± 0.98 (36%) Relative liver weight (%) 4.89 ± 1.35 5.61 ± 1.53 (15%) 7.83 ±5.04* (61%) 9.45 ±4.63* (93%) aMatsumoto et al. (2012). bNumber of animals survived/number of animals examined. °Mean ± standard deviation. dPercent change from control. * Significantly different from control at p< 0.01 by Dunnett's test performed by the study authors. 58 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-7. Hematology and Serum Chemistry in Male and Female CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Males Dose, mg/kg-d (as o-phenylenediamine) 0 27 56 106 Hematology Number of animals examined 36 38 42 39 RBC (106/|iL) 9.60 ± 0.91b 9.59 ± 1.28 (-0.1%)c 9.43 ± 1.92 (-2%) 9.10 ±0.84** (-5%) Hb (g/dL) 13.8 ± 1.1 13.6 ± 1.5 (-1%) 13.5 ± 2.3 (-2%) 13.3 ± 1.2** (-4%) MCV (fL) 45.8 ± 1.9 45.3 ± 2.9 (-1%) 46.9 ± 6.3 (2%) 46.9 ± 1.2** (2%) MCHC (g/dL) 31.5 ±0.7 31.4 ± 1.0 (-0.3%) 31.1 ± 1.3 (-1%) 31.1 ±0.6** (-1%) Platelet (107|iL) 1,911 ±411 1,985 ±418 (4%) 2,087 ± 470 (9%) 2,279 ± 303** (19%) WBC (103/|iL) 4.47 ±8.86 2.96 ± 1.61 (-34%) 3.05 ±2.90 (-32%) 2.00 ± 1.31** (-55%) Seram chemistry Number of animals examined 37 38 42 39 AST (IU/L) 88 ± 167 127 ± 224 (44%) 174 ± 355 (98%) 119 ±264 (35%) ALT (IU/L) 49 ±77 135 ±355 (176%) 178 ±514** (263%) 119 ±300** (143%) ALP (IU/L) 124 ± 28 220 ± 244** (77%) 337 ±396** (172%) 279 ±182** (125%) GGT (IU/L) 1± 1 2 ± 1 (100%) 2 ± 1 (100%) 2 ± 1 (100%) BUN (mg/dL) 23.4 ±9.4 22.8 ±3.3 (-3%) 28.4 ± 12.1** (21%) 30.9 ± 11.3** (32%) Females Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Hematology Number of animals examined 22 27 25 28 RBC (106/|iL) 9.82 ± 1.99 9.30 ± 1.22 (-5%) 8.94 ± 1.90 (-9%) 9.37 ± 0.85 (-5%) Hb (g/dL) 14.1 ±2.3 13.6 ± 1.7 (-4%) 12.9 ± 2.7 (-9%) 13.4 ± 1.2** (-5%) MCV (fL) 45.7 ±2.3 46.7 ± 2.8 (2%) 47.4 ±5.1 (4%) 46.7 ±2.1** (2%) MCHC (g/dL) 31.6 ± 1.1 31.5 ± 1.1 (-0.3%) 30.9 ± 1.6* (-2%) 30.7 ± 0.6** (-3%) Platelet (107|iL) 1,210 ±273 1,329 ±374 (10%) 1,399 ±453 (16%) 1,641 ± 454** (36%) WBC (103/|iL) 2.13 ± 1.63 4.50 ± 11.15 (111%) 4.17 ± 4.18 (96%) 1.94 ± 1.51 (-9%) 59 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-7. Hematology and Serum Chemistry in Male and Female CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Females Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Serum chemistry Number of animals examined 23 27 26 31 AST (IU/L) 87 ±36 130 ± 203 (49%) 133 ±151 (53%) 190 ±224 (118%) ALT (IU/L) 40 ±23 74 ± 123 (85%) 120 ± 189 (200%) 207 ±316** (418%) ALP (IU/L) 171 ±53 254 ± 88* (49%) 443 ± 468** (159%) 598 ± 559** (250%) GGT (IU/L) 2 ± 1 2 ± 1 (0%) 3 ± 3 (50%) 4 ± 6 (100%) BUN (mg/dL) 23.6 ±24.3 24.5 ± 10.6 (4%) 27.5 ± 11.9** (17%) 30.7 ± 13.9** (30%) "Matsimioto et al. (2012). bMean ± standard deviation. °Percent change from control. * Significantly different from control at p< 0.05 by Dunnett's test performed by the study authors. **Significantly different from control atp< 0.01 by Dunnett's test performed by the study authors. ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; GGT = y-glutamyl transferase; Hb = hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; RBC = red blood cell; WBC = white blood cell. 60 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-8. Incidences of Selected Non-neoplastic Lesions in Male and Female CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years" Males Dose, mg/kg-d (as o-phenylenediamine) 0 27 56 106 Liver Clear cell foci 3/50b 6/50 2/50 0/50 Acidophilic cell foci 2/50 10/50* 9/50* 5/50 Basophilic cell foci 2/50 5/50 9/50* 9/50* Gall bladder0 Papillary hyperplasia 0/46 13/50** 8/49** 8/47** Nasal cavity Eosinophilic change: olfactory epithelium 22/50 11/50* 22/50 24/50 Eosinophilic change: respiratory epithelium 32/50 27/50 28/50 45/50** Respiratory metaplasia: gland 31/50 25/50 25/50 34/50 Nasopharynx Eosinophilic change 2/50 2/50 1/50 4/50 Kidney Hydronephrosis 3/50 2/50 2/50 4/50 Inflammatory polyp: pelvis 2/50 2/50 2/50 3/50 Females Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Liver Clear cell foci 0/50 4/50 3/50 8/50** Acidophilic cell foci 2/50 4/50 3/50 18/50** Basophilic cell foci 1/50 7/50* 4/50 10/50** Gall bladder Papillary hyperplasia 0/50 2/50 14/50** 10/50** Nasal cavity Eosinophilic change: olfactory epithelium 7/50 1/50 11/50 18/50* Eosinophilic change: respiratory epithelium 37/50 45/50** 48/50** 