vvEPA United States Environmental Protection Agency EPA/690/R-21/003F | August 2021 | FINAL Provisional Peer-Reviewed Toxicity Values for 3,4-Toluenediamine (CASRN 496-72-0) U.S. EPA Office of Research and Development Center for Public Health and Environmental Assessment ------- A United States Environmental Protection %#UI JTT,Agency EPA/690/R-21/003F August 2021 https://www.epa.gov/pprtv Provisional Peer-Reviewed Toxicity Values for 3,4-Toluenediamine (CASRN 496-72-0) Center for Public Health and Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ii 3,4-T oluenediamine ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGER Lucina E. Lizarraga, PhD Center for Public Health and Environmental Assessment, Cincinnati, OH CONTRIBUTOR Grace Patlewicz, PhD Center for Computational Toxicology and Exposure, Research Triangle Park, NC SCIENTIFIC TECHNICAL LEADS Jeffry L. Dean II, PhD Center for Public Health and Environmental Assessment, Cincinnati, OH Q. Jay Zhao, PhD, MPH, DABT Center for Public Health and Environmental Assessment, Cincinnati, OH J. Phillip Kaiser, PhD, DABT Center for Public Health and Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212 PRIMARY INTERNAL REVIEWERS Q. Jay Zhao, PhD, MPH, DABT Center for Public Health and Environmental Assessment, Cincinnati, OH Ingrid Druwe, PhD Center for Public Health and Environmental Assessment, Cincinnati, OH PRIMARY EXTERNAL REVIEWERS Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421-3136 PPRTV PROGRAM MANAGEMENT Teresa L. Shannon Center for Public Health and Environmental Assessment, Cincinnati, OH J. Phillip Kaiser, PhD, DABT Center for Public Health and Environmental Assessment, Cincinnati, OH Questions regarding the content of this PPRTV assessment should be directed to the U.S. EPA Office of Research and Development (ORD) Center for Public Health and Environmental Assessment (CPHEA) website at http s: //ecomments. epa. gov/port v. in 3,4-T oluenediamine ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS AND ACRONYMS v BACKGROUND 1 QUALITY ASSURANCE 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVs 2 1. INTRODUCTION 3 2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 7 2.1. HUMAN STUDIES 10 2.1.1. Oral Exposures 10 2.1.2. Inhalation Exposures 10 2.2. ANIMAL STUDIES 10 2.2.1. Oral Exposures 10 2.2.2. Inhalation Exposures 10 2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 10 2.3.1. Genotoxicity 10 2.3.2. Short-Term and Acute Toxicity Studies 16 2.3.3. Reproductive/Developmental Studies of TDA Mixtures 19 2.3.4. Mode-of-Action/Mechanistic Studies 20 3. DERIVATION OI PROVISIONAL VALUES 21 3.1. DERIVATION OF PROVISIONAL REFERENCE DOSES 21 3.2. DERIVATION OF PROVISIONAL REFERENCE CONCENTRATION 21 3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES 21 3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 22 3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 22 APPENDIX A. SCREENING NONCANCER PROVISIONAL VALUES 23 APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING EVALUATION OF POTENTIAL CARCINOGENICITY 48 APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL CARCINOGENICITY 57 APPENDIX D. METHODOLOGY AND RESULTS FOR IN SILICO METABOLITE ANALYSIS OI TARGET AND ANALOGUES 72 APPENDIX E. REFERENCES 98 iv 3,4-T oluenediamine ------- COMMONLY USED ABBREVIATIONS AND ACRONYMS a2u-g alpha 2u-globulin LD50 median lethal dose ACGIH American Conference of Governmental LOAEL lowest-observed-adverse-effect level Industrial Hygienists MN micronuclei AIC Akaike's information criterion MNPCE micronucleated polychromatic ALD approximate lethal dosage erythrocyte ALT alanine aminotransferase MOA mode of action AR androgen receptor MTD maximum tolerated dose AST aspartate aminotransferase NAG 7V-acetyl-P-D-glucosaminidase atm atmosphere NCI National Cancer Institute ATSDR Agency for Toxic Substances and NO A F.I. no-observed-adverse-effect level Disease Registry NTP National Toxicology Program BMD benchmark dose NZW New Zealand White (rabbit breed) BMDL benchmark dose lower confidence limit OCT ornithine carbamoyl transferase BMDS Benchmark Dose Software ORD Office of Research and Development BMR benchmark response PBPK physiologically based pharmacokinetic BUN blood urea nitrogen PCNA proliferating cell nuclear antigen BW body weight PND postnatal day CA chromosomal aberration POD point of departure CAS Chemical Abstracts Service PODadj duration-adjusted POD CASRN Chemical Abstracts Service registry QSAR quantitative structure-activity number relationship CBI covalent binding index RBC red blood cell CHO Chinese hamster ovary (cell line cells) RDS replicative DNA synthesis CL confidence limit RfC inhalation reference concentration CNS central nervous system RfD oral reference dose CPHEA Center for Public Health and RGDR regional gas dose ratio Environmental Assessment RNA ribonucleic acid CPN chronic progressive nephropathy SAR structure-activity relationship CYP450 cytochrome P450 SCE sister chromatid exchange DAF dosimetric adjustment factor SD standard deviation DEN diethylnitrosamine SDH sorbitol dehydrogenase DMSO dimethylsulfoxide SE standard error DNA deoxyribonucleic acid SGOT serum glutamic oxaloacetic EPA Environmental Protection Agency transaminase, also known as AST ER estrogen receptor SGPT serum glutamic pyruvic transaminase, FDA Food and Drug Administration also known as ALT FEVi forced expiratory volume of 1 second SSD systemic scleroderma GD gestation day TCA trichloroacetic acid GDH glutamate dehydrogenase TCE trichloroethylene GGT y-glutamyl transferase TWA time-weighted average GSH glutathione UF uncertainty factor GST glutathione-S'-transfcrase UFa interspecies uncertainty factor Hb/g-A animal blood-gas partition coefficient UFC composite uncertainty factor Hb/g-H human blood-gas partition coefficient UFd database uncertainty factor HEC human equivalent concentration UFh intraspecies uncertainty factor HED human equivalent dose UFl LOAEL-to-NOAEL uncertainty factor i.p. intraperitoneal UFS subchronic-to-chronic uncertainty factor IRIS Integrated Risk Information System U.S. United States of America IVF in vitro fertilization WBC white blood cell LC50 median lethal concentration Abbreviations and acronyms not listed on this page are defined upon first use in the PPRTV document. v 3,4-T oluenediamine ------- EPA/690/R-21/003F PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR 3,4-TOLUENEDIAMINE (CASRN 496-72-0) 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 U.S. Environmental Protection Agency (U.S. EPA) guidance on human health toxicity value derivations. 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. Currently available PPRTV assessments can be accessed on the U.S. EPA's PPRTV website at https://www.epa.gov/pprtv. PPRTV assessments are eligible to be updated on a 5-year cycle and revised as appropriate to incorporate new data or methodologies that might impact the toxicity values or affect the characterization of the chemical's potential for causing adverse human-health effects. Questions regarding nomination of chemicals for update can be sent to the appropriate U.S. EPA Superfund and Technology Liaison (https://www.epa.gov/research/fact- sheets-regional-science). QUALITY ASSURANCE This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure data are of known and acceptable quality to support their intended use. Surveillance of the work by the assessment managers and programmatic scientific leads ensured adherence to QA processes and criteria, as well as quick and effective resolution of any problems. The QA manager, assessment managers, and programmatic scientific leads have determined under the QA program that this work meets all U.S. EPA quality requirements. This PPRTV was written with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP), the QAPP titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-Reviewed Toxicity Values (PPRTVs) and Related Assessments/Documents (L-CPAD-0032718-QP), and the PPRTV development contractor QAPP titled Quality Assurance Project Plan—Preparation of Provisional Toxicity Value (PTV) Documents (L-CPAD-0031971-QP). As part of the QA system, a quality product review is done prior to management clearance. A Technical Systems Audit may be performed at the discretion of the QA staff. All PPRTV assessments receive internal peer review by at least two CPHEA scientists and an independent external peer review by at least three scientific experts. The reviews focus on whether all studies have been correctly selected, interpreted, and adequately described for the purposes of deriving a provisional reference value. The reviews also cover quantitative and qualitative aspects of the provisional value development and address whether uncertainties associated with the assessment have been adequately characterized. 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 1 3,4-T oluenediamine ------- EPA/690/R-21/003F 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. 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 U.S. EPA ORD CPHEA website at https://ecomments.epa.gov/pprtv. 2 3,4-T oluenediamine ------- EPA/690/R-21/003F 1. INTRODUCTION 3,4-Toluenediamine (3,4-TDA), also known as 3,4-diaminotoluene (CASRN 496-72-0), belongs to the class of compounds known as anilines and is an ortho (o)-substituted compound. The principal commercial use for o-TDAs, including 3,4-TDA, is in the production of tolyltriazoles used in corrosion and nitrification inhibitors. 3,4-TDA is also used as an intermediate in the manufacture of urethane products, dyes, corrosion inhibitors, polyols, and benzimidazole thiol antioxidants and as a starting material for a pharmaceutical intermediate (Cartolano. 2005; HSDB. 2003). It is listed on U.S. EPA's Toxic Substances Control Act's public inventory (U.S. EPA. 2015). but it is not registered with Europe's Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) program (ECHA. 2016). TDA isomers, including 3,4-TDA, are produced by the catalytic hydrogenation of dinitrotoluenes under a variety of temperatures, pressures, and solvents. 3,4-TDA is then separated from meta (/«)-substituted TDAs by vacuum distillation (Cartolano. 2005). 3,4-TDA is one of six TDA isomers that are components of crude or commercial-grade mixtures used as intermediates in the production of dyes and pigments for commercial products (WHO. 1987). The crude mixture contains all six isomeric forms, while the two commercial mixtures are composed primarily of two isomers each. One commercial mixture, m-TDA, contains the 2,4- and 2,6- isomers (80:20 or 65:35), and the other, o-TDA, contains the 2,3- and 3,4- isomers (40:60). The empirical formula for 3,4-TDA is C7H10N2, and its structure is shown in Figure 1. Table 1 summarizes the physicochemical properties of 3,4-TDA. The compound is a light gray to purple solid at room temperature (Cartolano. 2005). The low vapor pressure and low estimated Henry's law constant for 3,4-TDA indicate that it is unlikely to volatilize from either dry or moist surfaces. 3,4-TDA has an estimated atmospheric half-life of 0.6 hours for the reaction with hydroxyl radicals, but this is not expected to be an important fate process because the compound is not likely to partition to the atmosphere. The estimated water solubility and low soil adsorption coefficient for 3,4-TDA indicate that it has the potential to leach to groundwater or undergo runoff after a rain event. However, given its acid dissociation constant (pKa), 3,4-TDA may exist partially as a cation in the environment, and cations generally adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts. Also, aromatic amines contain highly reactive amino groups that may cause strong bonding to soil organic matter. NH Figure 1. 3,4-Toluenediamine (CASRN 496-72-0) Structure 3 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 1. Physicochemical Properties of 3,4-Toluenediamine (CASRN 496-72-0) Property (unit) Value3 Physical state Solidb Boiling point (°C) 243 Melting point (°C) 88.8 Density (g/cm3) 1.13 (predicted) Vapor pressure (mm Hg) 6.29 x 10-4 pH (unitless) NV Acid dissociation constant (pKa) (unitless) 5.00 Solubility in water (mol/L) 2.39 (predicted) Octanol-water partition coefficient (log Kow) 0.66 Henry's law constant (atm-m3/mol) 5.59 x 10 x (predicted) Soil adsorption coefficient (Koc) (L/kg) 31.3 (predicted) Atmospheric OH rate constant (cm3/molecule-sec at 25°C) 1.43 x lO-10 (predicted) Atmospheric half-life (h) 0.6 (predicted)13 Relative vapor density (air = 1) NV Molecular weight (g/mol) 122 Flash point (°C) 137 (predicted) aData were extracted from the U.S. EPA CompTox Chemicals Dashboard (3,4-Toluenediamine, CASRN 496-72-0. https://comptox.epa.gov/dashboard/DTXSID9024930. Accessed on April 20, 2021). All values are experimental averages unless otherwise specified. bU.S. EPA (2012). NV = not available. No toxicity values for 3,4-TDA are available from U.S. EPA or other agencies/organizations searched, as shown in Table 2. 4 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 2. Summary of Available Toxicity Values for 3,4-Toluenediamine (CASRN 496-72-0) Source3 Value (applicability) Notes Referenceb Noncancer IRIS NV NA U.S. EPA (2020a) HEAST NV NA U.S. EPA (201 lb) DWSHA NV NA U.S. EPA (2018) ATSDR NV NA ATSDR (2018) IPCS NV NA IPCS (2020) CalFPA NV NA CalEPA (2019) OSHA NV NA OSHA (2020a): OSHA (2020b) NIOSH NV NA NIOSH (2016) ACGIH NV NA ACGIH (2020) Cancer IRIS NV NA U.S. EPA (2020a) HEAST NV NA U.S. EPA (2011b) DWSHA NV NA U.S. EPA (2018) NTP NV NA NTP (2016a) IARC NV NA IARC (2018) CalEPA NV NA CalEPA (2019) ACGIH NV NA ACGIH (2020) aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic Substances and Disease Registry; CalEPA = 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. bReference date is the publication date for the database and not the date the source was accessed. NA = not applicable; NY = not available. 5 3,4-T oluenediamine ------- EPA/690/R-21/003F Non-date-limited literature searches were conducted in November 2017 and updated in May 2020 and April 2021 for studies relevant to the derivation of provisional toxicity values for 3,4-toluenediamine (CASRN 496-72-0). Searches were conducted using U.S. EPA's Health and Environmental Research Online (HERO) database of scientific literature. HERO searches the following databases: PubMed, TOXLINE1 (including TSCATS1), and Web of Science. The following resources were searched outside of HERO for health-related values: American Conference of Governmental Industrial Hygienists (ACGIH), Agency for Toxic Substances and Disease Registry (ATSDR), California Environmental Protection Agency (CalEPA), Defense Technical Information Center (DTIC), European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), European Chemicals Agency (ECHA), U.S. EPA Chemical Data Access Tool (CDAT), U.S. EPA ChemView, U.S. EPA Integrated Risk Information System (IRIS), U.S. EPA Health Effects Assessment Summary Tables (HEAST), U.S. EPA Office of Water (OW), International Agency for Research on Cancer (IARC), U.S. EPA TSCATS2/TSCATS8e, U.S. EPA High Production Volume (HPV), Chemicals via IPCS INCHEM, Japan Existing Chemical Data Base (JECDB), Organisation for Economic Cooperation and Development (OECD) Screening Information Data Sets (SIDS), OECD International Uniform Chemical Information Database (IUCLID), OECD HPV, National Institute for Occupational Safety and Health (NIOSH), National Toxicology Program (NTP), Occupational Safety and Health Administration (OSHA), and World Health Organization (WHO). 'Note that this version of TOXLINE (https://www.nlm.nih.gov/databases/download/toxlinesiibset.html') is no longer updated; therefore, it was not included in the literature search updates from May 2020 and April 2021. 3,4-T oluenediamine 6 ------- EPA/690/R-21/003F 2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) As shown in Tables 3 A and 3B, there are no potentially relevant subchronic or chronic studies or developmental or reproductive toxicity studies of 3,4-TDA in humans or animals for n on cancer and cancer endpoints following oral or inhalation exposures. WHO (1987) described a small number of occupational health surveys of male workers exposed to diaminotoluene and dinitrotoluene mixtures; however, these studies are not useful for determining the effects of 3,4-TDA because they did not discuss or otherwise verify the presence of this isomer in the mixtures. The phrase "statistical significance" and term "significant," used throughout the document, indicate ap-value of < 0.05 unless otherwise specified. 7 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 3A. Summary of Potentially Relevant Noncancer Data for 3,4-Toluenediamine (CASRN 496-72-0) Number of Male/Female, Strain, Species, Study Category Type, Reported Doses, Study Duration Dosimetry Critical Effects Reference Notes Human 1. Oral (mg/kg-d) ND 2. Inhalation (mg/m3) ND Animal 1. Oral (mg/kg-d) ND 2. Inhalation (mg/m3) ND ND = no data. 8 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 3B. Summary of Potentially Relevant Cancer Data for 3,4-Toluenediamine (CASRN 496-72-0) Category Number of Male/Female, Strain, Species, Study Type, Reported Doses, Study Duration Dosimetry Critical Effects Reference Notes Human 1. Oral (mg/kg-d) ND 2. Inhalation (mg/m3) ND Animal 1. Oral (mg/kg-d) ND 2. Inhalation (mg/m3) ND ND = no data. 9 3,4-T oluenediamine ------- EPA/690/R-21/003F 2.1. HUMAN STUDIES 2.1.1. Oral Exposures No human studies following oral exposure to 3,4-TDA have been identified. 2.1.2. Inhalation Exposures No human studies following inhalation exposure to 3,4-TDA have been identified. 2.2. ANIMAL STUDIES 2.2.1. Oral Exposures No animal studies following oral exposure to 3,4-TDA have been identified. 2.2.2. Inhalation Exposures No animal studies following inhalation exposure to 3,4-TDA have been identified. 2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) Toxicity data available for the 3,4-TDA isomer are limited to genotoxicity studies and a 5-day oral study with few endpoints. Additionally, studies evaluating TDA mixtures containing the 3,4-TDA isomer are available, including a reproductive toxicity study, two developmental toxicity studies, several acute lethality studies, and eye and skin irritation assays. 2.3.1. Genotoxicity Available genotoxicity data for 3,4-TDA are summarized in Table 4. In vitro data indicate that 3,4-TDA has the potential to cause mutations in bacteria; however, evidence for mutation in mammalian cells is equivocal. In an NTP-sponsored study, 3,4-TDA was mutagenic to Salmonella typhimurium with, but not without, metabolic activation at concentrations as low as 333 |ig/plate, with cytotoxicity reported at >3,333 |ig/plate (Zeiger et al.. 1988). Other assays showed mutagenicity in S. typhimurium following exposure to 3,4-TDA with (but not without) metabolic activation. The number of revertants increased, but they were not dose related and/or observed only at high concentrations (-500 |ig/plate or higher) associated with >50% toxicity (Allied Chemical 1983a. b, c, 1979a. b; Litton Bionetics. 1979a. b). 3,4-TDA was not mutagenic to S. typhimurium in studies using lower concentrations of <366 |ig/plate (Watanabe et al., 1989; Florin et al.. 1980); toxicity was not reported in either study. Marginal evidence for mutagenicity in Chinese hamster ovary (CHO) cells and L5178Y/TK± mouse lymphoma cells was reported at concentrations associated with cytotoxicity (Allied Chemical. 1983a. b; Litton Bionetics. 1980b; Allied Chemical. 1979a. b; Litton Bionetics. 