48/50** Respiratory metaplasia: gland 14/50 19/50 27/50** 34/50** 61 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-8. Incidences of Selected Non-neoplastic Lesions Mice Administered o-Phenylenediamine Dihydrochloride in Male and Female CrjrBDFi in Drinking Water for 2 Years" Females Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Nasopharynx Eosinophilic change 3/50 5/50 3/50 13/50** Kidney Hydronephrosis 2/50 12/50** 13/50** 11/50** Inflammatory polyp: pelvis 2/50 9/50* 10/50* 6/50 aMatsimioto et at (2012). bNumber of animals affected/number of animals examined. °Number of males examined for non-neoplastic lesions of the gall bladder varied by dose group. * Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors. **Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors. 62 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-9. Selected Neoplastic Lesions in Male and Female CrjrBDFi Mice Administered £>-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3 Males Dose, mg/kg-d (as o-phenylenediamine) 0 27 56 106 Liver Hepatocellular adenoma 12/50##b 25/50** 34/50** 35/50** Hepatocellular carcinoma 6/50 9/50 12/50 10/50 Hepatocellular adenoma and/or carcinoma 18/50## 29/50** 39/50** 38/50** Gall bladder Papillary adenoma 0/46# 2/50 4/49 5/47* Females Dose, mg/kg-d (as o-phenylenediamine) 0 63.3 119 234 Liver Hepatocellular adenoma 6/50## 22/50** 23/50** 34/50** Hepatocellular carcinoma 1/50## 4/50 11/50** 17/50** Hepatocellular adenoma and/or carcinoma 6/50## 23/50** 31/50** 41/50** Gall bladder Papillary adenoma 0/50 1/50 5/50* 3/50 "Matsumoto et al. (2012). bNumber of animals affected/number of animals examined. * Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors. **Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors. "Significant dose-related positive trend (p < 0.05) by Peto's test performed by the study authors. ""Significant dose-related positive trend (p < 0.01) by Peto's test performed by the study authors. 63 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table B-10. Incidences of Tumors in Male CD Rats and Male and Female CD-I HaM/ICR Mice Exposed to o-Phenylenediamine Dihydrochloride in Food for 18 Months" Exposure Group, mg/kg-d (as o-phenylenediamine) Simultaneous control Pooled control Low High Male rats 0 0 83.6 167 Hepatocellular carcinoma 0/16b 2/111 0/14 5/16* Male mice 0 0 704 1,430 Hepatocellular carcinoma 0/14 7/99 5/17** 3/14 Female mice 0 0 717 1,450 Hepatocellular carcinoma 1/15 1/102 6/18*** 6/15** aWeisburger et at (1978). bNumber of animals affected/number of animals examined. * Significantly different from simultaneous and pooled controls (p < 0.025), as reported by the study authors. **Significantly different from simultaneous and pooled controls (p < 0.05), as reported by the study authors. ***Significantly different from pooled controls (p < 0.025), as reported by the study authors. 64 o-Phenyl enedi amine ------- FINAL 09-27-2016 APPENDIX C. BENCHMARK DOSE MODELING RESULTS MODELING OF NONCANCER ENDPOINTS As discussed in the body of the report in the "Derivation of a Chronic Provisional Reference Dose" section, the endpoints selected for benchmark dose (BMD) modeling were incidence of renal papillary mineralization in male rats, incidence of renal pelvis urothelial hyperplasia in female rats, and incidence of renal hydronephrosis in female mice (Nlatsumoto et al., 2012). The animal doses in the study, converted to equivalent doses of o-phenylenediamine, were used in the BMD modeling; the data are shown in Tables 7, B-4, B-7, and B-8. Modeling Procedure for Dichotomous Noncancer Data BMD modeling of dichotomous noncancer data was conducted with the EPA's Benchmark Dose Software (BMDS, Version 2.5). For these data, the Gamma, Logistic, Log-Logistic, Log-Probit, Multistage, Probit, and Weibull dichotomous models available within the software were fit using a benchmark response (BMR) of 10% extra risk. The Multistage model is run for all polynomial degrees up to n - 1, where n is the number of dose groups including control. Adequacy of model fit was judged based on the x2 goodness-of-fit p-value (p > 0.1), scaled residuals at the data point (except the control) closest to the predefined benchmark response (absolute value <2.0), and visual inspection of the model fit. In the cases where no best model was found to fit to the data, a reduced data set without the high-dose group was further attempted for modeling and the result was presented along with that of the full data set. Among all of the models providing adequate fit, the benchmark dose lower confidence limit (BMDL) from the model with the lowest Akaike's information criterion (AIC) was selected as a potential point of departure (POD) when BMDL values were sufficiently close. Otherwise, the lowest BMDL was selected as a potential POD. Model Predictions for Incidence of Renal Papillary Mineralization in Male Rats The procedure outlined above was applied to the data (see Table 7) on renal papillary mineralization in male rats exposed chronically to o-phenylenediamine dihydrochloride via drinking water for 2 years (Nlatsumoto et al.. 2012). All models provided adequate fit to the data set when assessed by the overall goodness-of-fit (p> 0.1) and scaled residuals (absolute value <2.0) (see Table C-l). The 10% benchmark dose lower confidence limit (BMDLio) from all models were sufficiently close; therefore, the Log-Logistic model providing the lowest AIC was selected as the best fitting. The BMDio and BMDLio values for incidence of renal papillary mineralization in male rats from this model were 7.60 and 4.80 mg/kg-day, respectively. Figure C-l shows the Log-Logistic model fit to the data. 65 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table C-l. Modeling Results for Renal Papillary Mineralization in Male Rats" Model Name AIC X2 Goodness-of-Fit /j-Valucb BMD io mg/kg-d BMDLio mg/kg-d Scaled Residual Gamma0 244.12 0.30 9.49 6.52 1.30 Logistic 245.13 0.18 14.86 11.68 1.49 Log-Logisticde 243.81 0.35 7.60 4.80 1.10 Log-Probit° 246.32 0.10 17.09 11.82 1.78 Multistage (2-degree/ 244.12 0.30 9.49 6.52 1.30 Multistage (3-degree/ 244.12 0.30 9.49 6.52 1.30 Probit 245.02 0.19 14.27 11.19 1.48 Weibull0 244.12 0.30 9.49 6.52 1.30 Quantal-Linear 244.12 0.30 9.49 6.52 1.30 aMatsumoto et al. (2012). bValues <0.1 fail to meet conventional goodness-of-fit criteria. Tower restricted to >1. dSlope restricted to >1. "Selected model. All models provided adequate fit to the data. BMDL values estimated from models providing adequate fit were sufficiently close; therefore, the model with the lowest AIC was selected (Log-Logistic). fBetas restricted to >0. AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR [i.e., io = dose associated with 10% extra risk]). 66 o-Phenyl enedi amine ------- FINAL 09-27-2016 Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B 0.7 0.6 0.5 T3 0) +-» £ 0.4 < c 0 1 0.3 LL 0.2 0.1 0 10 20 30 40 50 dose 10:41 04/21 2015 Figure C-l. Fit of Log-Logistic Model to Incidence of Renal Papillary Mineralization in Male Rats (Matsumoto et al., 2012) Text Output for Log-Logistic Model to Incidence of Renal Papillary Mineralization in Male Rats (Matsumoto et al., 2012) Logistic Model. (Version: 2.14; Date: 2/28/2013) BMDS Model Run The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))] Dependent variable = Effect Independent variable = Dose Slope parameter is restricted as slope >= 1 Total number of observations = 4 Total number of records with missing values = 0 Maximum number of iterations = 5 00 Relative Function Convergence has been set to: le-008 Log-Logistic BMDL BMD 67 o-Phenyl enedi amine ------- FINAL 09-27-2016 Parameter Convergence has been set to: le-008 User has chosen the log transformed model Default Initial Parameter Values background = 0.14 intercept = -4.11546 slope = 1 the user, background intercept Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -slope have been estimated at a boundary point, or have been specified by and do not appear in the correlation matrix ) background intercept 1 -0. 67 -0.67 1 Parameter Estimates Variable background intercept slope Estimate 0.154391 -4.22567 1 Std. Err. 0.0498843 0.322865 NA 95.0% Wald Confidence Interval Lower Conf. Limit Upper Conf. Limit 0.0566197 0.252162 -4.85847 -3.59286 NA - Indicates that this parameter has hit a bound implied by some ineguality constraint and thus has no standard error. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -118.88 -119.903 -127.533 # Param's 4 2 1 Deviance Test d.f. 2.04645 17.3055 P-value 0.3594 0.0006115 AIC: 243.806 Dose Est. Prob. Goodness of Fit Expected Observed Size Scaled Residual 0.0000 51.0000 25.0000 13.0000 0.1544 0.5155 0.3807 0.2894 7.720 25 .776 19.034 14.470 7.000 26.000 16.000 18.000 50 50 50 50 -0.282 0. 