1979a). Available data indicate that 3,4-TDA has the potential to cause cell transformation in mammalian cells at concentrations that were often associated with cytotoxicity. A significant enhancement of viral-induced cell transformation was observed in primary Syrian hamster embryo (SHE) cell cultures following exposure to 3,4-TDA at concentrations >10 |ag/mL either before or after inoculation with simian adenovirus SA7. Cell survival was generally <50% at all doses tested (Greene and Friedman. 1980). Evidence for induction of cell transformation in secondary SHE cell cultures was equivocal, with marginal increases in cell transformation observed in only two of five replicate assays. These findings were not dose related at noncytotoxic concentrations (<10 |ig/mL), and higher concentrations caused significant cytotoxicity (Greene and Friedman, 1980). Similarly, the number of transformed foci in Balb/3T3 cells exposed to 3,4-TDA was marginally increased only at the highest concentration (4 |.ig/mL), which produced 50% cytotoxicity (Litton Bionetics, 1980a, 1979a). 10 3,4-T oluenediamine ------- EPA/690/R-21/003F Limited data from in vivo studies indicate that intraperitoneal (i.p.) exposure to 3,4-TDA induces micronuclei (MN) formation in mouse bone marrow (Wild et al. 1980) and inhibits deoxyribonucleic acid (DNA) synthesis in mouse testes (Allied Chemical 1983a. d; Greene et al. 1981; Allied Chemical 1979a. b). 11 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Genotoxicity studies in prokaryotic organisms Mutation Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 0, 1,000, 1,710, 2,924, 5,000 ng/plate + TA98, TA1538 TA100, TA1535, TA1537 Plate incorporation assay. The number of revertants was increased at all doses in TA98 and TA1538 by 2- to 7-fold, but a dose-response relationship was not observed. Relative survival was <50% for TA98, TA100, and TA1538 at >1,000 ng/plate with or without metabolic activation and for TA1535 and TA1537 at 5,000 ng/plate without metabolic activation. Allied Chemical (1983a): Allied Chemical (1979a): Allied Chemical (1979b): Allied Chemical (1983b): Allied Chemical (1983c) Mutation S. typhimurium TA98 0, 0.5, 5.0, 50, 500 ng/plate Not tested + (rat S9 or mouse S9 pretreated with saline) (mouse S9 pretreated with 3,4-TDA) Plate incorporation assay. Cells were metabolically activated with rat S9 or S9 prepared from C57Bl/6xC3H mice pretreated with physiological saline or 3,4-TDA (i.p.). The number of revertants was increased 2- to 4-fold at 500 ng/plate in samples activated with rat S9 or S9 prepared from C57Bl/6xC3H mice pretreated with physiological saline. Toxicity was not reported. Allied Chemical (1983a): Allied Chemical (1979a): Allied Chemical (1979b): Allied Chemical (1983b): Allied Chemical (1983c) Mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 0, 0.5, 1.0, 10, 100, 500, 1,000, 2,500, 5,000 ng/plate + TA98, TA1538 TA100, TA1535, TA1537 Plate incorporation assay. The number of revertants was increased 6- to 20-fold in TA98 and 5- to 7-fold in TA1538 at >500 |ig/plate. Toxicity was observed at >500 ng/plate. Litton Bionetics (1979a): Litton Bionetics (1979b) Mutation S. typhimurium TA 97, TA98, TA100, TA1535 0, 33, 100, 333, 666, 1,000, 3,333, 6,666, 10,000 ng/plate + TA97, TA98, TA100 TA1535 Preincubation assay. The number of revertants was increased 7- to 20-fold in TA98 at >333 ng/plate (toxicity observed at >3,333 ng/plate) and at 2- to 4-fold in TA100 and TA97 at >1,000 |ig/plate (toxicity observed at >6,666 ng/plate). Zeieer et al. (1988) 12 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Mutation S. typhimurium TA98, TA100, TA1535, TA1537 3 |imol/platc (366 ng/plate) Spot test. The number of revertants was not increased by 3,4-TDA exposure. Toxicity was not reported. Florin etal. (1980) Mutation S. typhimurium TA98 0, 10, 30 ng/plate Plate incorporation assay. The mutagenic potency of 3,4-TDA was not enhanced by the addition of H2O2, indicating that oxidative products are not mutagenic. Toxicity was not reported. Watanabe et al. (1989) Genotoxicity studies in mammalian cells in vitro Mutation CHO cells Without S9: 0, 67, 100, 126, 149, 150, 158, 199, 223, 224, 250, 334, 447, 500 iig/mL With S9: 0, 200, 250, 299, 354, 447, 500, 669, 707, 1,000 ng/mL (+) (+) 3,4-TDA induced a dose-related increase in mutant frequency with or without metabolic activation in 1/4 replicate assays. The study authors note that cytotoxicity was higher without metabolic activation (no further details were provided). Allied Chemical (1983a): Allied Chemical (1979a): Allied Chemical (1979b): Allied Chemical (1983b) Mutation L5178Y/TK± mouse lymphoma cells Without S9: 0, 0.29, 4.69, 9.38, 13.8, 37.5 ng/mL With S9: 0, 0.29, 18.8, 37.5, 50, 75, 100, 150 |ig/mL + (+) Mutation frequency was increased 2- to 8-fold at >13.8 |ig/mL without metabolic activation and 2-fold at >18.8 ng/mL with metabolic activation. Moderate to high toxicity was observed at all concentrations. Without metabolic activation, high toxicity was observed at >9.38 ng/mL (93-98% growth inhibition). With metabolic activation, high toxicity was observed at 150 |ig/mL. Litton Bionetics (1980b): Litton Bionetics (1979a) 13 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Cell transformation assay Primary SHE cells inoculated with simian adenovirus SA7 Exposure prior to viral inoculation: 0, 12.5, 25, 50, 100, 200 iig/mL Exposure after viral inoculation: 0, 10, 18, 20, 32, 36, 56, 63, 100, 112, 200 ng/mL + Not tested 3,4-TDA significantly (p < 0.05) enhanced viral transformation of primary SHE cells at all concentrations by 2- to 19-fold when added before or after viral inoculation. Survival was generally <50% at all doses tested, and 100% toxicity was observed at >100 ng/mL with exposure after viral inoculation. Greene and Friedman (1980) Cell transformation assay Secondary SHE cells 0, 2.5, 5.0, 10, 15, 20, 22, 33, 50 ng/mL (+) Not tested 3,4-TDA induced cell transformation in two of five replicate assays. In positive replicates, 1-2 transformed loci were observed at noncytotoxic concentrations (<10 |ig/mL): findings were not dose related. The three negative replicates had "low activity" of positive control (BaP). Relative survival was decreased by approximately 50% or more at >15 |ig/mL. Greene and Friedman (1980) Cell transformation assay Balb/3 T3 cells 0, 0.05, 0.5, 1, 2, 4 ng/mL (+) Not tested A marginal increase in the total number of transformed foci was observed at 4 ng/mL, which produced 50% cytotoxicity. Litton Bionetics (1980a): Litton Bionetics (1979a) Genotoxicity studies in mammals in vivo Bone marrow micronucleus test NMRI mice treated with 3,4-TDA i.p.; 2 doses separated by 24 h; bone marrow isolated 6 h after second dose 0, 122, 244, 366 mg/kg-d + The percentage of micronucleated polychromatic erythrocytes in bone marrow was significantly (p < 0.01) increased by 4.4-8% at >244 mg/kg-d, compared with controls. Wild et al. (1980) 14 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity Endpoint Test System Doses/ Concentrations Tested Results without Activation3 Results with Activation3 Comments References Testicular DNA synthesis inhibition test C57Bl/6xC3H mice treated with 3,4-TDA i.p.; 3H-thymidine incorporation in testicular DNA was measured 0, 500 mg/kg + Significant decrease in radioactivity incorporated into DNA relative to controls (p < 0,01 in one replicate, p< 0.1 in a second replicate). Allied Chemical fl983a): Allied Chemical (1979a): Allied Chemical (1979b): Allied Chemical (1983d) Testicular DNA synthesis inhibition test C57Bl/6xC3H mice treated with 3,4-TDA i.p.; 3H-thymidine incorporation in testicular DNA was measured 0, 200, 299, 262, 300 mg/kg + Significant decrease in radioactivity incorporated into DNA relative to controls at all doses (p < 0.025). No significant change in rectal temperature (changes in rectal temperature can affect testicular DNA synthesis). Greene et al. (1981) a+ = positive result; (+) = weak positive result; - = negative result. BaP = benzo[a]pyrene; CHO = Chinese hamster ovary; DNA = deoxyribonucleic acid; 3H = hydrogen-3 isotope (tritium); H2O2 = hydrogen peroxide; i.p. intraperitoneal; SHE = Syrian hamster embryo; TDA = toluenediamine. 15 3,4-T oluenediamine ------- EPA/690/R-21/003F 2.3.2. Short-Term and Acute Toxicity Studies Available short-term and acute toxicity studies of 3,4-TDA or TDA mixtures containing 3,4-TDA are summarized in Table 5. Toxicity data for 3,4-TDA alone are limited to a short-term exposure study in which female Sprague Dawley rats were administered 3,4-TDA (97% purity) twice-daily via gavage in water at a dose of 500 mg/kg (1,000 mg/kg-day) (Selve. 1973). The study was terminated on the fifth day of exposure because 7/13 exposed rats had died (no further information on time of death was provided). Gross necropsy revealed that six of the dead animals had grossly observed perforated duodenal ulcers. Severe icterus (jaundice) was also reported, although further details were not provided. The study authors stated that comparable results were observed when males and females (10/sex) were similarly exposed via gavage in water, peanut oil, dimethylsulfoxide, or propylene glycol or via i.p. or subcutaneous (s.c.) injection (no further details were provided) (Selve. 1973). Oral median lethal dose (LDso) values in rats for TDA mixtures containing 3,4-TDA range from 660-1,760 mg/kg (WIL Research. 1978; Air Products and Chemicals. 1976; Carpenter et at.. 1974). An inhalation median lethal concentration (LC.mi) value >670 ppm was reported in rats exposed to o-TDA; the duration of exposure was not reported (Air Products and Chemicals. 1976). Exposure to concentrated o-TDA vapors was lethal after >8 hours of exposure (Carpenter et al.. 1974). Dermal LDso values for o-TDA in rabbits ranged from 1,120 to >5,750 mg/kg (Air Products and Chemicals. 1976; Carpenter et al.. 1974). o-TDA is slightly to moderately irritating to rabbit skin, and the undiluted liquid is irritating to the rabbit eye (Air Products and Chemicals. 1976; Carpenter et al.. 1974). Skin sensitization was "insignificant" in guinea pigs exposed to o-TDA (Air Products and Chemicals. 1976). 16 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 5. Short-Term and Acute Toxicity Studies for 3,4-Toluenediamine (CASRN 496-72-0) Test Materials and Methods Results Conclusions References Exposure to 3,4-TDA alone Short-term oral Main experiment: Female Sprague Dawley rats (,n = 13) were exposed to 3,4-TDA twice daily via gavage in water at 500 mg/kg per dose (1,000 mg/kg-d) for up to 5 d. The animals were observed for mortality, and gross necropsy was performed on the animals that died. Additional experiments: The main experiment was repeated with male and female rats (10/group) using water, peanut oil, dimethylsulfoxide, or propylene glycol as a vehicle, as well as with parenteral (i.p. or s.c.) exposure. Main experiment: 7/13 rats died within the first 5 d; 6/7 dead rats had "macroscopically obvious" and "usually perforated" duodenal ulcers. Severe icterus was also reported. Additional experiments: Results following oral exposure "essentially the same as in the main experiment." Result following parenteral exposure were "only doubtfully less efficacious." No further details were provided. Oral or parenteral exposure to high doses of 3,4-TDA caused perforating duodenal ulcers and icterus in rats. The study authors propose using 3,4-TDA to create an animal model of duodenal ulcers for testing of therapeutic interventions. Selve (1973) Exposure to TDA mixtures containing 3,4-TDA Acute oral lethality Rats were exposed to a mixture of 2,3- and 3,4-TDA in 4% aqueous solution by gavage at 6 doses. Study was reported in tabular form with no further details. LD5o = 660 mg/kg. No further details were provided. Rat oral LD5o = 660 mg/kg. Air Products and Chemicals (1976) Acute oral lethality Rats were exposed to a mixture of 2,3- and 3,4-TDA. No further details were provided. LD5o (95% CI) = 810 (590-1,120) mg/kg. No further details were provided. Rat oral LD5o = 810 mg/kg. Carpenter et al. (1974) Acute oral lethality Sprague Dawley rats (5/sex/group) were exposed to two mixtures containing 2,3-, 2,4-, and 3,4-TDA and 4,4-methylenedianiline at doses of 0, 310, 630, or 1,250 mg/kg via gavage in corn oil. One formulation contained 17.9% (wt) 3,4-TDA; the other was a similar formulation, but exact percentages were not "precisely known." The animals were observed hourly for the first 6 h for signs of toxicity, and subsequently for 14 d for mortality. Gross necropsies were performed. Most deaths occurred between 1 and 3 d postexposure. Surviving animals exhibited shallow respiration, depression, depressed righting and placement reflexes, excessive salivation, unkempt coats, piloerection, and yellowish-orange urine and mucoid diarrhea stains. All survivors were observed to be emaciated between the fourth and eighth day postexposure. Necropsies of decedents indicated congested lungs, adrenals, and kidneys; mottled livers; irritated GI tracts and peritoneal walls; and fluid-filled stomachs. Necropsies of survivors were unremarkable. LD50 = 1,100-1,760 mg/kg in male rats. LD50 = 1,080-1,220 mg/kg in female rats. WIL Research (1978) 17 3,4-T oluenediamine ------- EPA/690/R-21/003F Table 5. Short-Term and Acute Toxicity Studies for 3,4-Toluenediamine (CASRN 496-72-0) Test Materials and Methods Results Conclusions References Acute inhalation lethality Rats were exposed to a mixture of 2,3- and 3,4-TDA. Study was reported in tabular form with no further details. LC50 >670 ppm. No further details were provided. Rat inhalation LC50 >670 ppm. Air Products and Chemicals (1976) Acute inhalation lethality Rats were exposed to the concentrated vapors of a mixture of 2,3- and 3,4-TDA. No further details were provided. Maximum time producing no deaths was 8 h. No further details were provided. Exposure to the concentrated vapors of a TDA mixture is lethal after >8 h of exposure. Carpenter et al. (1974) Acute dermal lethality Rabbits were exposed dermally (abraded and nonabraded skin) to a mixture of 2,3- and 3,4-TDA in a 60% aqueous paste for 24 h and observed for 14 d. Study was reported in tabular form with no further details. LD50 >5,750 mg/kg. No further details were provided. Dermal LD50 >5,750 mg/kg. Air Products and Chemicals (1976) Acute dermal lethality Rabbits were exposed to a mixture of 2,3- and 3,4-TDA on shaved backs for 24 h. No details regarding concentration or occlusion were provided. LD50 (95% CI) = 1,120 (620-2,040) mg/kg. No further details were provided. Dermal LD50 = 1,120 mg/kg. Carpenter et al. (1974) Acute dermal irritation Rabbits were exposed to a mixture of 2,3- and 3,4-TDA on the uncovered skin of the belly. No details regarding concentration or duration of exposure were provided. Irritation score was 5/10. A mixture of TDA isomers is moderately irritating to the skin of rabbits. Carpenter et al. (1974) Skin sensitization Guinea pigs were exposed to a mixture of 2,3- and 3,4-TDA. The study was reported in tabular form with no further details. "Insignificant" sensitization. A mixture of TDA isomers is not a skin sensitizer in guinea pigs. Air Products and Chemicals (1976) Eye irritation Rabbits were exposed to a mixture of 2,3- and 3,4-TDA in 5% aqueous solution in eye. The study was reported in tabular form with no further details. No irritation after 72 h. A diluted mixture of TDAs is not irritating to the rabbit eye. Air Products and Chemicals (1976) Eye irritation Rabbits were exposed to a mixture of 2,3- and 3,4-TDA in a "suitable vehicle" in eye. Irritation score was 7/10. A mixture of TDA isomers is irritating to the rabbit eye. Carpenter et al. (1974) CI = confidence interval; GI = gastrointestinal; i.p. = intraperitoneal; LC50 = median lethal concentration; LD50 = median lethal dose; s.c. = subcutaneous; TDA = toluenediamine. 18 3,4-T oluenediamine ------- EPA/690/R-21/003F 2.3.3. Reproductive/Developmental Studies of TDA Mixtures Beccietal. (1983) Groups of 22 pregnant Sprague Dawley rats were administered o-TDA (a 40:60 mixture of 2,3- and 3,4-TDA) at doses of 0, 10, 30, 100, or 300 mg/kg-day via gavage in corn oil from Gestation Days (GDs) 6-15. Observations were conducted daily for general appearance, behavior, and mortality. Body weights of the dams were recorded on GDs 0, 6, 9, 12, 15, and 20. On GD 20, all dams were sacrificed, and uterine contents were removed and examined. One-half of the fetuses were examined for soft-tissue anomalies, and the remaining fetuses were examined for skeletal anomalies. No treatment-related effects on appearance or behavior were observed in treated dams, and all dams survived the duration of the study. There was a statistically significant decrease in weight gain (-20%) during gestation for treated dams receiving 300 mg/kg-day compared with controls. No significant differences in the number of live fetuses, implantation sites, or resorption sites were indicated. Fetal effects indicative of developmental delay included significant reductions in fetal body weight (-18%) in the 300-mg/kg-day group and significant increases in skeletal variations per litter (missing sternebrae at 300 mg/kg-day and incomplete ossification of vertebrae at 100 and 300 mg/kg-day), compared with controls. No exposure-related skeletal or soft-tissue malformations were observed. No maternal or developmental effects were seen at <30 mg/kg-day. Additionally, groups of 15 pregnant Dutch belted rabbits were exposed to o-TDA at doses of 0, 3, 10, 30, or 100 mg/kg-day via gavage in corn oil from GDs 6-18. Observations were conducted daily for general appearance, behavior, and mortality. Body weights of the does were recorded on GDs 0, 6, 9, 12, 15, 18, and 29. On GD 29, all does were sacrificed, and uterine contents were removed and examined. All of the fetuses were examined for both soft-tissue and skeletal anomalies. Appearance and behavior of does were unaffected by treatment. All does survived the duration of the study. Body-weight gain during gestation was significantly decreased (—60%) in treated does receiving 100 mg/kg-day compared with controls. Other observations at this dose included a significant 2.5-fold increase in the incidence of resorptions, a 16% decrease in the mean number of live fetuses/doe (reported as statistically significant in the text, but not in the table showing the data), and a significant 22% decrease in fetal body weight. No exposure-related skeletal or soft-tissue malformations or variations were observed. No maternal or developmental effects were seen at <30 mg/kg-day. BASF (2010) In an OECD 421 reproductive/developmental (R/D) study available only as an industry-submitted summary, groups of male and female Wistar rats (10/sex/group) were administered o-TDA (45:50 mixture of 2,3- and 3,4-TDA) at doses of 0, 10, 50, or 250 mg/kg-day via gavage (vehicle not reported) from premating through mating (males, at least 28 days) or premating through Postnatal Day (PND) 4 (up to 60 days for females). The pups were sacrificed and examined on PND 4 (endpoints examined at sacrifice were not reported). The available summary reported only "the most relevant results"; no statistics were provided, and the summary did not include the magnitude/incidence for many of the findings. Clinical signs of toxicity (reduced activity, eyelid drop, salivation, and/or piloerection) were observed in males and dams at >50 mg/kg-day. Decreased food consumption was observed during premating in males at 250 mg/kg-day and females at >50 mg/kg-day; decreased food consumption was also observed in dams during gestation at 250 mg/kg-day. Body-weight gain was decreased throughout the study in high-dose males, with a decreased terminal body weight compared with controls (magnitude not reported). Decreased body weight and body-weight gains 19 3,4-T oluenediamine ------- EPA/690/R-21/003F were observed during premating and gestation in dams at 250 mg/kg-day, with a decreased terminal body weight compared with controls (magnitude not reported). In males, a decrease in the number of spermatids/g testis was reported at 250 mg/kg-day; however, the study summary did not indicate whether decreased fertility was observed. Reproductive effects observed in high-dose dams included a 39% decrease in the number of implantation sites compared with controls, a 27.4% post implantation loss (control value not reported), and a 42% decrease in the number of delivered pups/litter. In offspring, a decreased viability index of 91% was observed at 250 mg/kg-day (viability index in controls was not reported). No effects were noted in males or dams administered 10 mg/kg-day. 2.3.4. Mode-of-Action/Mechanistic Studies Mechanistic data for 3,4-TDA are limited. Perkins and Green (1975) suggested that the duodenal ulcers observed by Solve (1973) may be a result of 3,4-TDA toxicity to Brunner's glands in the proximal duodenum. Brunner's glands function to secrete an alkaline mucoid material to protect the duodenum from the corrosive action of gastric juices. The volume of Brunner's glands' secretions was quantified in situ following single subcutaneous injections of 3,4-TDA in rats at 125 mg/kg (which caused minimal gastroduodenal damage), 350 mg/kg (which caused maximal duodenal damage and minimal mortality), and 500 mg/kg (which caused a low incidence of duodenal and a high incidence of gastric damage). The output of fluid from Brunner's glands was significantly decreased in all exposed groups compared with control, with lower inhibition at 125 mg/kg (25%) than 350 and 500 mg/kg (61 and 57%, respectively). 20 3,4-T oluenediamine ------- EPA/690/R-21/003F 3. DERIVATION OF PROVISIONAL VALUES 3.1. DERIVATION OF PROVISIONAL REFERENCE DOSES No subchronic or chronic studies have been located regarding the toxicity of 3,4-TDA to humans or animals via oral administration. Potentially relevant toxicity data for 3,4-TDA are limited to a 5-day gavage study that reported perforating duodenal ulcers in rats following exposure to 1,000 mg/kg-day (Selve. 1973). Additionally, gavage exposure studies evaluating TDA mixtures containing the 3,4-TDA isomer (approximately 50-60% 3,4-TDA) showed some evidence of potential R/D effects in rats at 250 mg/kg-day following premating through PND 4 and in rats and rabbits at >100 mg/kg-day following gestational exposure, primarily at doses associated with potential maternal toxicity (BASF. 2010; Becci et al.. 1983). The scope and design of these studies are inadequate to support the derivation of a subchronic or chronic provisional reference dose (p-RfD) for 3,4-TDA using chemical-specific data. Instead, screening p-RfDs are derived in Appendix A using an alternative, read-across approach. Based on the overall analogue approach presented in Appendix A, 2,5-toluenediamine was selected as the most appropriate analogue for 3,4-TDA for deriving a screening subchronic and chronic p-RfD (see Table 6). 3.2. DERIVATION OF PROVISIONAL REFERENCE CONCENTRATION The absence of relevant inhalation data precludes derivation of provisional reference concentrations (p-RfCs) for 3,4-TDA directly. An alternative read-across approach was attempted, but screening p-RfCs could not be derived due to a lack of inhalation toxicity values for analogues identified (see Appendix A). 3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES The noncancer provisional reference values for 3,4-TDA are summarized in Table 6. Table 6. Summary of Noncancer Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) Toxicity Type (units) Species/ Sex Critical Effect p-Reference Value POD Method POD UFc Principal Study Screening subchronic p-RfD (mg/kg-d) Rat/F Increased serum AST 1 x 1(T3 NOAEL (HED) 0.32 (based on analogue POD) 300 Hill (1997) as cited in SCCP (2007) and reDorted bv U.S. EPA (2013) Screening chronic p-RfD (mg/kg-d) Rat/F Increased serum AST 1 x 1(T4 NOAEL (HED) 0.32 (based on analogue POD) 3,000 Hill (1997) as cited in SCCP (2007) and reDorted bv U.S. EPA (2013) Subchronic p-RfC (mg/m3) NDr Chronic p-RfC (mg/m3) NDr AST = aspartate aminotransferase; F = female(s); HED = human equivalent dose; NDr = not determined; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose; UFC = composite uncertainty factor. 21 3,4-T oluenediamine ------- EPA/690/R-21/003F 3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR Under the U.S. EPA Cancer Guidelines (U.S. EPA. 2005a). there is "Inadequate Information to Assess the Carcinogenic Potential" of 3,4-TDA (see Table 7). No relevant studies are available in humans or animals. Within the current U.S. EPA Cancer Guidelines (U.S. EPA. 2005a). there is no standard methodology to support the identification of a weight-of-evidence (WOE) descriptor and derivation of provisional cancer risk estimates for data-poor chemicals using an analogue approach. In the absence of an established framework, a screening evaluation of potential carcinogenicity is provided using the methodology described in Appendix B. This evaluation determined that there was a concern for potential carcinogenicity of 3,4-TDA (see Appendix C). Table 7. Cancer WOE Descriptor for 3,4-Toluenediamine (CASRN 496-72-0) Possible WOE Descriptor Designation Route of Entry (oral, inhalation, or both) Comments "Carcinogenic to Humans" NS NA There are no human carcinogenicity data identified to support this descriptor. "Likely to Be Carcinogenic to Humans " NS NA There are no animal carcinogenicity studies identified to support this descriptor. "Suggestive Evidence of Carcinogenic Potential" NS NA There are no animal carcinogenicity studies identified to support this descriptor. "Inadequate Information to Assess Carcinogenic Potential" Selected Both This descriptor is selected due to the lack of adequate, chemical-specific data in humans or animals to evaluate the carcinogenic potential of 3,4-TDA. "Not Likely to Be Carcinogenic to Humans" NS NA No evidence of noncarcinogenicity is available. NA = not applicable; NS = not selected; TDA = toluenediamine; WOE = weight of evidence. 3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES The absence of suitable data precludes development of cancer risk estimates for 3,4-TDA (see Table 8). Table 8. Summary of Cancer Risk Estimates for 3,4-Toluenediamine (CASRN 496-72-0) Toxicity Type (units) Species/Sex Tumor Type Cancer Risk Estimate Principal Study p-OSF (mg/kg-d) 1 NDr p-IUR (mg/m3) 1 NDr NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor. 22 3,4-T oluenediamine ------- EPA/690/R-21/003F APPENDIX A. SCREENING NONCANCER PROVISIONAL VALUES Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity Value (PPRTV) document, it is inappropriate to derive provisional toxicity values for 3,4-toluenediamine (3,4-TDA). However, some information is available for this chemical, which although insufficient to support derivation of a provisional toxicity value under current guidelines, may be of limited use to risk assessors. In such cases, the Center for Public Health and Environmental Assessment (CPHEA) summarizes available information in an appendix and develops a "screening value." Appendices receive the same level of internal and external scientific peer review as the provisional reference values to ensure their appropriateness within the limitations detailed in the document. Users of screening toxicity values in an appendix to a PPRTV assessment should understand that there could be more uncertainty associated with deriving an appendix screening toxicity value than for a value presented in the body of the assessment. Questions or concerns about the appropriate use of screening values should be directed to the CPHEA. APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH The analogue read-across approach allows for the use of data from related compounds to calculate screening values when data for the compound of interest are limited or unavailable. Details regarding searches and methods for analogue analysis are presented in Wang et al. (2012). Three types of potential analogues (structural, metabolic, and toxicity-like) are identified to facilitate the final analogue chemical selection. The analogue approach may or may not be route specific or applicable to multiple routes of exposure. All information was considered together as part of the final weight-of-evidence (WOE) approach to select the most suitable analogue both toxicologically and chemically. Structural Analogues An initial analogue search focused on the identification of structurally similar chemicals with toxicity values from the Integrated Risk Information System (IRIS), PPRTV, Agency for Toxic Substances and Disease Registry (ATSDR), or California Environmental Protection Agency (CalEPA) databases to take advantage of the well-characterized chemical-class information. This was accomplished by searching structural similarity software tools, namely the National Library of Medicine's (NLM) ChemlDplus database (MM. 2019) and Organisation for Economic Co-operation and Development (OECD) quantitative structure-activity relationship (QSAR) Toolbox (OECD. 2019). These software tools employ slightly different quantitative methods to make similarity comparisons between chemical structures based on fingerprints; ChemlDplus uses a modified Tanimoto index and the OECD Toolbox uses the Dice index. Two TDA isomers that have oral noncancer toxicity values were identified as potential structural analogues of 3,4-TDA: 2,6- (U.S. HP A. 2005b) and 2,5-TDA (U.S. EPA. 2013) (see Table A-l). In addition, 2,3- (a compound being evaluated in a separate PPRTV assessment) and 2,4-TDA were included in the read-across analysis to provide information on the potential influence of the position of the amino groups (ortho [o-], me la \m-\ or para [/>]) on toxicity (note: these analogues do not have oral toxicity values; see Table A-l). Previous structure-activity relationship (SAR) analyses have suggested increased chemical reactivity and toxicity for o- and p- versus ///-substituted aromatic amines (Bajot et al., 2010). The target and 2,3-TDA are o- isomers, 2,5-TDA is ap- isomer, and 2,4- and 2,6-TDAs are m- isomers. 23 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-l summarizes the physicochemical properties and similarity scores for all analogues. 3,4-TDA and the identified analogues are aromatic amines that share a common basic structure, which consists of a benzene ring, two amino groups and a methyl group, differing only in the position of the amino functional groups. These compounds are major components of commercial grade TDA mixtures (WHO. 1987) and have physicochemical properties that are of the same relative order of magnitude; therefore, differences in the absorption and distribution between the analogues and the target are not expected to be significant or result in a preference in the selection of one analogue over another. These compounds are all weak bases and are expected to be substantially ionized at physiological pH values. Furthermore, their water solubility and their octanol-water partition coefficient (log Kow) values are consistent with a high degree of hydrophilicity (see Table A-l). Additionally, all of these diamines have low volatility and are not expected to be eliminated in exhaled breath. Structural alert (SA) predictions for relevant toxicity endpoints were generated for the TDA isomers using the OECD QSAR Toolbox rOECD (2019); see Table A-2], These included the repeated-dose profiler based on the Hazard Evaluation Support System (HESS) database and the developmental and reproductive toxicity (DART) scheme adapted from the Wu et al. (2013) framework for identifying chemicals with structural features associated with potential reproductive/developmental (R/D) toxicants. The model predictions suggest concern for hepatotoxicity, hemolytic anemia with methemoglobinemia, and for R/D toxicity for 3,4-TDA and all analogues. The predictions are based on SAs for aniline and toluene/small alkyl toluene derivatives, respectively. The HESS model also showed concern for renal toxicity for 3,4-, 2,3-, and 2,6-TDA based on the SA for toluene. In summary, the candidate analogues are considered suitable analogues for 3,4-TDA based on their similarities in structural and physicochemical properties and SA predictions. 24 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-l. Physicochemical Properties of 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues" Property 3,4-Toluenediamine 2,3-Toluenediamine 2,4-Toluenediamine 2,5-Toluenediamine 2,6-Toluenediamine Role Target Analogue Analogue Analogue Analogue Structure CASRN 496-72-0 2687-25-4 95-80-7 95-70-5 823-40-5 DTXSID 9024930 4027494 4020402 6029123 4027319 Molecular weight 122 122 122 122 122 ChemlDplus similarity score (%)b 100 70 64 75 55 OECD toolbox similarity score (%)° 100 67 78 78 78 Melting point (°C) 60.1 98.2 64.0 105 Boiling point (°C) 243 254 286 274 260 Vapor pressure (mm Hg) 6.29 x 10-4 5.53 x 10- 1.7 x KT 2.25 x 10 3 (predicted) 2.46 x 10-3 Henry's law constant (atm-m3/mol) 5.59 x 10-8 (predicted) 5.59 x 10-8 (predicted) 5.60 x 10-8 (predicted) 5.73 x 10-8 (predicted) 5.61 x 10-8 (predicted) Water solubility (mol/L) 2.39 (predicted) 2.36 (predicted) 2.57 (predicted) 2.73 (predicted) 2.52 (predicted) Octanol-water partition coefficient (Log Kow) 0.66 0.549 (predicted) 0.14 0.11 (predicted) 0.21 (predicted) Acid dissociation constant (pKa) (unitless) 5.00 4.91 (predicted)11 5.58 (predicted)6 6.52 (predicted)6 5.28 (predicted)6 'Data were extracted from the U.S. EPA CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard. Accessed on April 20, 2021). All values are experimental averages unless otherwise specified. ' ChemlDplus advanced similarity scores (NLM. 2019). °OECD QSAR Toolbox, similarity scores (OECD. 2019). dHSD6 (2013). eChemAxon (2017). OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity relationship. 25 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-2. Comparison of SAs for Relevant Endpoints for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues from the OECD QSAR Toolbox3 SA 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Repeated-dose toxicity (HESS) • Hepatotoxicity (anilines) • Hemolytic anemia with methemoglobinemia (anilines) • Renal toxicity (toluene) • Hepatotoxicity (anilines) • Hemolytic anemia with methemoglobinemia (anilines) • Renal toxicity (toluene) •Hepatotoxicity (anilines) •Hemolytic anemia with methemoglobinemia (anilines) • Hepatotoxicity (anilines) • Hemolytic anemia with methemoglobinemia (anilines) • Hepatotoxicity (anilines) • Hemolytic anemia with methemoglobinemia (anilines) • Renal toxicity (toluene) DART scheme • Known precedent reproductive and developmental toxic potential (toluene and small alkyl toluene derivatives) • Known precedent reproductive and developmental toxic potential (toluene and small alkyl toluene derivatives) • Known precedent reproductive and developmental toxic potential (toluene and small alkyl toluene derivatives) • Known precedent reproductive and developmental toxic potential (toluene and small alkyl toluene derivatives) • Known precedent reproductive and developmental toxic potential (toluene and small alkyl toluene derivatives) "OECD QSAR Toolbox (OECD. 20191. DART = developmental and reproductive toxicity; HESS = Hazard Evaluation Support System; OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity relationship; SA = structural alert. 26 3,4-T oluenediamine ------- EPA/690/R-21/003F Metabolic Analogues Table A-3 summarizes available toxicokinetic data in experimental animals for 3,4-TDA and the structurally similar compounds identified as candidate analogues. No toxicokinetic data has been identified for 3,4-TDA. Available information on the 2,4-, 2,5-, and 2,6-TDA analogues suggest that these compounds are rapidly and extensively absorbed following oral exposure, and are rapidly eliminated in the urine, which is a predominant route of excretion (see Table A-3). Major metabolic steps for the TDA analogues are acetylation of amino groups and ring hydroxylation, with some evidence for oxidation of the methyl groups in rats and mice exposed via intraperitoneal (i.p.) administration (see Table A-3). In the absence of in vivo toxicokinetic data on 3,4-TDA, a selection of available software tools, specifically the in vivo and in vitro rat metabolic simulators available within the Tissue Metabolism Simulator (TIMES) program (Dimitrov et al.. 2005; Mekenvan et al.. 2004) and Meteor Nexus (Marchant et al.. 2008) were used to predict metabolites for the target compound and analogues. Predicted metabolites for the TDA isomers are summarized in Table D-l and additional information on the in silico analysis can be found in Appendix D. The analysis reveals some overlap in terms of metabolites for the individual TDA compounds across the different tools and when comparing predictions with experimental data from in vivo rodent studies (captured in Table A-3), which increases confidence in the in silico results (see Table D-l). Furthermore, the corresponding metabolic pathway transformations were extracted from Meteor Nexus to allow for similarity comparisons across the TDAs. This level of information was not available from other tools (i.e., TIMES). Table A-4 shows a consistent pattern of pathway transformations among the TDA compounds, and Figure A-l confirms a high degree of similarity between 3,4-TDA and the candidate analogues with regards to the Meteor Nexus pathway predictions. There is also concordance between the in silico results (see Table A-4) and the major pathways expected for this group of compounds (A-acetylation, ring hydroxylation, and oxidation of methyl groups). Importantly, no outstanding differences in the predicted metabolic profiles between the target and analogues are noted. The metabolic tree for 2,4-TDA is displayed in Figure D-l to illustrate the relationship of the predicted metabolites for this specific analogue that correspond to the pathway transformations shared among the TDAs (see Appendix D for more details). In summary, in vivo data demonstrate toxicokinetic commonalities among the analogues, particularly with respect to metabolism pathways, and according to in silico predictions, a similar metabolism pattern is expected for 3,4-TDA. Therefore, the candidate analogues are considered suitable analogues for 3,4-TDA based on their toxicokinetic properties. 