063 -0.884 1.101 Chi^2 =2.08 d.f. = 2 P-value = 0.3542 68 o-Phenyl enedi amine ------- FINAL 09-27-2016 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 7.60222 BMDL = 4.79952 Model Predictions for Incidence of Renal Pelvis Urothelial Hyperplasia in Female Rats The procedure outlined above was applied to the data (see Table 7) on renal pelvis urothelial hyperplasia in female rats exposed chronically to o-phenylenediamine dihydrochloride via drinking water for 2 years (Nlatsumoto et aL 2012). The Gamma, Log-Logistic, Multistage, Weibull, and Quantal-Linear models provided adequate fit to the data when assessed by the overall goodness-of-fit (p> 0.1) and scaled residuals (absolute value <2.0) (see Table C-2). The BMDLio from models providing adequate fit were sufficiently close; therefore, the Log-Logistic model providing the lowest AIC was selected as the best fitting. The BMDio and BMDLio values for incidence of renal pelvis urothelial hyperplasia in female rats from this model were 8.32 and 5.58 mg/kg-day, respectively. Figure C-2 shows the Log-Logistic model fit to the data. 69 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table C-2. Modeling Results for Renal Pelvis Urothelial Hyperplasia in Female Rats3 Model Name AIC X2 Goodness-of-Fit />-Valucb BMD io mg/kg-d BMDLio mg/kg-d Scaled Residual Gamma0 192.81 0.23 9.49 6.68 1.49 Logistic 195.11 0.08 16.17 12.93 -0.25 Log-Logisticde 192.40 0.29 8.32 5.58 1.31 Log-Probitc 196.30 0.04 15.59 11.04 -0.45 Multistage (2-degree/ 192.81 0.23 9.49 6.68 1.49 Multistage (3-degree/ 192.81 0.23 9.49 6.68 1.49 Probit 194.82 0.09 15.27 12.12 1.76 Weibull0 192.81 0.23 9.49 6.68 1.49 Quantal-Linear 192.81 0.23 9.49 6.68 1.49 aMatsumoto et al. (2012). bValues <0.1 fail to meet conventional goodness-of-fit criteria. Tower restricted to >1. dSlope restricted to >1. "Selected model. All models, except Logistic, Log-Probit, and Probit provided adequate fit to the data. BMDL values estimated from models providing adequate fit were sufficiently close; therefore, the model with the lowest AIC was selected (Log-Logistic). fBetas restricted to >0. AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR [i.e., io = dose associated with 10% extra risk]). 70 o-Phenyl enedi amine ------- "O CD -t—' o < c o '¦4—' o 03 FINAL 09-27-2016 Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B 0.5 0.4 0.3 0.2 0.1 Log-Logistic BMDL 14:27 04/21 2015 Figure C-2. Fit of Log-logistic Model to Incidence of Renal Pelvis Urothelial Hyperplasia in Female Rats (Matsumoto et al., 2012) Text Output for Log-Logistic Model to Incidence of Renal Pelvis Urothelial Hyperplasia in Female Rats (Matsumoto et al., 2012) Logistic Model. (Version: 2.14; Date: 2/28/2013) BMDS_Model_Run The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))] Dependent variable = Effect Independent variable = Dose Slope parameter is restricted as slope >= 1 Total number of observations = 4 Total number of records with missing values Maximum number of iterations = 5 00 = 0 71 o-Phenyl enedi amine ------- FINAL 09-27-2016 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 User has chosen the log transformed model Default Initial Parameter Values background = 0.04 intercept = -4.16226 slope = 1 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -slope have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) background intercept background 1 -0.6 intercept -0.6 1 Parameter Estimates Interval Variable Limit background intercept slope Estimate 0.0486009 -4.31559 1 Std. Err. Indicates that this value is not calculated. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -93.0231 -94.198 -101.451 # Param's 4 2 1 Deviance Test d.f. 2 .34973 16.8563 P-value 0.3089 0.0007565 AIC: 192.396 Dose Est. Prob. Goodness of Fit Expected Observed Size Scaled Residual 0.0000 11.0000 20.0000 35.0000 0. 0486 0.1705 0.2492 0.3517 2.430 8 .525 12.460 17.586 2.000 12.000 10.000 17.000 50 50 50 50 -0.283 1.307 -0.804 -0.173 Chi^2 =2.46 d.f. = 2 P-value = 0.2916 72 o-Phenyl enedi amine ------- FINAL 09-27-2016 Benchmark Dose Computation Specified effect = 0.1 Risk Type Extra risk Confidence level 0. 