27 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 nh2 Jr nh2 &l; ch3 nh2 ch3 X.nh2 h2n^^ CH3 H2N^^^NH2 U Target Analogue Analogue Analogue Analogue DTXSID 9024930 DTXSID 4027494 DTXSID 4020402 DTXSID 6029123 DTXSID 4027319 Absorption ND ND Rats cx nosed orallv (sinule dose of Rats cxnoscd orallv (sinule dose of Rats cxnoscd orallv (sinele dose of 3 or 60 me/ke): 2.5 or 25 me/ke): 10 ma/anima 1: aooroximatelv 57 to • 70% of administered dose based on recovered radioactivity in the urine and tissue/carcass over 48 h Rats cx nosed via i.D. iniection (sinele • Blood radioactivity peaked at 1 h after dosing (as sulfate) • Time to Cmax in blood 0.5-1 h after dosing (as sulfate) 67 me/ke based on a reported bodv weishts of 150-175 s): • Rapidly and extensively absorbed 77-ms/ks dose): • Rapid absorption; levels peaked in blood and plasma 1 h after dosing 28 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Distribution ND ND Rats cxDoscd orallv (sinele dose of ND Rats exposed orallv (sinsle dose of 3 or 60 me/ke): 10 me/animal: aroroximatelv 57 to • 2-5% in tissue and carcass (after 48 h) Rats and mice exposed via i.D. iniection (sinele 77-me/ke dose): • Liver and kidney > blood > muscle; high levels also in GI tract, spleen, heart, testes, lymph nodes, eyes, and lungs • Mouse tissues showed lower level of radioactivity than rat tissues 67 me/ke based on a reported bodv weiehts of 150-175 e): Wide distribution following single dose (% of administered dose) • 3.6% large intestine • 1% muscle • 0.6% liver • 0.5% skin • 0.2% blood • 0.1% small intestine • >0.1% for perirenal fat, stomach contents, brain, spleen, testis, heart, and lungs Metabolites ND ND Rats exposed orallv (sinele 50-me/ke Rats exposed orallv to sulfate salt Rats exposed orallv (sinele dose): (sinele dose of 2.5 or 25 me/ke): 10 me/animal: aroroximatelv 57 to Urinary over 48 h (% dose excreted) • 3-Hydroxy-4-acetylamino- 2-aminotoluene (18%) • 5-Hydroxy-4-acetylamino- 2-aminotoluene (14%) • 5-Hydroxy-2,4-diaminotoluene (12%), 3-hydroxy-2,4- diaminotoluene (8%) and 6-hydroxy-2,4-diaminotoluene (5%) • Other (unidentified) conjugates (16-34% of all acid-labile conjugates) Urinary over 96 h • Y,Y'-Diacctyl-tolucnc-2.5-diaminc • Two unidentified mono-Y-acetylated metabolites Rats cxDoscd bv i.v. iniection (sinsle 2.5-me/ke dose): Urine and feces over 96 h • 2,5-Diacetylamino toluene was a major metabolite 67 me/ke based on a reported bodv weiehts of 150-175 e): Urinary over 24 h • 3-Hydroxy-2,6-toluenediamine • 5-Hydroxy-2-acetylamino- 6-aminotoluene • 2-Acetylamino-6-aminotoluene • 2,6-Diacetylamino toluene • No parent compound detected 29 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Continued: Continued: Continued: Continued: Continued: Rats exposed via i.p. injection (single 77-mg/kg dose): Urinary over 24 h (% dose) • Free metabolites (20.9%), including 4-acetylamino-2-aminotoluene, (5.7%), 4-acetyl amino-2-amino benzoic acid (2.7%), and 2,4-diacetylamino toluene (2.6%) • Glucuronide conjugates (7.5%) • Sulfate conjugates (10.1%) • Water soluble (30.9%) Mice exposed via i.p. injection (single 77-mg/kg dose): Urinary over 24 h (% dose) • Free metabolites (20.2%), including 4-acetylamino-2-aminobenzoic acid (5.4%), 4-acetylamino-2-aminotoluene (2.1%), and 2,4-diacetyl aminobenzoic acid (1.1%) • Glucuronide conjugates (17.4%) • Sulfate conjugates (9.6%) • Water soluble (35.3%) 30 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Excretory pattern after oral exposure (unless otherwise indicated) ND ND Rats cxooscd orallv (sinele dose of Rats cxooscd orallv (sinele dose of Rats cxooscd orallv (sinsle dose of 3 or 60 me/ke): 2.5 or 25 me/ke): 10 me/animal: aroroximatelv 57 to Urine (% dose): >60% (within 48 h) Feces (% dose): 23-31 (within 48 h) Urine (% dose): >60 (within 98 h) Feces (% dose): 22-31 (within 98 h) 67 me/ke based on a reported bodv weiehts of 150-175 e): Urine (% dose): 85 (within 24 h) Feces (% dose): 10 (within 72 h) Exhaled air (% dose): 0 References NA NA Titnchalk et al. (1994); Grantham et al. W eaker (2005b). Weaker (2005a). Cunningham et al. (1989) (1979); Waring and Pheasant (1975) Weaker (2005c). and Charles River Laboratories (2010) as cited in SCCS (2012). pages 50-52 and 56-57 ADME = absorption, distribution, metabolism, and excretion; Cmax = maximum concentration; GI = gastrointestinal; i.p. = intraperitoneal; i.v. = intravenous; NA = not applicable; ND = no data. 31 3,4-T oluenediamine ------- EPA 690 R-21 003F Table A-4. Comparison of Metabolic Pathway Transformations for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues from Meteor Nexus3'b Pathway 3,4-TDA 2,3-TDA 2,4-TDA 2,5-TDA 2,6-TDA 5-Hydroxy lation of 1,2,4-trisubstituted benzenes 1 0 1 1 0 Hydroxylamines from primary aromatic amines 1 1 1 1 1 Hydroxylation of methyl carbon next to an aromatic ring 1 1 1 1 1 Y-Accty lation of primary aromatic amines 1 1 1 1 1 O-Sulfonation of aromatic alcohols 0 1 0 O-Sulfonation of Y-hvdroxy compounds 1 1 1 1 1 Oxidation of primary alcohols 1 1 1 1 1 aMeteor Nexus (Dimitrov et al.. 2005: Mekenvan et al.. 2004). bl/0 denotes whether pathway transformation was identified/not identified. TDA = toluenediamine. < n fN < Q fN ,x < Q ^ rn fN < Q in fN < O m" 1 0.71 0.71 1 1 0.86 0.83 0.83 1 0.86 0.83 0.83 1 -0.95 £ £ 2r4-TDA 2,6-TDA 2r3-TDA 2,5-TDA 3,4-TDA DTXSID Figure A-l. Metabolic Pathway Similarities for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues. Heatmap displays Jaccard pairwise similarities rounded to two decimal places for the TDA compounds, comparing metabolic pathway transformations from Meteor Nexus (Dimitrov et al., 2005; Mekenvan et al., 2004). 32 3,4-T oluenedi amine ------- EPA/690/R-21/003F Toxicity-Like Analogues Table A-5 summarizes subchronic and chronic oral toxicity data for 3,4-TDA and the compounds identified as candidate analogues. None of the analogues had subchronic or chronic inhalation toxicity values from U.S. EPA, ATSDR, or CalEPA. Relevant toxicity data in animals for 3,4-TDA alone comes from a single 5-day study in rats with limited information on toxicity endpoints and associated effect levels (increased mortality and incidence of perforated duodenal ulcers in dead animals at 1,000 mg/kg-day) (Selve. 1973). Oral toxicity values are available for 2,5- and 2,6-TDA. Hepatic effects were the basis for the subchronic and chronic provisional reference doses (p-RfDs) for 2,5-TDA using a point of departure (POD) of 2.5 mg/kg-day (1.4 mg/kg-day for the free base estimated for this assessment) (U.S. HP A. 2013). Thyroid and body-weight effects were the basis for the subchronic p-RfD for 2,6-TDA using a POD of 62 mg/kg-day (U.S. EPA. 2005b). The chronic p-RfD for 2,6-TDA was derived based on a POD of 25 mg/kg-day for no adverse effects (U.S. HP A. 2005b). Although thyroid toxicity was only noted following 2,6-TDA exposure, liver effects (ranging from changes in serum biomarkers and liver weight to gross and histopathological lesions) were reported after exposure to 2,4- (>6 mg/kg-day), 2,5- (>3 mg/kg-day), and 2,6-TDA (692 mg/kg-day) (U.S. HP A. 2013. 2005b; Criteria Group for Occupational Standards. 2001; WHO. 1987). R/D toxicity was commonly seen with exposure to TDA compounds, but these endpoints were generally less sensitive than the systemic effects described above. Impaired male fertility and sperm damage were reported in male rats orally exposed to 2,4-TDA at 15 mg/kg-day (Criteria Group for Occupational Standards. 2001). Developmental effects were observed in laboratory animals orally exposed to 2,6- (>100 mg/kg-day) or 2,5-TDA (>44 mg/kg-day) during gestation, primarily at doses associated with potential maternal toxicity (U.S. HP A. 2013; WHO. 1987). Data on the o-TDA mixture (40:60 or 45:50 mixture of 2,3- and 3,4-TDA) showed possible evidence of R/D effects in rats and rabbits exposed to >100 mg/kg-day via gavage, including alterations in sperm measures (decreased spermatid number) and/or fetal viability and growth (decreased implantation sites, litter size, pup viability, and fetal weight, as well as increased post implantation loss, resorptions and skeletal variations) often accompanied by reductions in maternal body-weight gain (BASF. 2010; Bccci et at.. 1983). These findings suggest that the reproductive system and developing embryo/fetus may be toxicity targets of 3,4- and 2,3-TDA. Acute lethality studies via different exposure routes were available for o- (2,3- and 3,4-) and m- (2,4- and 2,6-) TDA mixtures and individual TDA isomers (see Table A-6). The oral median lethal dose (LDso) value for 2,5-TDA (102 mg/kg) in rats was lower than the oral LDso values for o- (660 and 810 mg/kg) and m-TDA (270 and 300 mg/kg) mixtures. In mice, the i.p. LDso values for 2,3-TDA (286 mg/kg) and a m-TDA mixture (240 mg/kg) were similar. Likewise, the rabbit dermal LD50 value for a o-TDA mixture (1,120 mg/kg) was similar to the rat dermal LD50 value for a m-TDA mixture (1,200 mg/kg). Central nervous system depression and methemoglobinemia were associated with high-dose, acute TDA toxicity in animals (WHO. 1987). SAR evaluations have suggested increased chemical reactivity for o- andsubstituted aromatic amines such as 3,4-, 2,3-, and 2,5-TDA based on their oxidation potential into reactive qui nones that can interact with glutathione to produce reactive oxygen species (ROS) (Baiot et at.. 2010). In contrast, /w-subsituted aromatic amines such as 2,4- and 2,6-TDA are less likely to 33 3,4-T oluenediamine ------- EPA/690/R-21/003F form qui nones and are therefore expected to have decreased chemical reactivity (Baiot et al.. 2010). The o- andp- substituents were also associated with enhanced, acute aquatic toxicity compared to m- substituents (Baiot et al.. 2010). No experimental data was found to evaluate potential differences in chemical reactivity for the TDA isomers. The available evidence in animals shows consistency with respect to toxicity targets (primarily liver and R/D effects) among this group of compounds and although potency differences are apparent in some cases, there is no clear pattern with respect to the position of the amino groups. In summary, limited toxicity data for 3,4-TDA from mixture studies reveals similarities in acute toxicity potency and R/D outcomes between the target and analogues. As such, the candidate analogues are considered suitable analogues for 3,4-TDA on the basis of toxicity similarity comparisons. 34 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Role Target Analogue Analogue Analogue Analogue DTXSID 9024930 4027494 4020402 6029123 4027319 Structure Repeated-dose toxicity—short-term and subchronic studies Effects In a 5-d study in rats 0n = 13), exposure to 3,4-TDA (97% purity) at a dose of 1,000 mg/kg-d for 5 d resulted in 7/13 deaths; 6 animals had grossly observed perforated duodenal ulcers (refer to Table 5 in the main document for additional details). NA In a 14-d study in female mice (n = 6-8/group) exposed to 25, 50, or 100 mg/kg-d of 2,4-TDA (98.4% purity), increased B cells in spleens, and lower spleen weights were observed at >25 mg/kg-d and changes in hepatic enzymes in serum and elevated liver weights were observed at 100 mg/kg-d. Unclear whether organ-weight changes were absolute and/or relative. A 7-wk study exposed rats (n = 5/sex/group) to 0, 250, 500, 1,000, 2,000, or 3,000 ppm and mice Groups of 15 male and female rats were gavaged with doses of 0, 2.5, 5, 10, or 20 mg/kg-d 2,5-TDA sulfate (99.7% purity) for 13 weeks. Increased serum AST in females at 5 mg/kg-d (3 mg/kg-d as free base), increased urine with decrease in specific gravity at >10 mg/kg-d (6 mg/kg-d as free base) and abnormally shaped pituitary glands at 20 mg/kg-d (11 mg/kg-d as free base) were observed (refer to the "Noncancer Oral Toxicity Values" section below for additional details). A 91-d dietary exposure study in rats (n = 10/sex/group) dosed with 0, 100, 300, 1,000, 3,000, or 10,000 ppm of 2,6-TDA dihydrochloride (>99% purity). Thyroid hyperplasia in males and decreased body weight (both sexes) were observed at 192 mg/kg-d (as free base); thyroid hyperplasia was observed in females at 767 mg/kg-d (highest dose as free base). Other effects observed at the high dose (692 mg/kg-d in males and 767 mg/kg-d in females as free base) included thyroid enlargement; 35 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Continued: Continued: Continued: Continued: 0n = 5/sex/group) to 0, 100, 200, 300, 500, 700, or 1,000 ppm of 2,4-TDA (>99.9% purity) via the diet. Decreased body weights, elevated hematopoiesis, and histopathological liver changes were observed in rats at 1,000 ppm (~75 mg/kg-d). The same diet resulted only in decreased body weights in mice. Continued: In an accompanying range-finding study in rats (n = 10/sex/group) gavaged withO, 7.5, 15, 30, or 60 mg/kg-d of 2,5-TDA sulfate, variations in serum CPK, AST, and LDH and increased absolute and relative liver weight were seen at >30 mg/kg-d (20 mg/kg-d as free base). Continued: darkening of spleen, lymph nodes, liver, kidney, adrenals, and nasal turbinates; histopathological lesions in the kidney (nephrosis) and bone marrow (hyperplasia); and death. In the companion mouse study, reduced body weight was also observed; however, exposure levels were unclear. No pathological changes were noted. Sources Solve (1973) NA Bums et al. (1994) and NCI (1979) as cited in Criteria Grouo for Occupational Hill (1997. 1994) as cited in U.S. EPA (2013) NTP (1980) as cited in U.S. EPA (2005b) Standards (2001) Repeated-dose toxicity—chronic studies Effects NA NA A 2-yr NTP bioassay exposed groups of 20-50 rats (TWA doses of 0, 79, or 171-176 ppm) and mice (0, 100, or 200 doses) to 2,4-TDA (>99.9% purity). Reduced body weight and histopathological changes in the liver (lesions ranged from cellular alterations to severe In a 78-wk cancer bioassay, rats and mice were exposed to 2,5-TDA toluene sulfate (>99% purity) at TWA concentrations of 0.06 or 0.2% and 0.06 or 0.1% in the diet, respectively (n = 50/species/sex/group); no exposure-related changes in clinical signs, survival, In a 103-wk study in rats (,n = 50/sex/group) exposed to 0, 250, or 500 ppm of 2,6-TDA dihydrochloride (>99%) in the diet, no adverse effects were reported up to 500 ppm (25 and 30 mg/kg-d in males and females, respectively as free base). 36 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Continued: Continued: Continued: Continued: degenerative changes in rats and hyperplasia in mice) were observed in exposed animals [at doses ~>6 mg/kg-d in rats and >17 mg/kg-d in mice estimated for this analysis usine U.S. EPA (1988) default values for B W and food intake]. Decreased survival, kidney histopathology (chronic renal disease) and secondary hyperthyroidism associated with renal disease were also reported in rats at same doses. Continued: growth, or non-neoplastic histology were observed up to the highest doses tested (~95 mg/kg-d as free base). Continued: In the companion mouse study (n = 50/sex/group) with exposures to 0, 50, or 100 ppm, no adverse effects were observed at doses up to 100 ppm 2,6-TDA dihydrochloride (10 mg/kg-d for both males and females as free base). Source NA NA NCI (1979) as cited in Criteria Grout) for NTP (1978) as cited in U.S. EPA (2013) NTP (1980) as cited inU.S. EPA (2005b) Occupational Standards (2001) and WHO (1987) 37 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Reproductive/developmental studies Effects An R/D screen in rats (n = 10/sex/group) exposed to a 45:50 mixture of 2,3- and 3,4-TDA (0, 10, 50, or 250 mg/kg-d doses) reported clinical signs, decreased food consumption and body weight, decreased number of spermatids/g testis, decreased number of implantation sites, increased post implantation loss, decreased litter size, and decreased pup viability at 250 mg/kg-d. Gestational exposure studies in pregnant rats (n = 22/group) and rabbits (n = 15/group) with exposure to a 40:60 mixture of 2,3- and 3,4-TDA (0, 10, 30, 100, or 300 mg/kg-d and 0, 3, 10, 30, or 100 mg/kg-d for rats and rabbits, respectively) reported increased incidences of skeletal variations at >100 mg/kg-d and reduced maternal body-weight gain and reduced fetal weight at 300 mg/kg-d in rats. Reduced maternal body-weight gain, reduced fetal weight, increased number of resorption sites, and decreased numbers of live fetuses/litter were observed at 100 mg/kg-d in rabbits. Refer to the "Reproductive/Developmental Studies of TDA Mixtures" section in the main document for additional study details. Three studies evaluated male reproductive effects in rats (n = 8-10/group) after a 10-wk exposure in the diet containing 0, 0.01, or 0.03% 2,4-TDA (98% purity); they reported decreased fertility and inhibition of sperm production, altered serum reproductive hormones, and histological changes in the reproductive organs at the highest exposure level (-15 mg/kg-d). Gestational exposure studies in animals (n = 16-30/group) gavaged with 2,5-TDA sulfate (purity unspecified) reported decreased maternal body weight at >50 mg/kg-d (28 mg/kg-d as free base) and increased resorptions at 80 mg/kg-d in rats (44 mg/kg-d as free base). Increased maternal and neonatal mortality were observed at 160 mg/kg-d in mice (88 mg/kg-d as free base), and no effects were observed in rabbits at doses up to 50 mg/kg-d (28 mg/kg-d as free base). Exposure doses were 0, 10, 50, or 80 mg/kg-d in rats (GD 6-15), 0, 10,35, or 50 mg/kg-d in rabbits (GD 6-18) and 160 mg/kg-d in mice (GD 8-12). A gestational exposure study (GDs 6-15) in rats (number of animals and 2,6-TDA purity not specified) gavaged with 0, 10, 30, 100, or 300 mg/kg-d showed increased incidence of hemorrhagic abdomens at >30 mg/kg-d and skeletal variations at >100 mg/kg-d following exposure to 2,6-TDA, with reduced maternal weight gain at 300 mg/kg-d. An accompanying study in rabbits (GDs 6-18) exposed to 0, 3, 10, 30, or 100 mg/kg-d showed reduced maternal weight, increased resorptions, decreased fetal weight, and decreased neonatal survival following gavage exposure to 2,6-TDA at 100 mg/kg-d. 38 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Continued: Continued: Continued: Continued: A 2-generation reproductive toxicity study in rats (n = 24/sex/group) exposed to 0, 5, 15, or 45 mg/kg-day 2,5-TDA sulfate (98.2% purity) reported no effects in clinical signs, body-weight gain, food consumption, male and female fertility or fetal growth and survival up to the highest dose (~25 mg/kg-d as free base). Continued: Source BASF (2010); Becci et al. (1983) Yarma et al. (1988); Thy sen et al. (1985a) and (1985b) as Bornatowicz (1986). Osterburg (1982). Seidenberg Knickerbocker et al. (1980) as cited in WHO (1987) cited in Criteria Grout) for Occupational Standards (2001) et al. (1986). and Kavlock et al. (1987) as cited in U.S. EPA (2013) Health reference values—subchronic POD (mg/kg-d) NA NA NA 2.5 (as 2,5-TDA sulfate); 1.4 (as free base estimated for this assessment) 62 (as free base) POD type NA NA NA NOAEL NO AFT UFC NA NA NA 1,000 (UFa, UFd, UFh) 1,000 (UFa, UFd, UFh) p-RfD (mg/kg-d) NA NA NA 3 x 10 3 (as 2,5-TDA sulfate); 2 / 10 3 (as free base)3 Note: screening value owing to use of secondary source. 6 x 10 2 (as free base) 39 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Critical effects NA NA NA Increased serum AST in females. Thyroid hyperplasia (males) and decreased body weight (both sexes). Species NA NA NA Rat Rat Duration NA NA NA 13 wk (91 d) 91 d Route (method) NA NA NA Oral (gavage) Oral (diet) Source NA NA NA Hill (1997) as cited in U.S. EPA (2013) NTP (1980) as cited in U.S. EPA (2005b) Health reference values—chronic POD (mg/kg-d) NA NA NA 2.5 (as 2,5-TDA sulfate); 1.4 (as free base estimated for this assessment) 25 (as free base) POD type NA NA NA NOAEL NOAEL UFC NA NA NA 10,000 (UFa, UFd, UFh, UFs) 1,000 (UFa, UFd, UFh) p-RfD (mg/kg-d) NA NA NA 3 x 10"4 (as 2,5-TDA sulfate); 2 / 10 4 (as free base)3 Note: screening value owing to the use of secondary source andUFc >3,000. 3 x 10 2 (as free base) Critical effects NA NA NA Increased serum AST in females. None Species NA NA NA Rat Rat Duration NA NA NA 13 wk 103 wk 40 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,4-Toluenediamine CASRN 95-80-7 2,5-Toluenediamine CASRN 95-70-5 2,6-Toluenediamine CASRN 823-40-5 Route (method) NA NA NA Oral (gavage) Oral (diet) Source NA NA NA Hill (1997) as cited in U.S. EPA (2013) NTP (1980) as cited in U.S. EPA (2005b) aThe screening p-RfD values for 2,5-TDA as free base were calculated as follows: p-RfD for 2,5-TDA sulfate x (MW of 2,5-TDA as free base [122.17] MW of 2,5-TDA sulfate [220.25] = 0.55) (U.S. EPA. 20131. AST = aspartate aminotransferase; BW = body weight; CPK = creatine phosphokinase; GD = gestation day; LDH = lactate dehydrogenase; MW = molecular weight; NA = not applicable; NOAEL = no-observed-adverse-effect level; NTP = National Toxicology Program; POD = point of departure; p-RfD = provisional reference dose; R/D = reproductive/developmental; TDA = toluenediamine; TWA = time-weighted average; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFd = database uncertainty factor; UFH = intraspecies uncertainty factor; UFS = subchronic-to-chronic uncertainty factor. 41 3,4-T oluenediamine ------- EPA/690/R-21/003F Table A-6. Comparison of Available Acute Lethality Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues Parameter 3,4-Toluenediamine CASRN 496-72-0 2,3-Toluenediamine CASRN 2687-25-4 2,5-Toluenediamine CASRN 95-70-5 2,4-Toluenediamine CASRN 95-80-7 2,6-Toluenediamine CASRN 823-40-5 Role Target Analogue Analogue Analogue Analogue DTXSID 9024930 4027494 6029123 4020402 4027319 Structure Rat oral LD50 (mg/kg) 660 (mix of 2,3- and 3,4-); 810 (mix of 2,3- and 3,4-) 102 270 and 300 (mix of 2,4- and 2,6-) Mouse oral LD50 (mg/kg) NV NV 350 (mix of 2,4- and 2,6-) Rat i.p. LD5o (mg/kg) NV NV 230 (mix of 2,4- and 2,6-) 325 (2,4-TDA technical grade) Mouse i.p. LD5o (mg/kg) NV 286 NV 240 (mix of 2,4- and 2,6-) 90-480 (2,4-TDA technical grade) Rabbit dermal LD50 (mg/kg) 1,120 (mixof2,3-and3,4-) NV NV Rat dermal LD50 (mg/kg) NV NV 1,200 (mix of 2,4- and 2,6-) Source Air Products and Chemicals (1976): Carpenter et al. (1974): NLM (2019) NLM (2019) Izmerov et al. (1982). Weisbrod and Stephan (1983). Grantham et al. (1979). and Weisburger et al. (1978) as cited in WHO (1987) i.p. = intraperitoneal; LD50 = median lethal dose; NV = not available; TDA = toluenediamine. 42 3,4-T oluenediamine ------- EPA/690/R-21/003F Weight-of-Evidence Approach A WOE approach is used to evaluate information from candidate analogues as described by Wang et al. (2012). Commonalities in structural/physicochemical properties, toxicokinetics, metabolism, toxicity, or mode of action (MOA) between candidate analogues and chemical(s) of concern are identified. Emphasis is given to toxicological and/or toxicokinetic similarity over structural similarity. Analogues are excluded if they do not have commonality or demonstrate significantly different physicochemical properties and toxicokinetic profiles that set them apart from the pool of analogues and/or chemical(s) of concern. From the remaining analogues, the most appropriate analogue (most biologically or toxicologically relevant analogue chemical) with the highest structural similarity and/or most conservative toxicity value is selected. Oral 2,5- and 2,6-TDA were identified as structural analogues of 3,4-TDA with available noncancer oral toxicity values. Two additional structural analogues were included in the read-across analysis, 2,3- and 2,4-TDA, to provide information on the potential influence of the position of the amino groups on toxicity. The analogues share a basic structure with the target compound (a benzene ring, two amino groups, and a methyl group, differing only in the position of the amino functional groups) and have similar physicochemical properties (i.e., water solubility, log Kow, volatility, etc.; see Table A-l) important for bioavailability. 3,4-TDA and its analogues also showed similar SA predictions for repeated-dose toxicity and R/D endpoints (see Table A-2). Evidence from oral and i.p. exposure studies in rodents suggests that the TDA analogues are predominantly metabolized via acetylation of amino groups, ring hydroxylation, and potential oxidation of methyl groups (see Table A-3). A comparative analysis of metabolite predictions across different software tools revealed a similar metabolic profile for the target compound and analogues and confirmed observations from in vivo studies (see "Metabolic Analogues" section above and Appendix D for more details). Oral exposure studies in animals showed commonalities in target tissues for the TDA analogues, most notably, liver and R/D toxicities (see Table A-5). No adequate toxicity data is available for 3,4-TDA; however, studies evaluating TDA mixtures containing 3,4-TDA suggest similarities between the target and analogues with respect to acute toxicity potency and R/D outcomes (see Tables A-5 and A-6). Similarities in structure, physicochemical properties, SA, and metabolite predictions and limited toxicity data support the suitability of both 2,5- and 2,6-TDA (the two analogues with available toxicity values) as analogues of 3,4-TDA. 2,5-TDA is selected as the most appropriate analogue for deriving screening p-RfDs based on mechanistic considerations and health protectiveness. Although it is unclear how the position of the amino groups could affect the repeated-dose toxicity of TDA compounds, the o- andp- isomers (3,4- and 2,5-TDA, respectively) are expected to have greater chemical reactivity (related to quinone formation) than the m- isomer (2,6-TDA). Furthermore, the POD values for 2,5-TDA (1.4 mg/kg-day for both the subchronic and chronic p-RfDs) are more than an order of magnitude lower than the POD values for 2,6-TDA (62 and 25 mg/kg-day for the subchronic and chronic p-RfDs, respectively). Inhalation None of the candidate analogues have repeated-dose inhalation toxicity values, precluding derivation of screening provisional reference concentrations (p-RfCs). 43 3,4-T oluenediamine ------- EPA/690/R-21/003F NONCANCER ORAL TOXICITY VALUES Derivation of a Screening Subchronic Provisional Reference Dose Based on the overall analogue approach presented in this PPRTV assessment, 2,5-TDA is selected as the most appropriate analogue for 3,4-TDA for deriving a screening subchronic p-RfD. The principal study used for the U.S. EPA screening subchronic p-RfD for 2,5-TDA was a 13-week rat study [Hill (1997) as cited in SCCP (2007) and reported by U.S. EPA (2013)1. U.S. EPA (2013) described the study as follows: Hill (1997, as cited in SCCP, 2007) administered toluene-2,5-diamine sulfate (99.7% pure) via gavage in deionized water to Sprague-Dawley rats (15/sex/dose) at 0, 2.5, 5, 10, or 20 mg/kg-day for 13 weeks. The original report for this study is not available; SCCP briefly described the study. Animals were observed daily for mortality and clinical signs. Body weights andfood intake were recorded weekly. Ophthalmoscopic examinations were performed on all animals before the initiation of treatment and during Week 13. Blood and urine samples were collected during Week 4 and during Week 12 or 13. Following treatment, all animals were sacrificed and necropsied. Organ weights were recorded, and tissues were subjected to microscopic examination. No dose-related changes in mortality, clinical signs, body weights, body-weight gains, or food consumption were reported (data not shown). The researchers did not consider hematological variations (not further described) to be treatment-related. Aspartate aminotransferase (AST) levels were statistically significantly (p < 0.05) increased in females at doses of >5 mg/kg-day (data not shown). Increased urine levels, associated with a statistically (p < 0.05) significant decrease in specific gravity, were observed at >10 mg/kg-day (females) or 20 mg/kg-day (males) (data not shown). Although retinopathy was observed in some animals, a pathology peer review concluded that the incidence of these effects in the treatment groups was similar to the spontaneous incidence for Sprague-Dawley rats. At 20 mg/kg-day, an increased incidence of abnormally shaped pituitary glands was reported. The SCCP (2007) identified a NOAEL of 2.5 mg/kg-day for toluene-2,5-diamine sulfate in this study based on significantly elevated AST levels at 5 mg/kg-day. However, experimental data were not presented in the summary, and the adversity of the reported effects has not been demonstrated (there was no mention of the magnitude or dose-response of the observed change in AST, or corresponding changes in other serum enzymes or liver pathology). The available description of this study lacked information to support independent evaluation of the study. The apparent NOAEL of 2.5 mg/kg-day and LOAEL of 5 mg/kg-day for increased serum AST levels in rats treated with toluene-2,5-diamine sulfate by gavage in water for 13 weeks (Hill, 1997, as cited in SCCP, 2007) can be used as the basis for derivation of screening provisional toxicity values for toluene-2,5-diamine sulfate and toluene-2,5-diamine. Based on available information, this appeared to be the most sensitive endpoint identified in the available studies. The choice of endpoint was supported by the results of the 14-day range-finding study, which reported changes in AST and other clinical chemistry measures at 30 mg/kg-day (Hill, 1994, as cited in SCCP, 2007). 44 3,4-T oluenediamine ------- EPA/690/R-21/003F Reproductive and developmental toxicity studies reported effects only at higher doses (80-160 mg/kg-day) (Kavlock et al., 1987; Seidenburg et al., 1986; Osterberg, 1982a,b, as cited in SCCP, 2007 and reviewed in Pang, 1992). The critical effect identified in the U.S. EPA (2013) assessment for 2,5-TDA was increased serum aspartate aminotransferase (AST) in female rats exposed for 13 weeks (Hill. 1997). A no-observed-adverse-effect level (NOAEL) of 2.5 mg/kg-day for increased AST was selected as the POD in the screening subchronic p-RfD for 2,5-TDA sulfate (U.S. EPA. 2013). The corresponding POD for the free base is calculated by multiplying the POD for the sulfate by the ratio of the molecular weights (MW of 2,5-TDA as free base [122.17] MW of 2,5-TDA sulfate [220.25] = 0.55). The resulting NOAEL of 1.4 mg/kg-day for 2,5-TDA is adopted as the POD for deriving the screening subchronic p-RfD for 3,4-TDA. The NOAEL of 1.4 mg/kg-day is not adjusted for molecular-weight differences between 3,4- and 2,5-TDA (both as free base), because the molecular weights are identical. The NOAEL of 1.4 mg/kg-day is converted to a human equivalent dose (HED) according to current (U.S. EPA. 201 lc) guidance. In Recommended Use of Body Weight3 4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lc). the Agency endorses body-weight scaling to the 3/4 power (i.e., BW3'4) as a default to extrapolate toxicologically equivalent doses of orally administered agents from all laboratory animals to humans for the purpose of deriving an RfD from effects that are not portal-of-entry effects. Following U.S. EPA (2011c) guidance, the POD for increased serum AST in female rats is converted to an HED through the application of a dosimetric adjustment factor (DAF) derived as follows: DAF = (BWa1 4 - BWh14) where DAF = dosimetric adjustment factor BWa = animal body weight BWh = human body weight Using a reference BWa of 0.204 kg for female Sprague Dawley rats in a subchronic study and a reference BWh of 70 kg for humans (U.S. EPA, 1988), the resulting DAF is 0.23. Applying this DAF to the NOAEL of 1.4 mg/kg-day yields a POD (HED) as follows: POD (HED) = NOAEL (mg/kg-day) x DAF = 1.4 mg/kg-day x 0.23 = 0.32 mg/kg-day In deriving a screening p-RfD for 3,4-TDA, a composite uncertainty factor (UFc) of 300 is applied, based on a 3-fold uncertainty factor value for interspecies extrapolation (interspecies uncertainty factor [UFa], reflecting use of a dosimetric adjustment) and 10-fold uncertainty factor values for both intraspecies variability (UFh) and database deficiencies (database uncertainty factor [UFd], reflecting lack of adequate repeated dose toxicity information for 3,4-TDA). The screening subchronic p-RfD for 3,4-TDA is derived as follows: 45 3,4-T oluenediamine ------- EPA/690/R-21/003F Screening Subchronic p-RfD = Analogue POD (HED) UFc = 0.32 mg/kg-day -^300 = 1 x 10"3 mg/kg-day Table A-7 summarizes the uncertainty factors for the screening subchronic p-RfD for 3,4-TDA. Table A-7. Uncertainty Factors for the Screening Subchronic p-RfD for 3,4-Toluenediamine (CASRN 496-72-0) UF Value Justification UFa 3 A UFa of 3 (100 5) is applied to account for residual uncertainty, including toxicodynamic differences, between rats and humans following 3,4-TDA exposure. The toxicokinetic uncertainty has been accounted for by calculation of an HED through application of a DAF in extrapolating from animals to humans (U.S. EPA. 20110). UFd 10 A UFd of 10 is applied owing to the absence of adequate repeated-dose toxicity studies for 3,4-TDA alone and the use of a read-across approach to derive the screening p-RfD. UFh 10 A UFh of 10 is applied for interindividual variability to account for human-to-human variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and toxicodynamics of 3,4-TDA in humans. UFl 1 A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL. UFS 1 A UFS of 1 is applied because a subchronic study was selected as the principal study. UFC 300 Composite uncertainty factor = UFA x UFD x UFH x UFL x UFS. DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose; TDA = toluenediamine; UF = uncertainty factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor; UFl = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor. Derivation of a Screening Chronic Provisional Reference Dose 2,5-TDA is also selected as the most appropriate analogue for 3,4-TDA for deriving the screening chronic p-RfD. U.S. EPA (2013) used the critical effect of increased AST levels in female rats and associated POD of 2.5 mg/kg-day (HED of 0.32 mg/kg-day estimated for this assessment) identified in the 13-week rat study (Hill, 1997) to derive a screening chronic p-RfD for 2,5-TDA. The principal study and calculation of the POD (HED) is described above. Although a cancer bioassay in rats and mice exposed to 2,5-TDA for 78 weeks was available (N I P. 1978). the U.S. EPA (2013) assessment concluded that the study was inadequate in scope and design for evaluating noncancer oral toxicity based on the following: (1) evaluations were limited to measures of body weight, food consumption, clinical signs, and non-neoplastic histopathology; (2) histopathological examinations were conducted after a lengthy recovery period (28-31 weeks); and (3) treatment was initiated at different times for the low- and high-dose groups (~11 months apart). 46 3,4-T oluenediamine ------- EPA/690/R-21/003F In deriving the screening chronic p-RfD for 3,4-TDA, the POD (HED) of 0.32 mg/kg-day from the 13-week rat study with 2,5-TDA is selected, applying an additional uncertainty factor of 10 to account for increased uncertainty associated with extrapolating from a subchronic to a chronic exposure (UFs). A UFc of 3,000 was derived, reflecting a 3-fold UFa, and 10-fold uncertainty factor values for UFh, UFs, and UFd. Finally, the screening chronic p-RfD for 3,4-TDA is derived as follows: Screening Chronic p-RfD = Analogue POD (HED) UFc = 0.32 mg/kg-day ^ 3,000 = 1 x 10"4 mg/kg-day Table A-8 summarizes the uncertainty factors for the screening chronic p-RfD for 3,4-TDA. Table A-8. Uncertainty Factors for the Screening Chronic p-RfD for 3,4-Toleuenediamine (CASRN 496-72-0) UF Value Justification UFa 3 A UFa of 3 (10°5) is applied to account for residual uncertainty, including toxicodynamic differences, between rats and humans following 3,4-TDA exposure. The toxicokinetic uncertainty has been accounted for by calculation of an HED through application of a DAF in extrapolating from animals to humans (U.S. EPA. 201 lc). UFd 10 A UFd of 10 is applied owing to the absence of adequate repeated-dose toxicity studies for 3,4-TDA alone and the use of a read-across approach to derive the screening p-RfD. UFh 10 A UFh of 10 is applied for interindividual variability to account for human-to-human variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and toxicodynamics of 3,4-TDA in humans. UFl 1 A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL. UFS 10 A UFS of 10 is applied because a subchronic study was selected as the principal study. UFC 3,000 Composite uncertainty factor = UFA x UFD x UFH x UFL x UFS. DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose; TDA = toluenediamine; UF = uncertainty factor; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor; UFl = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor. 47 3,4-T oluenediamine ------- EPA 690 R-21 003F APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING EVALUATION OF POTENTIAL CARCINOGENICITY For reasons noted in the main Provisional Peer-Reviewed Toxicity Value (PPRTV) document, there is inadequate information to assess the carcinogenic potential of 3,4-toluenediamine (3,4-TDA). However, information is available for this chemical which, although insufficient to support a weight-of-evidence (WOE) descriptor and derivation of provisional cancer risk estimates under current guidelines, may be of use to risk assessors. In such cases, the Center for Public Health and Environmental Assessment (CPHEA) summarizes available information in an appendix and develops a "screening evaluation of potential carcinogenicity." Appendices receive the same level of internal and external scientific peer review as the provisional cancer assessments in PPRTVs to ensure their appropriateness within the limitations detailed in the document. Users of the information regarding potential carcinogenicity in this appendix should understand that there could be more uncertainty associated with this evaluation than for the cancer WOE descriptors presented in the body of the assessment. Questions or concerns about the appropriate use of the screening evaluation of potential carcinogenicity should be directed to the CPHEA. The screening evaluation of potential carcinogenicity includes the general steps shown in Figure B-l. The methods for Steps 1 through 8 apply to any target chemical and are described in this appendix. Chemical-specific data for all steps in this process are summarized in Appendix C. STEP 1 Use automated tools to identify an initial list of structural analogues with genotoxicity and/or carcinogenicity data STEP 2 Apply expert judgment to refine the list of analogues (based on physicochemical properties, ADME, and mechanisms of toxicity) STEP 3 Compare experimental genotoxicity data (if any) for the target and analogue compounds STEP 4 Summarize ADME data from targeted literature searches. Identify metabolites likely related to genotoxic and/or carcinogenic alerts STEP 5 Summarize cancer data and MOA information for analogues. STEP 8 Assign qualitative level of concern for carcinogenicity based on evidence integration (potential concern or inadequate information) Use computational tools to identify common structural alerts and SAR predictions for genotoxicity and/or carcinogenicity Integrate evidence streams Figure B-l. Steps Used in the Screening Evaluation of Potential Carcinogenicity 48 3,4-T oluenediamine ------- EPA 690 R-21 003F STEP 1. USE OF AUTOMATED TOOLS TO IDENTIFY STRUCTURAL ANALOGUES WITH CARCINOGENICITY AND/OR GENOTOXICITY DATA ChemACE Clustering The U.S. EPA's Chemical Assessment Clustering Engine [ChemACE; U.S. EPA (2011a)1 is an automated tool that groups (or clusters) a user-defined list of chemicals based on chemical structure fragments. The methodology used to develop ChemACE was derived from U.S. EPA's Analog Identification Methodology (AIM) tool, which identifies structural analogues for a chemical based on common structural fragments. ChemACE uses the AIM structural fragment recognition approach for analogue identification and applies advanced queries and user-defined rules to create the chemical clusters. The ChemACE cluster outputs are available in several formats and layouts (i.e., Microsoft Excel, Adobe PDF) to allow rapid evaluation of structures, properties, mechanisms, and other parameters which are customizable based on an individual user's needs. ChemACE clustering has been successfully used with chemical inventories for identifying trends within a series of structurally similar chemicals, demonstrating structural diversity in a chemical inventory, and detecting structural analogues to fill data gaps and/or perform read-across. For this project, ChemACE is used to identify potential structural analogues of the target compound that have available carcinogenicity assessments and/or carcinogenicity data. An overview of the ChemACE process in shown in Figure B-2. Create and curate an inventory of chemicals with carcinogenicity assessments and/or cancer data Cluster the target compound with the chemical inventory using ChemACE Identify structural analogues for the target compound from specific ChemACE clusters Figure B-2. Overview of ChemACE Clustering Process The chemical inventory was populated with chemicals from the following databases and lists: • Carcinogenic Potency Database [CPDB; CPDB (2011)1 • Agents classified by the International Agency for Research on Cancer (IARC) monographs (IARC. 2018) • National Toxicology Program (NTP) Report on Carcinogens [ROC; NTP (2016a)1 • NTP technical reports (NTP. 2017) • Integrated Risk Information System (IRIS) carcinogens (U.S. EPA, 2017) • California EPA (CalEPA) Prop 65 list (CalEPA. 