95 BMD 8 .31753 BMDL 5 .57528 Model Predictions for Incidence of Renal Hydronephrosis in Female Mice The procedure outlined above was applied to the data (see Table 7) on renal hydronephrosis in female mice exposed chronically to o-phenylenediamine dihydrochloride via drinking water for 2 years (Nlatsumoto et al.. 2012). No models provided adequate fit to the data when assessed by the overall goodness-of-fit (p< 0.1) (see Table C-3). The data were then modeled without the high-dose group; using the reduced data set, the Gamma, Log-Logistic, Multistage (2-degree), Weibull and Quantal-Linear models provided adequate fit to the data (p > 0.1 and scaled residuals <2.0; see Table C-3). The BMDLio from these models providing adequate fit were sufficiently close; therefore, the Log-Logistic model providing the lowest AIC was selected as the best fitting. The BMDio and BMDLio values for incidence of renal hydronephrosis in female mice from this model were 36.41 and 23.08 mg/kg-day, respectively. Figure C-3 shows the Log-Logistic model fit to the data. 73 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table C-3. Modeling Results for Renal Hydronephrosis in Female Mice" Model Name AIC X2 Goodness-of-Fit />-Valucb BMD io mg/kg-d BMDLio mg/kg-d Scaled Residual Gamma0 192.94 0.03 89.46 52.77 1.71 Logistic 194.80 0.02 155.31 102.58 1.19 Log-Logisticd 192.21 0.04 73.05 42.26 1.67 Log-Probitd 197.01 0.01 191.61 102.21 -0.80 Multistage (2-degree)6 192.94 0.03 89.46 52.77 1.71 Multistage (3-degree)6 192.94 0.03 89.46 52.77 1.71 Probit 194.62 0.02 147.42 96.46 1.17 Weibull0 192.94 0.03 89.46 52.77 1.71 Quantal-Linear 192.94 0.03 89.46 52.77 1.71 High Dose Dropped Gamma0 134.40 0.27 40.22 27.05 0.89 Logistic 136.56 0.07 64.94 50.77 1.44 Log-Logisticd'f 134.12 0.34 36.41 23.08 0.75 Log-Probitd 136.37 0.07 57.13 42.45 1.43 Multistage (2-degree)0 134.40 0.27 40.22 27.05 0.89 Probit 136.22 0.08 61.31 47.58 1.40 Weibull0 134.40 0.27 40.22 27.05 0.89 Quantal-Linear 134.40 0.27 40.22 27.05 0.89 aMatsimioto et al. (2012). bValues <0.1 fail to meet conventional goodness-of-fit criteria. Tower restricted to >1. dSlope restricted to >1. "Betas restricted to >0. Selected model. No models fit the full data set. After dropping the high dose, the Gamma, Log-Logistic, Multistage (2-degree), Weibull and Quantal-Linear models provided adequate fit to the data. BMDL values estimated from models providing adequate fit were sufficiently close; therefore, the model with the lowest AIC was selected (Log-Logistic). AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR [i.e., io = dose associated with 10% extra risk]). 74 o-Phenyl enedi amine ------- FINAL 09-27-2016 Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the T3 0) -t—' o a= < c o '¦4—' o <0 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Log-Logistic BMDL BMD 20 40 60 dose 80 100 120 09:20 04/22 2015 Figure C-3. Fit of Log-Logistic Model to Incidence of Renal Hydronephrosis in Female Mice (Matsumoto et al., 2012) Text Output for Log-Logistic Model to Incidence of Renal Hydronephrosis in Female Mice (Matsumoto et al., 2012) Logistic Model. (Version: 2.14; Date: 2/28/2013) BMDS_Model_Run The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))] Dependent variable = Effect Independent variable = Dose Slope parameter is restricted as slope >= 1 Total number of observations = 3 Total number of records with missing values = 0 Maximum number of iterations = 5 00 Relative Function Convergence has been set to: le-008 75 o-Phenyl enedi amine ------- FINAL 09-27-2016 Parameter Convergence has been set to: le-008 User has chosen the log transformed model Default Initial Parameter Values background = 0.04 intercept = -5.43194 slope = 1 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -slope have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) background intercept background 1 -0.5 intercept -0.5 1 Parameter Estimates Interval Variable Limit background intercept slope Estimate 0.0436682 -5.79203 1 Std. Err. Indicates that this value is not calculated. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -64.604 -65.0595 -70.709 # Param's 3 2 1 Deviance Test d.f. 0.910965 12.2099 P-value 0.3399 0.002232 AIC: 134.119 Dose Goodness of Fit Est._Prob. Expected Observed Size Scaled Residual 0.0000 63.0000 119.0000 0.0437 0.1979 0.2984 2.183 9.894 14.922 2.000 12.000 13.000 50 50 50 -0.127 0.747 -0.594 Chi^2 =0.93 d.f. = 1 P-value = 0.3354 Benchmark Dose Computation 76 o-Phenylenediamine ------- FINAL 09-27-2016 Specified effect = 0.1 Risk Type Extra risk Confidence level 0. 95 BMD 36.4085 BMDL 23.0781 MODELING OF CANCER ENDPOINTS As discussed in the body of the report in the "Derivation of a Provisional Oral Slope Factor" section the tumor types selected for BMD modeling were hepatocellular adenomas and carcinomas in male and female rats and mice, urinary bladder transitional cell papillomas and carcinomas and thyroid follicular adenomas in male rats, and papillary adenomas of the gall bladder in male and female mice exposed to o-phenylenediamine via drinking water for 2 years (Nlatsumoto et al., 2012). The tumor incidences and associated human equivalent doses (HEDs) used in the modeling are shown in Tables 12, B-5, and B-9. Modeling Procedure for Cancer Incidence Data BMD modeling of dichotomous cancer data was conducted with the EPA's BMDS (Version 2.6). The Multistage-Cancer model was fit to the incidence data using the extra risk option and a BMR of 10% extra risk. The Multistage-Cancer model was run for all polynomial degrees up to n - 1 (where n is the number of dose groups including control). Adequacy of model fit was judged based on the x2 goodness-of-fitp-walue (p > 0.1), magnitude of scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. Among all of the models providing adequate fit, the BMDL from the model with the lowest AIC was selected as a potential POD when BMDL values were within a factor of 2-3. When BMDL values from models providing adequate fit varied more than 2- or 3-fold, the lowest BMDL was selected as a potential POD. Model Predictions for Hepatocellular Adenomas and/or Carcinomas in Male Mice The procedure outlined above was applied to the data (see Table 12) on hepatocellular adenomas and/or carcinomas in male mice exposed chronically to o-phenylenediamine dihydrochloride via drinking water for 2 years (Nlatsumoto et al.. 2012). The software converged on the 1-degree model, which provided adequate fit (p > 0.05); thus, it was selected as the best-fitting model (see Table C-4). The BMDio (FLED) and BMDLio (FLED) values from this model were 1.26 and 0.90 mg/kg-day, respectively. Figure C-4 shows the model fit to the data. 77 o-Phenyl enedi amine ------- FINAL 09-27-2016 Table C-4. Modeling Results for Hepatocellular Adenoma and/or Carcinoma in Male Micea Model DF x2 X2 Goodness-of-Fit />-Valueb Scaled Residual AIC BMDio (HED) mg/kg-d BMDLio (HED) mg/kg-d Multistage Cancer (1-degree)0'1 2 4.14 0.13 -0.54 249.3 1.26 0.90 Multistage Cancer (2-degree)° 2 4.14 0.13 -0.54 249.3 1.26 0.90 Multistage Cancer (3-degree)0 2 4.14 0.13 -0.54 249.3 1.26 0.90 aMatsumoto et al. (2012). bValues <0.05 fail to meet conventional goodness-of-fit criteria. °Betas restricted to >0. dSelected model. All models provided adequate fit to the data. AIC = Akaike's information criterion; BMDio (HED) = 10% benchmark dose human equivalent dose; BMDLio (HED) = 10% benchmark dose lower confidence limit human equivalent dose; DF = degree(s) of freedom. 78 o-Phenyl enedi amine ------- FINAL 09-27-2016 Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL Multistage Cancer Linear extrapolation - 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 BMDL BMD 0 2 4 6 8 10 12 14 dose 09:36 07/22 2015 Figure C-4. Fit of Multistage-Cancer (1-Degree) Model to Incidence of Hepatocellular Adenoma and/or Carcinoma in Male Mice (Matsumoto et al., 2012) Text Output for Multistage-Cancer (1-Degree) Model for Incidence of Hepatocellular Adenoma and/or Carcinoma in Male Mice (Matsumoto et al., 2012) Multistage Model. (Version: 3.4; Date: 05/02/2014) Input Data File: C:/Users/bowens/BMDS260/Data/msc_livercancer_Opt.(d) Gnuplot Plotting File: C:/Users/bowens/BMDS260/Data/msc_livercancer_Opt.pit Wed Jul 22 09:36:30 2015 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose 79 o-Phenyl enedi amine ------- FINAL 09-27-2016 Total number of observations = 4 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 = 5 00 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.4 64296 Beta(1) = 0.0663523 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(l) Background 1 -0.63 Beta (1) -0.63 1 Parameter Estimates Variable Background Beta(1) Estimate 0.397509 0.0833613 95.0% Wald Confidence Interval Std. Err. 0.0646602 0.0196939 Lower Conf. Limit 0.270777 0. 