2017) • European Chemicals Agency (ECHA) carcinogenicity data available in the Organisation for Economic Co-operation and Development (OECD) Quantitative Structure-Activity Relationship (QSAR) Toolbox (OECD. 2017) • PPRTVs for Superfund (U.S. EPA. 2020b) In total, 2,123 distinct substances were identified from the sources above. For the purpose of ChemACE clustering, each individual substance needed to meet the following criteria: 49 3,4-T oluenediamine ------- EPA/690/R-21/003F 1) Substance is not a polymer, metal, inorganic, or complex salt because ChemACE is not designed to accommodate these substances; 2) Substance has a CASRN or unambiguous chemical identification; and 3) Substance has a unique Simplified Molecular Input Line Entry System (SMILES) notation (encoded molecular structure format used in ChemACE) that can be identified from one of these sources: a. Syracuse Research Corporation (SRC) and Distributed Structure-Searchable Toxicity (DSSTox) database lists of known SMILES associated with unique CASRNs (the combined lists contained >200,000 SMILES) or b. ChemlDplus, U.S. EPA CompTox Chemicals Dashboard, or internet searches. Of the initial list of 2,123 substances, 201 were removed because they did not meet one of the first two criteria, and 155 were removed because they did not meet the third. The final inventory of substances contained 1,767 unique compounds. Two separate ChemACE approaches were compared for clustering of the chemical inventory. The restrictive clustering approach, in which all compounds in a cluster contain all of the same fragments and no different fragments, resulted in 208 clusters. The less restrictive approach included the following rules for remapping the chemical inventory: • treat adjacent halogens as equivalent, allowing fluorine (F) to be substituted for chlorine (CI), CI for bromine (Br), Br for iodine (I); • allow methyl, methylene, and methane to be equivalent; • allow primary, secondary, and tertiary amines to be equivalent; and • exclude aromatic thiols (removes thiols from consideration). Clustering using the less restrictive approach (Pass 2) resulted in 284 clusters. ChemACE results for clustering of the target chemical within the clusters of the chemical inventory are described in Appendix C. Analogue Searches in the OECD QSAR Toolbox (Dice Method) The OECD QSAR Toolbox (Version 4.1) is used to search for additional structural analogues of the target compound. There are several structural similarity score equations available in the Toolbox (Dice, Tanimoto, Kulczynski-2, Ochiai/Cosine, and Yule). Dice is considered the default equation. The specific options that are selected for the performance of this search include a comparison of molecular features (atom-centered fragments) and atom characteristics (atom type, count hydrogens attached, and hybridization). Chemicals identified in these similarity searches are selected if their similarity scores exceeded 50%. The OECD QSAR Toolbox Profiler is used to identify those structural analogues from the Dice search that have carcinogenicity and/or genotoxicity data. Nine databases in the OECD QSAR Toolbox (Version 4.1) provide data for carcinogenicity or genotoxicity (see Table B-l). Analogue search results for the target chemical are described in Appendix C. 50 3,4-T oluenediamine ------- EPA/690/R-21/003F Table B-l. Databases Providing Carcinogenicity and Genotoxicity Data in the OECD QSAR Toolbox (Version 4.1) Database Name Toolbox Database Description3 CPDB The CPDB provides access to bioassay literature with qualitative and quantitative analysis of published experiments from the general literature (through 2001) and from the NCI/NTP (through 2004). Reported results include bioassays in rats, mice, hamsters, dogs, and nonhuman primates. A calculated carcinogenic potency (TD50) is provided to standardize quantitative measures for comparison across chemicals. The CPDB contains 1,531 chemicals and 3,501 data points. ISSCAN The ISSCAN database provides information on carcinogenicity bioassays in rats and mice reported in sources including NTP, CPDB, CCRIS, and IARC. This database reports a carcinogenicity TD5o. There are 1,149 chemicals and 4,518 data points included in the ISSCAN database. ECHA CHEM The ECHA CHEM database provides information on chemicals manufactured or imported in Europe from registration dossiers submitted by companies to ECHA to comply with the REACH Regulation framework. The ECHA database includes 9,229 chemicals with almost 430,000 data points for a variety of endpoints including carcinogenicity and genotoxicity. ECHA does not verily the information provided by the submitters. ECVAM Genotoxicity and Carcinogenicity The ECVAM Genotoxicity and Carcinogenicity database provides genotoxicity and carcinogenicity data for Ames positive chemicals in a harmonized format. ECVAM contains in vitro and in vivo bacteria mutagenicity, carcinogenicity, CA, CA/aneuploidy, DNA damage, DNA damage and repair, mammalian culture cell mutagenicity, and rodent gene mutation data for 744 chemicals and 9,186 data points. ISSCTA ISSCTA provides results of four types of in vitro cell transformation assays including Syrian hamster embryo cells, mouse BALB/c 3T3, mouse C3H/10T1/2 and mouse Bhas 42 assays that inform nongenotoxic carcinogenicity. ISSCTA consists of 352 chemicals and 760 data points. Bacterial mutagenicity ISSSTY The ISSSTY database provides data on in vitro Salmonella typhimurium Ames test mutagenicity (positive and negative) taken from the CCRIS database in TOXNET. The ISSSTY database provides data for 7,367 chemicals and 41,634 data points. Genotoxicity OASIS The Genotoxicity OASIS database provides experimental results for mutagenicity results from "Ames tests (with and without metabolic activation), in vitro chromosomal aberrations and MN and MLA evaluated in vivo and in vitro, respectively." The Genotoxicity OASIS database consists of 7,920 chemicals with 29,940 data points from 7 sources. 51 3,4-T oluenediamine ------- EPA/690/R-21/003F Table B-l. Databases Providing Carcinogenicity and Genotoxicity Data in the OECD QSAR Toolbox (Version 4.1) Database Name Toolbox Database Description3 Micronucleus OASIS The Micronucleus OASIS database provides experimental results for in vivo bone marrow and peripheral blood MNT CA studies in blood erythrocytes, bone marrow cells, and polychromatic erythrocytes of humans, mice, rabbits, and rats for 557 chemicals. ISSMIC The ISSMIC database provides data on the results of in vivo MN mutagenicity assay to detect CAs in bone marrow cells, peripheral blood cells, and splenocytes in mice and rats. Sources include TOXNET, NTP, and the Leadscope FDA CRADA Toxicity Database. The ISSMIC database includes data for 563 chemicals and 1,022 data points. 'Descriptions were obtained from the OECD QSAR Toolbox documentation | Version 4.1; OECD (2017)1. CA = chromosomal aberration; CCRIS = Chemical Carcinogenesis Research Information System; CPBD = Carcinogenic Potency Database; CRADA = cooperative research and development agreement; DNA = deoxyribonucleic acid; ECHA = European Chemicals Agency; ECVAM = European Centre for the Validation of Alternative Methods; FDA = Food and Drug Administration; IARC = International Agency for Research on Cancer; ISSCAN = Istituto Superiore di Sanita Chemical Carcinogen; ISSCTA = Istituto Superiore di Sanita Cell Transformation Assay; ISSMIC = Istituto Superiore di Sanita Micronucleus; ISSSTY = Istituto Superiore di Sanita Salmonella typhimurium; ML A = mouse lymphoma gene mutation assay; MN = micronuclei; MNT = micronucleus test; NCI = National Cancer Institute; NTP = National Toxicology Program; OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity relationship; REACH = Registration, Evaluation, Authorization and Restriction of Chemicals; TD5o = median toxic dose. STEPS 2-5. ANALOGUE REFINEMENT AND SUMMARY OF EXPERIMENTAL DATA FOR GENOTOXICITY, TOXICOKINETICS, CARCINOGENICITY, AND MODE OF ACTION The outcome of the Step 1 analogue identification process using ChemACE and the OECD QSAR Toolbox is an initial list of structural analogues with genotoxicity and/or carcinogenicity data. Expert judgment is applied in Step 2 to refine the list of analogues based on physicochemical properties; absorption, distribution, metabolism, and excretion (ADME); and mechanisms of toxicity. The analogue refinement process is chemical-specific and is described in Appendix C. Steps 3, 4, and 5 (summary of experimental data for genotoxicity, toxicokinetics, carcinogenicity, and mode of action [MOA]) are also chemical specific (see Appendix C for further details). STEP 6. STRUCTURAL ALERTS AND STRUCTURE-ACTIVITY RELATIONSHIP PREDICTIONS FOR 3,4-TDA AND ANALOGUES Structural alerts (SAs) and predictions for genotoxicity and carcinogenicity are identified using six freely available structure-based tools (described in Table B-2). 52 3,4-T oluenediamine ------- EPA/690/R-21/003F Table B-2. Tools Used to Identify SAs and Predict Carcinogenicity and Genotoxicity Name Description3 OECD QSAR Toolbox (Version 4.1) Seven OECD QSAR Toolbox profiling methods were used, including: • Carcinogenicity (genotox and nongenotox) alerts by ISS (Version 2.3); updated version of the module originally implemented in Toxtree. It is a decision tree for estimating carcinogenicity, based on 55 SAs (35 from the Toxtree module and 20 newly derived). • DNA alerts for Ames by OASIS (Version 1.4); based on the Ames mutagenicity TIMES model, uses 85 SAs responsible for interaction of chemicals with DNA. • DNA alerts for CA and MNT by OASIS (Version 1.1); based on the DNA reactivity of the CAs TIMES model, uses 85 SAs for interaction of chemicals with DNA. • In vitro mutagenicity (Ames test) alerts by ISS (Version 2.3); based on the Mutagenicity module in Toxtree. ISS is a decision tree for estimating in vitro (Ames test) mutagenicity, based on a list of 43 SAs relevant for the investigation of chemical genotoxicity via DNA adduct formation. • In vivo mutagenicity (MN) alerts by ISS (Version 2.3); based on the ToxMic rulebase in Toxtree. The rulebase has 35 SAs for in vivo MN assay in rodents. • OncoLogic Primary Classification (Version 4.0); "developed by LMC and OECD to mimic the structural criteria of chemical classes of potential carcinogens covered by the U.S. EPA's OncoLogic Cancer Expert System for Predicting the Carcinogenicity Potential" for categorization purposes only, not for predicting carcinogenicity. It is applicable to organic chemicals with at least one of the 48 alerts specified. • Protein binding alerts for CAs by OASIS (Version 1.3); based on 33 SAs for interactions with specific proteins including topoisomerases, cellular protein adducts, etc. OncoLogic (Version 7) OncoLogic is a tool for predicting the potential carcinogenicity of chemicals based on the application of rules for SAR analysis, developed by experts. Results may range from "low" to "high" concern level. ToxAlerts ToxAlerts is a platform for screening chemical compounds against SAs, developed as an extension to the OCHEM system (https://ochem.eu). Only "approved alerts" were selected, which corresponds to a moderator approved the submitted data. A list of the ToxAlerts found for the chemicals screened in the preliminary batch is below: • Genotoxic carcinogenicity, mutagenicity o Aliphatic halide (general) o Aliphatic halide (specific) o Aliphatic halogens o Aromatic amine (general) o Aromatic amine (specific) o Aromatic amines o Aromatic and aliphatic substituted primary alkyl halides o Aromatic nitro (general) o Aromatic nitro (specific) o Aromatic nitro groups o Nitroarenes o Nitro-aromatic o Primary and secondary aromatic amines o Primary aromatic amine, hydroxyl amine, and its derived esters or amine generating group • Nongenotoxic carcinogenicity o Aliphatic halogens 53 3,4-T oluenediamine ------- EPA/690/R-21/003F Table B-2. Tools Used to Identify SAs and Predict Carcinogenicity and Genotoxicity Name Description3 ToxRead (Version 0.9) ToxRead is a tool designed to assist in making read-across evaluations reproducible. SAs for mutagenicity are extracted from similar molecules with available experimental data in its database. Five similar compounds were selected for this project. The rule sets included: • Benigni/Bossa as available in Toxtree (Version 1) • S ARpy rules extracted by Politecnico di Milano, with the automatic tool SARpy • IRFMN rules extracted by human experts at Istituto di Ricerche Farmacologiche Mario Negri • CRS4 rules extracted by CRS4 Institute with automatic tools Toxtree (Version 2.6.13) Toxtree estimates toxic hazard by applying a decision tree approach. Chemicals were queried in Toxtree using the Benigni/Bossa rulebase for mutagenicity and carcinogenicity. If a potential carcinogenic alert based on any QSAR model or if any SA for genotoxic and nongenotoxic carcinogenicity was reported, then the prediction was recorded as a positive carcinogenicity prediction for the test chemical. The output definitions from the tool manual are listed below: • SA for genotoxic carcinogenicity (recognizes the presence of one of more SAs and specifies a genotoxic mechanism) • SA for nongenotoxic carcinogenicity (recognizes the presence of one or more SAs, and specifies a nongenotoxic mechanism) • Potential Salmonella typhimurium TA100 mutagen based on QSAR • Unlikely to be a S. typhimurium TA100 mutagen based on QSAR • Potential carcinogen based on QSAR (assigned according to the output of QSAR8 aromatic amines) • Unlikely to be a carcinogen based on QSAR (assigned according to the output of QSAR8 aromatic amines) • Negative for genotoxic carcinogenicity (no alert for genotoxic carcinogenicity) • Negative for nongenotoxic carcinogenicity (no alert for nongenotoxic carcinogenicity) 54 3,4-T oluenediamine ------- EPA/690/R-21/003F Table B-2. Tools Used to Identify SAs and Predict Carcinogenicity and Genotoxicity Name Description3 VEGA VEGA applies several QSARs to a given chemical, as described below: • Mutagenicity (Ames test) CONSENSUS model: a consensus assessment is performed based on predictions of the VEGA mutagenicity models (CAESAR, SARpy, ISS, and /i-NN) • Mutagenicity (Ames test) model (CAESAR): integrates two models, one is a trained SVM classifier, and the other is for FN removal based on SAs matching • Mutagenicity (Ames test) model (SARpy/IRFMN): rule-based approach with 112 rules for mutagenicity and 93 for nonmutagenicity, extracted with SARpy software from the original training set from the CAESAR model; includes rules for both mutagenicity and nonmutagenicity • Mutagenicity (Ames test) model (ISS): rule-based approach based on the work of Benigni and Bossa (ISS) as implemented in the software Toxtree Version 2.6 • Mutagenicity (Ames test) model (/i-NN/rcad-across): performs a read-across analysis and provides a qualitative prediction of mutagenicity on S. typhimurium (Ames test) • Carcinogenicity model (CAESAR): Counter Propagation Artificial neural network developed using data for carcinogenicity in rats extracted from the CPDB database • Carcinogenicity model (ISS): built implementing the same alerts Benigni and Bossa (ISS) implemented in the software Toxtree 2.6 • Carcinogenicity model (IRFMN/ANTARES): a set of rules (127 SAs), extracted with the SARpy software from a data set of 1,543 chemicals obtained from the carcinogenicity database of European Union-funded project ANT ARES • Carcinogenicity model (IRFMN/ISSCAN-CGX): based on a set of rules (43 SAs) extracted with the SARpy software from a data set of 986 compounds; the data set of carcinogenicity of different species was provided bv Kirkland et al. (2005) aThere is some overlap between the tools. For example, OncoLogic classification is provided by the QS AR Toolbox, but the prediction is available only through OncoLogic, and alerts or decision trees were used in or adapted from several models (e.g., Benigni and Bossa alerts and Toxtree decision tree) (OECD. 20171. ANT ARES = Alternative Non-Testing Methods Assessed for REACH Substances; CA = chromosomal aberration; CAESAR = Computer Assisted Evaluation of industrial chemical Substances According to Regulations; CONSENSUS = consensus assessment based on multiple models (CAESAR, SARpy, ISS, and &-NN); CRS4 = Center for Advanced Studies, Research and Development in Sardinia; CPDB = Carcinogenic Potency Database; DNA = deoxyribonucleic acid; FN = false negative; IRFMN = Istituto di Ricerche Farmacologiche Mario Negri; ISS = Istituto Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di Sanita Chemical Carcinogen; /i-NN = ^-nearest neighbor; LMC = Laboratory for Mathematical Chemistry; MN = micronucleus; MNT = micronucleus test; OCHEM = Online Chemical Monitoring Environment; OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity relationship; REACH = Registration, Evaluation, Authorisation and Restriction of Chemicals; SA = structural alert; SAR = structure-activity relationship; SVM = support vector machine; TIMES = The Integrated MARKEL-EFOM System; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture. The tool results for the target and analogue compounds are provided in Appendix C. 55 3,4-T oluenediamine ------- EPA/690/R-21/003F STEP 7. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF 3,4-TDA CARCINOGENICITY Data identified across multiple lines of evidence from Steps 1-6 (outlined above) are integrated to determine the qualitative level of concern for potential carcinogenicity of the target compound (Step 8). In the absence of information supporting carcinogenic portal-of-entry effects, the qualitative level of concern for the target chemical should be considered applicable to all routes of exposure. Evidence integration for the target compound is provided in Appendix C. 56 3,4-T oluenediamine ------- EPA 690 R-21 003F APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL CARCINOGENICITY STEP 1. USE OF AUTOMATED TOOLS TO IDENTIFY STRUCTURAL ANALOGUES WITH CARCINOGENICITY AND/OR GENOTOXICITY DATA U.S. EPA's Chemical Assessment Clustering Engine (ChemACE) clustering was performed as described in Appendix B. The cluster containing 3,4-toluenediamine (3,4-TDA; less restrictive approach; Cluster 71) also contains 2,3-toluenediamine (2,3-TDA; an additional target compound being evaluated in a separate Provisional Peer-Reviewed Toxicity Value [PPRTV] document) and 13 structural analogues. The 15 cluster members all contain a benzene ring substituted with one or more amino groups (-NR2) and one or more methyl groups (-CH3). The methyl groups are present on the ring or the nitrogen substituent (-N(CFb)2) (see Figure C-l). The Organisation for Economic Co-operation and Development (OECD) Quantitative Structure-Activity Relationship (QSAR) Toolbox Profiler was used to identify structural analogues from the Dice analogue search with carcinogenicity and/or genotoxicity data (see Step 1 methods in Appendix B). This process identified an additional 49 compounds to be considered as potential analogues for 3,4-TDA. Refinement of selection of final analogues is described below. STEP 2. ANALOGUE REFINEMENT USING EXPERT JUDGMENT Expert judgment was applied to refine the initial list of 62 potential analogues based on physicochemical properties; absorption, distribution, metabolism, and excretion (ADME); and mechanisms of toxicity. Compounds were considered potential analogues if they had (1) one aromatic ring (benzene) substituted with (2) two unsubstituted amines on the ring, in a meta (m)- or para (p)-substitution pattern, (3) a methyl group on the ring, and (4) no other functional group. Such compounds are similar to the target chemical in all attributes except for the proximity of the two amine substituents to one another on the aromatic ring. The closest analogue structurally for o Amine group O Methyl group o Benzene group Figure C-l. Illustration of Common Fragments in Cluster 71 57 3,4-T oluenediamine ------- EPA/690/R-21/003F 3,4-TDA would be 2,3-TDA because of the similar ortho (o)-substitution pattern; however, 2,3-TDA does not have adequate experimental data for evaluating potential carcinogenicity and was not considered further as an analogue. Simple salts (e.g., hydrochlorides or sulfates) of the m- and /^-substituted diamines are also considered as potential analogues. Of the 62 chemicals identified as potential analogues by ChemACE clustering and the OECD Toolbox analogue selection tool (Dice), 54 were not selected for further review. Common rationales for not selecting these chemicals included the presence of polycyclic aromatics or ring systems other than toluene; lack of two amine substituents; occurrence of functional groups not present in the target chemicals (e.g., phenols, halogens, carboxylic acids); A-alkyl-substituted amines and acetamide derivatives of aromatic amines. In addition, nitro amines and dinitro compounds were not selected. Each of these attributes introduce significant differences in bioavailability, reactivity, and applicable metabolic pathways relative to 3,4-TDA. Additionally, ar-methyl-l,3-benzenediamine (CASRN 25376-45-8) was not selected for further review because it can exist as a mixture of two TDA isomers, in which the location of the methyl on the aromatic ring is not defined. The remaining nine possible analogues for 3,4-TDA are listed in Table C-l. The existence of a cancer risk estimate and/or a weight-of-evidence (WOE) determination for cancer is indicated for each analogue. Compounds are grouped with their respective simple salts, which were identified by Dice only. Salts did not cluster with free acids in ChemACE because it is fragment-based; therefore, salts and free acids have different fragments and will not cluster without special treatment (i.e., modify the Simplified Molecular Input Line Entry System [SMILES] being clustered so that representative free acid structures are entered for salts). The analogue results from Dice are based on SMILES arbitrary target specification (SMARTS) substructure searching, allowing for identification of both free acid and respective salt analogues. 58 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-l. Summary of Cancer Assessment Information for Analogues of 3,4-Toluenediamine (CASRN 496-72-0) Analogue Name (CASRN) Cancer Risk Estimates (if available) WOE Determinations 2,6-TDA (823-40-5)a b 2,6-TDA dihydrochloride (15481-70-6)b None U.S. EPA (2005b)—inadeauate information 2,5-TDA (95-70-5)a b 2,5-TDA dihydrochloride (615-45-2)b 2,5-TDA sulfate (6369-59-1 and 615-50-9)b U.S. EPA (2013)—screening d-OSF U.S. EPA (2013)—suggestive I ARC (1987)—not classifiable 2,4-TDA (95-80-7)a b 2,4-TDA dihydrochloride (636-23-7)b CalEPA (2011a)—OSF. IUR I ARC (1987)—vossiblv NTP (2016b)—reasonably anticipated ¦ '; ] i H i5 A f ?i) j j;j!—kno 11II 2. '-I)iniclh> llvii/ciic-1.4-diamiiic n i()<) None None 2.5-1 )iniclli> llvii/ciic-l .4-diamiiic None None aFound by ChemACE. bFound by Dice. IUR = inhalation unit risk; OSF = oral slope factor; p-OSF = provisional oral slope factor; TDA = toluenediamine; WOE = weight of evidence. 2.3-Dimethylbenzene-l,4-diamine and 2,5-dimethylbenzene-l,4-diamine, which lack cancer risk estimates or WOE determinations (highlighted in gray in Table C-l), were not further considered as potential analogues for the screening evaluation of potential carcinogenicity of 3,4-TDA. Compounds selected for further consideration were 2,4-TDA, 2,5-TDA, and 2,6-TDA and their simple salts. STEP 3. COMPARISON OF THE EXPERIMENTAL GENOTOXICITY DATA FOR 3,4-TDA AND ANALOGUES The available genotoxicity data for 3,4-TDA are described in detail in the "Other Data" section in the main body of this report. Briefly, the data indicate that 3,4-TDA is mutagenic in bacterial systems with metabolic activation; however, evidence for mutation in mammalian cells is equivocal. 3,4-TDA induces cell transformation in mammalian cells at cytotoxic concentrations and also induces micronuclei (MN) and inhibits deoxyribonucleic acid (DNA) synthesis in vivo. A summary of the genotoxicity data for the structural analogues, 2,4-, 2,5-, and 2,6-TDA, is provided below for comparative purposes. 2.4-, 2,5-, and 2,6-TDA are mutagenic to Salmonella typhimurium in the presence of metabolic activation (U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b). Sex-linked recessive mutations were observed in Drosophila melanogaster exposed to 2,4-TDA (ECHA. 2008). However, 2,4-, 2,5-, and 2,6-TDA were generally nonmutagenic to mammalian cells in vitro or in vivo (U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b). 59 3,4-T oluenediamine ------- EPA/690/R-21/003F 2,4-, 2,5-, and 2,6-TDA show evidence of in vitro clastogenicity in mammalian cells, both with and without metabolic activity. Chromosomal aberrations (CAs) were induced by 2,4- and 2,5-TDA, sister chromatid exchanges (SCEs) were induced by 2,4-TDA, and MN were induced by 2,6-TDA (U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b). Induction of MN in bone marrow or hepatocytes was generally not observed following in vivo exposure to 2,4-, 2,5-, or 2,6-TDA. However, weak induction of MN in bone marrow following exposure to 2,4- or 2,6-TDA was reported in some studies (Takasawa et at.. 2013; U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b). The majority of in vitro studies indicate that 2,4-, 2,5-, and 2,6-TDA are capable of damaging mammalian DNA. Results were most consistent with 2,4-TDA, which induced DNA damage and/or unscheduled DNA synthesis (UDS) in human skin fibroblasts, human hepatocytes, and primary rat hepatocytes, and formed DNA adducts in rat hepatocytes and purified calf thymus DNA (ECHA. 2008). 2,5-TDA also induced DNA damage in rat and hamster hepatocytes (U.S. EPA. 2013). UDS was observed in primary cultured human hepatocytes exposed to 2,6-TDA, but not primary rat hepatocytes (U.S. EPA. 2005b). Low levels of covalent binding to DNA were observed for 2,6-TDA (U.S. EPA. 2005b). DNA strand breaks and UDS were consistently reported in rodents following in vivo exposure to 2,4-TDA (ECHA. 2008). but results in rodents exposed to 2,5- or 2,6-TDA were mixed (U.S. EPA. 2013. 2005b). DNA adducts were observed in multiple organs following in vivo exposure to 2,4-, but not 2,6-TDA (ECHA. 2008; U.S. EPA. 2005b). 2,5- and 2,6-TDA induced cell transformation in hamster embryo cells (U.S. EPA. 2013. 2005b). In summary, the available genotoxicity data suggest some commonalities between the target compound and TDA analogues. Like 3,4-TDA, the TDA analogues are mutagenic in bacterial systems with metabolic activation and show some evidence of genotoxicity in mammalian cells, including clastogenic effects and DNA damage under certain conditions. STEP 4. TOXICOKINETICS OF 3,4-TDA AND ANALOGUES The toxicokinetics of 3,4-, 2,4-, 2,5-, and 2,6-TDA are briefly described in Table C-2 (see additional information in Table A-3). Experimental data indicate that 2,4-, 2,5-, and 2,6-TDA are rapidly absorbed following oral exposure and excreted in the urine (see Table A-3). The primary metabolic pathways for 2,4-, 2,5-, and 2,6-TDA include acetylation of the amino groups and ring hydroxylation with some evidence of oxidation of the methyl group (see Table A-3). No toxicokinetic data are available for 3,4-TDA, but similar metabolic pathways are expected for the target compound based on a comparative in silico metabolism analysis (see section on "Metabolic Analogues" in Appendix A and Appendix D for additional details). 60 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-2. Summary of Toxicokinetic Data for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Compound Absorption, Distribution, Excretion Metabolism References 3,4-TDA ND ND NA 2,4-TDA • Rapid and extensive absorption • Wide distribution • Primarily excreted in urine, with small amounts in feces • Primary pathways include acetylation of the amino groups and ring hydroxylation • Primary urinary metabolites: 3-hydroxy- 4-acetylamino-2-aminotoluene, 5-hydroxy-2,4-diaminotoluene, and 5-hydroxy-4-acetylamino-2-aminotoluene Titnchalk et al. (1994); Grantham et al. (1979); Waring and Pheasant (1975) 2,6-TDA • Rapid and extensive absorption • Wide distribution • Primarily excreted in urine, with small amounts in feces • Primary pathways include acetylation of the amino groups and ring hydroxylation • Primary urinary metabolites: 3 -hydroxy-2,6-toluenediamine, 5 -hydroxy -2 -acety lamino -6 -amino toluene, 2-acetylamino-6-aminotoluene, and 2,6-diacetylamino-toluene Cunningham et al. (1989) 2,5-TDA • Rapid and extensive absorption • Wide distribution • Primarily excreted in urine, with small amounts in feces • Primary pathways include acetylation of the amino groups and ring hydroxylation • Primary urinary metabolites: .Y,.V'-diacctyl-tolucnc-2.5-diamine Weaker (2005a). Weaker (2005b). Weaker (2005c) and Charles River Laboratories (2010) as cited in SCCS (2012). pages 50-52 and 56-57 NA = not applicable; ND = no data; TDA = toluenediamine. STEP 5. CARCINOGENICITY OF 3,4-TDA ANALOGUES AND MODE-OF-ACTION DISCUSSION U.S. EPA cancer WOE descriptors for 3,4-TDA and its analogue compounds are shown in Table C-3. As noted in the main PPRTV document, there is inadequate information to assess the carcinogenic potential of 3,4-TDA. The analogue 2,5-TDA is characterized by U.S. EPA as having evidence of carcinogenic potential. Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a). there is "Suggestive Evidence of Carcinogenic Potential" for 2,5-TDA (U.S. EPA. 2013). The U.S. EPA has not assessed the potential carcinogenicity of 2,4-TDA (U.S. EPA. 1991); however, this compound is listed as a carcinogen by CalEPA (201 la), considered possibly carcinogenic to humans by IARC (1987) and reasonably anticipated to be a human carcinogen by NTP (2016a). The U.S. EPA determined that there is "Inadequate Information to Assess Carcinogenic Potential" for 2,6-TDA (U.S. EPA. 2005b). Oral slope factor (OSF) values varied by an order of magnitude, with the highest potency value calculated for 2,4-TDA (4 x 10° [mg/kg-day] ') (CalEPA. 2011b) and the lowest (screening) potency value for 2,5-TDA (1.8 x 10 1 [mg/kg-day] 1 as a sulfate) (U.S. EPA. 2013). Exposure-related increases were observed in liver tumors in male and female rats and female mice, subcutaneous fibromas in male rats, mammary tumors in female rats, and lymphoma in female mice following dietary 2,4-TDA exposure (NTP. 2016a; CalEPA. 2011a; IARC. 1987). Testicular tumors were observed in male rats and lung tumors were observed in female mice following dietary exposure to 2,5-TDA (U.S. EPA. 2013). Potential carcinogenic effects of 61 3,4-T oluenediamine ------- EPA/690/R-21/003F 2,6-TDA were evaluated in rats and mice in 2-year feeding studies (U.S. EPA. 2005b; N I P. 1980). Dose-related trends for increased incidence of hepatocellular carcinomas and islet-cell adenomas of the pancreas were observed in male rats, a slight increase in vascular neoplasm of the spleen and liver and a significant trend in increased lymphomas were observed in male mice, and a significant trend for increased hepatocellular carcinomas was reported in female mice (U.S. HP A. 2005b). The study authors did not consider the neoplastic lesions observed with exposure to 2,6-TDA to be treatment related due to the absence of statistically significant effects in any treatment group compared to controls, but it was unclear whether exposure levels were adequate to assess carcinogenic potential (U.S. HP A. 2005b). The carcinogenic mode of action (MOA) has not been established for 2,4- or 2,5-TDA, although both compounds (along with 2,6-TDA and the target compound, 3,4-TDA) exhibit some evidence of genotoxicity (see "Step 3. Comparison of the Experimental Genotoxicity Data for 3,4-TDA and Analogues" above for more information). 62 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-3. Comparison of Available Oral Carcinogenicity Data for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Type of Data 3,4-TDA CASRN 496-72-0 2,4-TDA CASRN 95-80-7 2,6-TDA CASRN 823-40-5 2,5-TDA CASRN 95-70-5 Role Target Analogue Analogue Analogue Structure nh2 1^nh2 ch3 X/NH2 ch3 CH3 X/NH2 HSC^ T nh2 U1 h2n^ U.S. EPA WOE characterization "Inadequate Information to Assess Carcinogenic Potential" (see Table 7) NAa "Inadequate Information to Assess Carcinogenic Potential" "Suggestive Evidence of Carcinogenic Potential" Oral slope factor (mg/kg-dT1 NA 4 x 10ob ND Screening p-OSF: 1 x 10 1 (as sulfate); screening p-OSF: 1.8 x io_1 (as free base) Data set(s) used for slope factor derivation NA NTP (1978): mammary gland tumors in female F344 rats NTP (1980) studies were considered insufficient to assess carcinogenic potential; results were not considered treatment related but doses were too low, and a maximum tolerated dose was not achieved NTP (1978): interstitial-cell tumors of the testis in male F344 rats Other tumors observed in animal bioassays NA Liver tumors in rats and mice; subcutaneous fibroma in male rats; lymphoma in female mice NA Lung tumors in female mice Study doses (mg/kg-d) NA 0, 3.2, 7.0 (M); 0, 3.95, 8.55 (F) NA Adjusted daily dose: 0, 47, 158 (M); 0, 55, 183 (F) 63 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-3. Comparison of Available Oral Carcinogenicity Data for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Type of Data 3,4-TDA CASRN 496-72-0 2,4-TDA CASRN 95-80-7 2,6-TDA CASRN 823-40-5 2,5-TDA CASRN 95-70-5 Route (method) NA Diet Diet Diet Duration NA 103 wk 2 yr 78 wk POD type NA BMDLio NA BMDLio (HED) Source NA CalEPA (2011a): CalEPA (2009) U.S. EPA (2005b) U.S. EPA (2013) "There is no U.S. EPA WOE descriptor for 2.4-TDA; however, this compound is listed as a carcinogen by CalEPA (2011a). considered possibly carcinogenic to humans by IARC (1987) and reasonably anticipated to be a human carcinogen by NTP (2016b). 'OSF derived by CalEPA (2011a). BMDLio = 10% benchmark dose lower confidence limit; F = female(s); HED = human equivalent dose; M = male(s); NA = not applicable; ND = no data; OSF = oral slope factor; POD = point of departure; p-OSF = provisional oral slope factor; TDA = toluenediamine; WOE = weight of evidence. STEP 6. STRUCTURAL ALERTS AND STRUCTURE-ACTIVITY RELATIONSHIP PREDICTIONS FOR 3,4-TDA AND ANALOGUES Structural alerts (SAs) and predictions for genotoxicity and carcinogenicity were identified using computational tools as described in Appendix B. The model results for 3,4-TDA and its analogue compounds are shown in Table C-4. Concerns for carcinogenicity and/or mutagenicity of 3,4-TDA and its analogues were indicated by several models within each predictive tool (see Table C-4). Table C-5 provides a list of the specific SAs that underlie the findings of a concern for carcinogenicity or mutagenicity in Table C-4. OECD QSAR Toolbox models showed a concern for mutagenicity, CAs, MN, and protein binding for 3,4-TDA and all analogues based on SAs (see Tables C-4 and C-5). The ToxRead and VEGA models also indicated a concern for mutagenicity for 3,4-TDA and all analogues. The Toxtree tool indicated a concern for 3,4-, 2,4-, and 2,5-TDA mutagenicity in S. typhimurium TA100, but indicated that 2,6-TDA was unlikely to be mutagenic in S. typhimurium TA100. The Toxtree results for 2,6-TDA are inconsistent with positive experimental data (see "Step 3. Comparison of the Experimental Genotoxicity Data for 3,4-TDA and Analogues" above for more information) and the results of the other QSAR models. 