0447619 Upper Conf. Limit 0.52424 0.121961 Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) # Param' -120.585 4 -122.662 2 -132.813 1 Deviance Test d.f. 4.15331 24.4558 P-value 0.1253 <.0001 AIC: 249.323 Dose Goodness of Fit Est. Prob. Expected Observed Size Scaled Residual 0.0000 3.8000 7.7000 15.0000 Chi^2 =4.14 0.3975 0.5611 0.6829 0.8275 d.f. = 2 19.875 18.000 50.000 -0.542 28.054 29.000 50.000 0.269 34.145 39.000 50.000 1.475 41.373 38.000 50.000 -1.262 P-value = 0.12 64 Benchmark Dose Computation Specified effect = 0.1 80 o-Phenyl enedi amine ------- FINAL 09-27-2016 Risk Type = Extra risk Confidence level = 0.95 BMD = 1.2 639 BMDL = 0.902339 BMDU = 2 . 03423 Taken together, (0.902339, 2.03423) is a 90 % two-sided confidence interval for the BMD Cancer Slope Factor = 0.110823 Model Predictions for Papillary Adenomas of the Gall Bladder in Male Mice The procedure outlined above was applied to the data (see Table 12) on papillary adenomas of the gall bladder in male mice exposed chronically to o-phenylenediamine dihydrochloride via drinking water for 2 years (Nlatsumoto et al.. 2012). The software converged on the 1-degree Multistage-Cancer model, which provided adequate fit (p > 0.05) and scaled residuals <2.0; thus, it was selected as the best-fitting model (see Table C-5). The BMDio (HED) and BMDLio (HED) values from this model were 11.6 and 7.4 mg/kg-day, respectively. Figure C-5 shows the model fit to the data. Table C-5. Modeling Results for Papillary Adenoma of the Gall Bladder in Male Mice" Model DF x2 X2 Goodness-of-Fit />-Valueb Scaled Residuals AIC BMDio (HED) mg/kg-d BMDLio (HED) mg/kg-d Multistage Cancer (1-degree)0'1 3 0.40 0.94 -0.43 78.8 11.6 7.4 Multistage Cancer (2-degree)° 3 0.40 0.94 -0.43 78.8 11.6 7.4 Multistage Cancer (3-degree)0 3 0.40 0.94 -0.43 78.8 11.6 7.4 aMatsimioto et al. (2012). bValues <0.05 fail to meet conventional goodness-of-fit criteria. °Betas restricted to >0. dSelected model. All models provided adequate fit to the data. AIC = Akaike's information criterion; BMDio (HED) = 10% benchmark dose human equivalent dose; BMDLio (HED) = 10% benchmark dose lower confidence limit human equivalent dose; DF = degree(s) of freedom. 81 o-Phenyl enedi amine ------- FINAL 09-27-2016 Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL Multistage Cancer Linear extrapolation - 0.25 0.2 0.15 0.1 0.05 0 BMDL BMD 0 2 4 6 8 10 12 14 dose 09:45 07/22 2015 Figure C-5. Fit of Multistage-Cancer (1-Degree) Model to Incidence of Papillary Adenoma of the Gall Bladder in Male Mice (Matsumoto et al., 2012) Text Output for Multistage-Cancer (1-Degree) Model for Incidence of Papillary Adenoma of the Gall Bladder in Male Mice (Matsumoto et al., 2012) Multistage Model. (Version: 3.4; Date: 05/02/2014) Input Data File: C:/Users/bowens/BMDS260/Data/msc_gallbladder cancer_Opt.(d) Gnuplot Plotting File: C:/Users/bowens/BMDS260/Data/msc_gallbladder cancer_Opt.pit Wed Jul 22 09:45:00 2015 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Effect 82 o-Phenyl enedi amine ------- FINAL 09-27-2016 Independent variable = Dose Total number of observations = 4 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 = 5 00 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.0101723 Beta(1) = 0.0074551 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 Variable Estimate Std. Err. Background 0 NA Beta(1) 0.00905685 0.00273192 NA - Indicates that this parameter has hit implied by some ineguality constraint has no standard error. Estimates 95.0% Wald Confidence Interval Lower Conf. Limit Upper Conf. Limit 0.00370238 0.0144113 a bound and thus Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -38.179 -38.3762 -42.1343 # Param's 4 1 1 Deviance Test d.f. 0.394311 7.91053 P-value 0.9414 0.0479 AIC: 78.7524 Dose Est. Prob. Goodness of Fit Expected Observed Size Scaled Residual 0.0000 3.8000 7.7000 15.0000 0.0000 0.0338 0.0674 0.1270 0.000 1.692 3.301 5.970 0.000 2.000 4.000 5.000 46.000 50.000 49.000 47.000 0. 000 0.241 0.399 -0.425 83 o-Phenyl enedi amine ------- FINAL 09-27-2016 Chi^2 = 0.40 d.f. = 3 P-value = 0.9407 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 11.6332 BMDL = 7.35584 BMDU = 25.1514 Taken together, (7.35584, 25.1514) is a 90 % two-sided confidence interval for the BMD Cancer Slope Factor = 0.0135946 Model Predictions for MS_ Combo-Multiple Tumor Model for All Tumor Types in Male Mice MSCombo-multiple tumor BMD modeling was used to combine tumor incidence data for combined hepatocellular adenomas and/or carcinomas and papillary adenomas of the gall bladder in male mice. For each tumor type, the best-fitting Multistage model (i.e., the degree of Poly setting) was maintained in the MSCombo model run. The calculated combined tumor BMDLio (HED) based on the MS Combo model is 0.84 mg/kg-day. This BMDLio (HED) is used as the POD to derive the provisional oral slope factor (p-OSF). Text Output for MS COMBO Multiple Tumor Model for Combined Tumors in Male Mice MS_COMBO. (Version: 1.9; Date: 05/20/2014) Input Data File: C:\Users\bowens\BMDS260\Data\multi_test.(d) Gnuplot Plotting File: C:\Users\bowens\BMDS2 60\Data\multi_test.plt Wed Jul 22 09:13:52 2015 BMDS Model Run 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 Dependent variable = Effect Independent variable = Dose Data file name = livercancer.dax Total number of observations = 4 84 o-Phenyl enedi amine ------- FINAL 09-27-2016 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 = 5 00 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.4 64296 Beta(1) = 0.0663523 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(l) Background 1 -0.7 Beta (1) -0.7 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.397509 * * * Beta(1) 0.0833613 * * * * - Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) # Param's Deviance Test d.f. P-value -120.585 -122.662 -132.813 4.15331 24.4558 0.1253 <.0001 AIC: 249.323 Log-likelihood Constant 112.23100406060614 Dose Est. Prob. Goodness of Fit Expected Observed Size Scaled Residual 0.0000 3.8000 7.7000 15.0000 0.3975 0.5611 0.6829 0.8275 19.875 28.054 34.145 41.373 18.000 29.000 39.000 38.000 50.000 50.000 50.000 50.000 -0.542 0.269 1. 475 -1.262 Chi^2 =4.14 d.f. = 2 P-value = 0.12 64 85 o-Phenyl enedi amine ------- FINAL 09-27-2016 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 1.2 639 BMDL = 0.902339 BMDU = 2 . 03423 Taken together, (0.902339, 2.03423) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.110823 MS_COMBO. (Version: 1.9; Date: 05/20/2014) Input Data File: C:\Users\bowens\BMDS260\Data\multi_test.(d) Gnuplot Plotting File: C:\Users\bowens\BMDS2 60\Data\multi_test.plt Wed Jul 22 09:13:52 2015 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose Data file name = gallbladdercancer.dax Total number of observations = 4 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 = 5 00 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.0101723 Beta(1) = 0.0074551 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Background 86 o-Phenyl enedi amine ------- FINAL 09-27-2016 the user, Beta(1) have been estimated at a boundary point, or have been specified by and do not appear in the correlation matrix ) Beta(1) 1 Interval Variable Limit Background Beta(1) Estimate Parameter Estimates 95.0% Wald Confidence Std. Err. Lower Conf. Limit Upper Conf. 0 0.00905685 * * - Indicates that this value is not calculated. Model Full model Fitted model Reduced model AIC: Analysis of Deviance Table Log(likelihood) # Param's Deviance Test d.f. P-value -38.179 -38.3762 -42.1343 78.7524 Log-likelihood Constant 0.394311 7.91053 33.617802094648134 0.9414 0.0479 Dose Est. Prob. Goodness of Fit Expected Observed Size Scaled Residual 0.0000 3.8000 7.7000 15.0000 Chi^2 = 0.40 0.0000 0.0338 0.0674 0.1270 d.f. = 3 0.000 0.000 46.000 1.692 2.000 50.000 3.301 4.000 49.000 5.970 5.000 47.000 P-value = 0.94 07 0. 000 0.241 0.399 -0.425 Benchmark Dose Computation Specified effect Risk Type Confidence level BMD BMDL BMDU 0.1 Extra risk 0. 95 11.6332 7.35584 25.1514 Taken together, (7.35584, 25.1514) is a 90 interval for the BMD two-sided confidence 87 o-Phenyl enedi amine ------- Multistage Cancer Slope Factor = 0.0135946 **** Start of combined BMD and BMDL Calculations.**** Combined Log-Likelihood -161.03774986868211 Combined Log-likelihood Constant 145.84880615525427 Benchmark Dose Computation Specified effect Risk Type Confidence level BMD BMDL 0.1 Extra risk 0. 95 1.14004 0. 835366 Multistage Cancer Slope Factor = 0.119708 FINAL 09-27-2016 88 o-Phenyl enedi amine ------- FINAL 09-27-2016 APPENDIX D. 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