64 3,4-T oluenediamine ------- EPA 690 R-21 003F Table C-4. Heat Map Illustrating the Structural Alert and SAR Prediction Results for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Tool Model" 3,4-TDA 2,6-TDA 2,6-TDA dihydrochloride 2,5-TDA 2,5-TDA dihydrochloride 2,5-TDA sulfate 2,4-TDA 2,4-TDA dihydrochloride Mutagenicity/genotoxicity alerts OECD QSAR Toolbox DNA alerts for Ames by OASIS DNA alerts for CA and MNT by OASIS In vitro mutagenicity (Ames test) alerts by ISS In vivo mutagenicity (micronucleus) alerts by ISS Protein binding alerts for CA by OASIS ToxRead ToxRead (mutagenicity) VEGA Mutagenicity (Ames test) CONSENSUS model—assessment Mutagenicity (Ames test) model (CAESAR)—assessment Mutagenicity (Ames test) model (SARpy/IRFMN)—assessment Mutagenicity (Ames test) model (ISS)—assessment Mutagenicity (Ames test) model (A-NN/read-across)—assessment Toxtree Potential Salmonella tvphimurium TA100 mutagen based on QSAR Carcinogenicity alerts OECD QSAR Toolbox Carcinogenicity (genotoxicity and nongenotoxicity) alerts by ISS OncoLogic OncoLogic (prediction of the carcinogenic potential of the chemical) VEGA Carcinogenicity model (CAESAR)—assessment Carcinogenicity model (ISS)—assessment Carcinogenicity model (IRFMN/ANTARES)—assessment Carcinogenicity model (IRFMN/ISSCAN-CGX)—assessment Toxtree Potential carcinogen based on QSAR Nongenotoxic carcinogenicity 65 3,4-T oluenediamine ------- EPA 690 R-21 003F Table C-4. Heat Map Illustrating the Structural Alert and SAR Prediction Results for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Tool Model" 3,4-TDA 2,6-TDA 2,6-TDA dihydrochloride 2,5-TDA 2,5-TDA dihydrochloride 2,5-TDA sulfate 2,4-TDA 2,4-TDA dihydrochloride Combined alerts ToxAlerts Aromatic amine (general) (for genotoxic carcinogenicity, mutagenicity) Aromatic amine (specific) (for genotoxic carcinogenicity, mutagenicity) Aromatic amines (for genotoxic carcinogenicity, mutagenicity) Primary and secondary aromatic amines (for genotoxic carcinogenicity, mutagenicity) Primary ar. amine, hydroxyl amine and its derived esters or amine generating group (genotoxicity, carcinogenicity, mutagenicity) Toxtree Structural alert for genotoxic carcinogenicity Model results or alerts indicating no concern for carcinogenicity/mutagenicity. Model results outside the applicability domain for carcinogenicity/mutagenicity. Model results or alerts indicating concern for carcinogenicity/mutagenicity. aAll tools and models described in Appendix B were used. Models with results or alerts are presented in the heat map (models without results were omitted). ANT ARES = Alternative Non-Testing Methods Assessed for REACH Substances; CA = chromosomal aberration; CAESAR = Computer-Assisted Evaluation of industrial chemical Substances According to Regulations; CONSENSUS = consensus assessment based on multiple models (CAESAR, SARpy, ISS, and A-NN); DNA = deoxyribonucleic acid; IRFMN = Istituto di Ricerche Fannacologiche Mario Negri; ISS = Istituto Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di Sanita Chemical Carcinogen; A-NN = A-nearest neighbor; OECD = Organisation for Economic Co-operation and Development; SAR = structure-activity relationship; QSAR = quantitative structure-activity relationship; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture. 66 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-5. SAs for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues SA Tools Compounds Aromatic amine OncoLogic ToxAlerts 3,4-TDA; 2,4-TDA; 2.4-TDA dihydrochloride; 2.5-TDA; 2,5-TDA dihydrochloride; 2.5-TDA sulfate; 2.6-TDA; 2,6-TDA dihydrochloride3 Primary aromatic amine, hydroxyl amine, and its derived esters Toxtree OECD QSAR Toolbox Primary aromatic amine, hydroxyl amine, and its derived esters or amine generating group ToxAlerts Substituted anilines OECD QSAR Toolbox Single ring-substituted primary aromatic amines OECD QSAR Toolbox aThe SA in OncoLogic for 2,6-TDA dihydrochloride was reported as "marginal." OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity relationship; SA = structural alert; TDA = toluenediamine. OECD QSAR Toolbox models showed a concern for carcinogenicity for 3,4-TDA and all analogues based on SAs (see Tables C-4 and C-5). The level of carcinogenicity concern in OncoLogic for 3,4-TDA was "high-moderate" based on structure-activity relationship (SAR) analysis only (aromatic amine with amino groups ortho to one another). OncoLogic indicated the level of concern for carcinogenicity as "moderate" for 2,4-TDA based on animal carcinogenicity data and SAR analysis (aromatic amine with amino groups meta to one another). The level of carcinogenicity concern in OncoLogic for 2,6-TDA, 2,5-TDA, 2,5-TDA hydrochloride, 2,5-TDA sulfate, and 2,4-TDA dihydrochloride was "moderate" based on SAR analysis only (aromatic amine with amino groups meta or para to one another). OncoLogic reported a "marginal" level of concern for 2,6-TDA dihydrochloride (shown as no results for models in Table C-4) based on a lack of evidence of carcinogenicity from animal studies and SAR analysis (aromatic amine with amino groups meta to one another). VEGA showed concern for carcinogenicity of 3,4-TDA using the CAESAR and ISS models (no data for the IRFMN/ANTARES or IRFMN/ISSCAN-CGX models). All four VEGA models showed concern for carcinogenicity for 2,4-TDA and 2,4-TDA dihydrochloride. Carcinogenicity models in VEGA produced inconsistent results for 2,5- and 2,6-TDA (and their salts). While the CAESAR model showed concern for both compounds (and their salts), the ISS model showed concern only for 2,5-TDA (and its salts), and the IRFMN/ISSCAN-CGX model did not show concern for either compound (or their salts). There were no data for the IRFMN/ANTARES model for 2,5- or 2,6-TDA (or their salts). The Toxtree tool indicated that 2,4- and 2,6-TDA were potential carcinogens based on QSAR, but that 3,4- and 2,5-TDA were not. The Toxtree tool showed there was no concern for nongenotoxic carcinogenicity for 3,4-TDA or any of its analogues. The ToxAlerts tool showed a concern for genotoxic carcinogenicity and/or mutagenicity for 3,4-TDA and all analogues based on various SAs (see Tables C-4 and C-5). The Toxtree models also suggest a concern for genotoxic carcinogenicity for 3,4-TDA and all analogues based on SAs. 67 3,4-T oluenediamine ------- EPA/690/R-21/003F In general, SAR predictions indicate a concern for genotoxicity and carcinogenicity for 3,4-TDA and all TDA analogues across several software systems evaluated. Moreover, a clear pattern or relationship between the position of the amino groups (3,4-TDA is a o- isomer, 2,5- is ap- isomer, and 2,4- and 2,6- are m- isomers) and potential differences in SAR predictions are not apparent for the TDA compounds. Previous SAR evaluations have suggested enhanced chemical reactivity for the o- and /^-substituted aromatic amines due to qui none formation (Baiot et al.. 2010). However, based on the available experimental and in silico data discussed above, the influence of the position of the amino groups on the potential genotoxicity and carcinogenicity of the TDA compounds is unclear. STEP 7. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF 3,4-TDA CARCINOGENICITY Table C-6 presents the data for multiple lines of evidence pertinent to the screening evaluation of the carcinogenic potential of 3,4-TDA. 68 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-6. Integration of Evidence for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Evidence Stream 3,4-TDA CASRN 496-72-0 2,4-TDA CASRN 95-80-7 2,6-TDA CASRN 823-40-5 2,5-TDA CASRN 95-70-5 Role Target Analogue Analogue Analogue Structure nh2 X/NH2 JJ ch3 A ,NH, mh2 ch3 ,,X ,,_nh2 L CH 3 H Analogue selection and evaluation (see Steps 1 and 2) Target compound: contains (1) one aromatic ring (benzene) substituted with (2) two unsubstituted amines on the ring, in an o-substitution pattern, (3) a methyl group on the ring, and (4) no other functional group Isomer: contains (1) one aromatic ring (benzene) substituted with (2) two unsubstituted amines on the ring, in a ///-substitution pattern, (3) a methyl group on the ring, and (4) no other functional group Isomer: contains (1) one aromatic ring (benzene) substituted with (2) two unsubstituted amines on the ring, in a ///-substitution pattern, (3) a methyl group on the ring, and (4) no other functional group Isomer: contains (1) one aromatic ring (benzene) substituted with (2) two unsubstituted amines on the ring, in a ^-substitution pattern, (3) a methyl group on the ring, and (4) no other functional group Experimental genotoxicity data (see Step 3) Mutagenic in Salmonella, induces MN in vivo; inhibits DNA synthesis in vivo; induces cell transformation in mammalian cells Mutagenic in Salmonella, clastogenic in mammalian cells; DNA damaging in mammalian cells in vitro and in vivo; forms DNA adducts in vivo Mutagenic in Salmonella, clastogenic in mammalian cells; inconsistent evidence for DNA damage in mammalian cells in vitro and in vivo; induces cell transformation in hamster embryo cells Mutagenic in Salmonella, clastogenic in mammalian cells; inconsistent evidence for DNA damage in mammalian cells in vitro and in vivo; induces cell transformation in hamster embryo cells ADME evaluation (see Step 4) ND; metabolic pathways expected to be similar to other TDA isomers based on metabolite prediction data Common metabolic pathways with other TDA isomers (acetylation of the amino groups, ring hydroxylation and potential oxidation of methyl groups) Common metabolic pathways with other TDA isomers (acetylation of the amino groups, ring hydroxy lation and potential oxidation of methyl groups) Common metabolic pathways with other TDA isomers (acetylation of the amino groups, ring hydroxy lation and potential oxidation of methyl groups) Cancer data andMOA (see Step 5) ND Liver tumors in rats and mice, subcutaneous fibromas in male rats, mammary tumors in rats, lymphoma in female mice; MO A not established No significant evidence of carcinogenicity (but doses may have been too low); MO A not established Testicular tumors in rats, lung tumors in mice; MO A not established 69 3,4-T oluenediamine ------- EPA/690/R-21/003F Table C-6. Integration of Evidence for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues Evidence 3,4-TDA 2,4-TDA 2,6-TDA 2,5-TDA Stream CASRN 496-72-0 CASRN 95-80-7 CASRN 823-40-5 CASRN 95-70-5 Common SA ALERTS ALERTS ALERTS ALERTS and SAR • Aromatic amine • Aromatic amine • Aromatic amine • Aromatic amine predictions • Primary aromatic amine, • Primary aromatic amine, • Primary aromatic amine, • Primary aromatic amine, (see Step 6) hydroxyl amine and its derived hydroxyl amine and its derived hydroxyl amine and its derived hydroxyl amine and its derived esters esters esters esters • Primary aromatic amine, • Primary aromatic amine, • Primary aromatic amine, • Primary aromatic amine, hydroxyl amine and its derived hydroxyl amine and its derived hydroxyl amine and its derived hydroxyl amine and its derived esters or amine generating group esters or amine-generating esters or amine-generating esters or amine-generating • Substituted anilines group group group • Single ring-substituted primary • Substituted anilines • Substituted anilines • Substituted anilines aromatic amines • Single ring-substituted primary • Single ring-substituted primary • Single ring-substituted primary aromatic amines aromatic amines aromatic amines SAR PREDICTIONS: Concerns for mutagenicity and SAR PREDICTIONS: SAR PREDICTIONS: SAR PREDICTIONS: carcinogenicity in most models; not Concerns for mutagenicity and Concerns for mutagenicity and Concerns for mutagenicity and likely to be a carcinogen based on carcinogenicity in most models; carcinogenicity in most models; carcinogenicity in most models; QSAR in Toxtree; no concern for no concern for nongenotoxic no concern for carcinogenicity in no concern for carcinogenicity in nongenotoxic carcinogenicity in carcinogenicity in Toxtree two of three VEGA models and one of three VEGA models; not Toxtree no concern for nongenotoxic carcinogenicity in Toxtree likely to be a carcinogen based on QSAR in Toxtree; no concern for nongenotoxic carcinogenicity in Toxtree ADME = absorption, distribution, metabolism, and excretion; DNA = deoxyribonucleic acid; m = meta; MN = micronuclei; MOA = mode of action; ND = no data; o = ortho; p=para; QSAR = quantitative structure-activity relationship; SA = structural alert; SAR = structure-activity relationship; TDA = toluenediamine; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture. 70 3,4-T oluenediamine ------- EPA/690/R-21/003F STEP 8. QUALITATIVE LEVEL OF CONCERN FOR 3,4-TDA POTENTIAL CARCINOGENICITY A concern for potential carcinogenicity for 3,4-TDA is identified based on multiple lines of evidence, including similarities in structural features, in silico metabolism profiles, SAs and SAR predictions, and experimental data for carcinogenicity and/or genotoxicity for the target and analogues (see Table C-7 for additional details). Because of the lack of information supporting carcinogenic portal-of-entry effects, the qualitative level of concern for this chemical is considered to be applicable to all routes of exposure. Table C-7. Qualitative Level of Concern for Carcinogenicity of 3,4-Toluenediamine (CASRN 496-72-0) Level of Concern Designation Comments Concern for potential carcinogenicity Selected 2,4-, 2,5-, and 2,6-TDA were identified as structural analogues of 3,4-TDA for evaluating carcinogenic potential. These compounds share a basic chemical structure (benzene ring, two amino groups, and a methyl group), differing only in the position of the amino functional groups. The analogues exhibit commonalities in toxicokinetic properties, including common metabolic pathways, which are expected to be similar for 3,4-TDA based on metabolite predictions. Two of three analogues have carcinogenic potential based on tumors observed in rodent studies (2,4- and 2,5-TDA); the third analogue (2,6-TDA) has not been adequately assessed for carcinogenicity. Although the carcinogenic MOA for 3,4-, 2,4-, 2,5- and 2,6-TDA is not known, all compounds appear to be mutagenic in bacterial systems with metabolic activation and show some evidence of genotoxicity in mammalian test models. Furthermore, the target compound and analogues have identical SAs (e.g., aromatic amine) and similar SAR predictions showing concern for carcinogenicity/genotoxicity. Inadequate information for assigning qualitative level of concern Not selected NA MOA = mode of action; NA = not applicable; SA = structural alert; SAR = structure-activity relationship; TDA = toluenediamine. 71 3,4-T oluenediamine ------- EPA/690/R-21/003F APPENDIX D. METHODOLOGY AND RESULTS FOR IN SILICO METABOLITE ANALYSIS OF TARGET AND ANALOGUES An in silico analysis of metabolism was conducted for 3,4-toluenediamine (3,4-TDA) and its analogues using different software tools. The main objective of this analysis is to provide a qualitative comparison of metabolite predictions for TDA compounds in the absence of experimental data for the target. The focus is on the major metabolism pathways characterized in the literature, highlighting any notable differences between the target and analogues. Chemical structures were extracted from the U.S. EPA CompTox Chemicals Dashboard for 3,4-TDA and the identified structural analogues [2,3-, 2,4-, 2,5-, and 2,6-TDA; U.S. EPA (2019)1. The metabolite predictions for the chemicals of interest were generated using commercially available software systems, including the Tissue Metabolism Simulator (TIMES) (Dimitrov et al.. 2005; Mekenvan et al.. 2004) and Meteor Nexus (Marchant et al.. 2008). A structure data file (SDF) was imported into the TIMES program (Version 2.29.1; http://oasis-lmc.org/products/software/times.aspx). using the in vitro rat S9 metabolic simulator (Version 11.16) and the rat in vivo metabolic simulator (Version 07.12) to make predictions of likely metabolites. The predictions were exported as a .txt file for subsequent processing. For the Meteor Nexus predictions, the SDF was split into separate molecular data (MOL) files for batch processing in Meteor Nexus. A python script (Python; Version 3.6.5; python.org) was used to split the SDF, and a second script was used to concatenate the individual substance prediction files that were created as separate excel workbooks. Default settings were used in Meteor Nexus (Version 3.1.0) developed by Lhasa Limited (https://www.lhasalimited.org/librarv/publishing.htm). The settings were for a maximum depth of tree to be 3, for the maximum number of metabolites to be capped at 1,000 and for the scoring method to be Site of Metabolism Scoring (with Molecular Mass Variance). The results are described as a score that uses experimental data for compounds that match the same biotransformation, have similar molecular weights and are structurally similar around the site of metabolism to the query compound (for more details, see https://www.lhasalimited.org/products/meteor-reasoning-methodologies.htm). The prediction files were then processed further within a Jupyter notebook (iupvter.org) imported with python libraries RDKit (Version 2018.03.2.0; RDKit.org), Pandas (Version 0.23.1; pandas.pydata.org), NumPy (Version 1.14.3; numpv.org), and Matplotlib (Version 2.2.2; matplotlib.org). The software systems provided Simplified Molecular Input Line Entry System (SMILES) representations for the predicted metabolites. These were converted into RDKit mol objects and exported as a Pandas Tools worksheet that provided depictions of chemical structure. International Union of Pure and Applied Chemistry (IUPAC) International Chemical Identifier (InChI™) keys were created using RDKit because SMILES representations are not unique. The structures of the predicted metabolites from each of the tools evaluated are presented in Table D-l, which compares metabolites identified across the different software tools and experimental data from in vivo animal studies captured in Table A-3 of the "Metabolic Analogues" section in Appendix A. Additionally, pathway transformations corresponding to the metabolite predictions were extracted from Meteor Nexus to facilitate similarity comparisons between the target and candidate analogues. Other software tools (i.e., TIMES) did not provide the same level of information. The pathway transformations for target and candidate analogues were extracted from the Meteor Nexus summary report, then grouped by substance and pivoted 72 3,4-T oluenediamine ------- EPA/690/R-21/003F to provide a representation of the TDA compounds as rows and the unique pathways as columns (see Table A-4 in Appendix A). A pairwise distance matrix was then computed using the Jaccard distance as a metric, which was then transformed to a similarity matrix (see Figure A-l in Appendix A). A metabolic tree for the 2,4-TDA was constructed to highlight the relationships of predicted metabolites for this specific analogue that correspond to the pathway transformations shared among the TDA compounds (see Figure D-l). Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod 3,4-Toluenediamine (CASRN 496-72-0) A, jrs Nh^ CDOUPQQ J GF C ACL- UHFFFAOYSA-N CC(=0)Nc 1 cc(C)ccc IN 1 1 1 NDr 0 ^ AJQr nh2 JBJRVPVZADJOOX- UHFFFAOYSA-N CC(=0)Nc lccc(C)cc IN 1 1 1 NDr H2N^^OH LXBXLRPRAMALBT - UHFFFAOYSA-N Cc 1 cc(N)c(N)cc 10 1 1 1 NDr NH, (a AMUSQFJRRCHIDJ- UHFFFAOYSA-N Cc 1 ccc(N)c(NO)c 1 1 0 1 NDr 5 o / * NH, HO / ^ —NHj HEMGYNNCNNODN X-UHFFFAOYSA-N Nc lccc(C(=0)0)cc IN 1 0 1 NDr 73 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod KjN V y y W OH h2n HMVJXTUUQJUYJI- UHFFFAOYSA-N Nclccc(CO)cclN 1 0 1 NDr h3n HN- / \ / \\ // HO V V LQKOQGZAAWXAJO -UHFFF AOY S A-N Cc 1 ccc(NO)c(N)c 1 1 0 1 NDr HO H2N ^ h2n FQQXRJHNPCQKQB - UHFFF AOYSA-N Cc lcc(N)c(N)c(0)c 1 0 1 1 NDr A Xj H 1 HN. OH AELQHYALRZNYNG -UHFFF AOY S A-N CC(=0)Nc lccc(C)cc IN 0 1 0 0 NDr CDBLIIZYT JQRIR- UHFFF AOYSA-N Cc lccc(N0S(=0)(=0)0) c(N)cl 1 0 0 NDr m2 CFFKPFQMLZQSMC- UHFFF AOYSA-N CC(=0)Nclccc(CO)ccl N 1 0 0 NDr 74 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod NH, DMIWVGYENDrUAG J-UHFFFAOYSA-N Cclccc(N)c(NOS(=0)(= 0)0)cl 1 0 0 NDr <\ // V -0H \* OYMWICMADIVGJD- UHFFFAOYSA-N Nclccc(CO)cclNO 1 0 0 NDr ^ c> OYXHIB S VHUIIHN - UHFFFAOYSA-N CC(=0)Nc 1 cc(C)ccc IN 0 1 0 0 NDr HN-. OH OH HOv /T~\ / \ // \\ NH YPMPQTKAGF CMHX -UHFFFAOYSA-N Nclcc(CO)ccclNO 1 0 0 NDr nh2 NH, Z SHDMSD VKO VL AA -UHFFFAOYSA-N CC(=0)Nc lcc(CO)ccc 1 N 1 0 0 NDr HQ—S // 0 OH P / HN-—V / CFNITIXCKKIDKK- UHFFFAOYSA-N CC(=0)Nc lcc(0)c(C)cc IN 0 1 0 NDr H-,N 75 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod 0 J -—U —OH HjN IHUHNLWFEHPZOR- UHFFFAOYSA-N CC(=0)Nc lcc(C)c(0)cc IN 0 1 0 NDr X o -/iL* \ /—~~Z' RHCSARHFCNPXNQ- UHFFFAOYSA-N CC(=0)Nc lcc(C)cc(0)c IN 0 1 0 NDr X ° / VRFLCZIWWHOCPA- UHFFFAOYSA-N CC(=0)Nc lc(N)cc(C)cc 10 0 1 0 NDr AIYGLIJSQZZWPP- UHFFFAOYSA-N Cc lccc(N=0)c(N)c 1 0 0 1 NDr CFDDU ZIK VUIONZ - UHFFFAOYSA-N Cclccc(N(0)SCC(NC(= 0)CCC(N)C(=0)0)C(= 0)NCC(=0)0)c(N)c 1 0 0 1 NDr L FGMJURDRCOTDCZ- UHFFFAOYSA-N Cclccc(N)c(N(0)SCC(N C(=0)CCC(N)C(=0)0)C (=0)NCC(=0)0)c 1 0 0 1 NDr 76 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod GMFRNXFZQNBPLZ- UHFFFAOYSA-N CC1=CC(=N)C(N)=CC1 =0 0 0 1 NDr nh2 GMPOLBFESRQGCU - UHFFFAOYSA-N Cc 1 ccc(N)c(N=0)c 1 0 0 1 NDr & NAGMCFVTHPBBFR- UHFFFAOYSA-N Cclc(0)cc(N)c(N)clSC C(NC(=0)CCC(N)C(=0) 0)C(=0)N CC(=0)0 0 0 1 NDr h2n—(V V)— NSILMBMDJGSYNS- UHFFFAOYSA-N Nclccc(C=0)cclN 0 0 1 NDr QAFMNLZTMYPOOY -UHFFFAOYSA-N CC1(SCC(NC(=0)CCC( N)C(=0)0)C(=0)NCC(= 0)0)CC(=N)C(N)=CC 1 =0 0 0 1 NDr OH o. f \_-~NH, m, UVIYTNUYRRBTME- UHFFFAOYSA-N Nc lcc(C=0)cc(0)c IN 0 0 1 NDr 77 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod sf z 4/ o X YXTRRMZKHFFNPW -UHFFFAOYSA-N Nc lcc(C0)cc(0)c IN 0 0 1 NDr 2,3-Toluenediamine (CASRN 2687-25-4) o H I NH2 CDQDPNFLCSCJCH- UHFFFAOYSA-N CC(=0)Nclc(C)cccclN 1 1 1 NDr o nh2 LQAAALNVGAFVJD- UHFFFAOYSA-N CC(=0)Nc 1 cccc(C)c IN 1 1 1 NDr nh2 H i N-. OH FQKUNAYBPMGVRP -UHFFFAOYSA-N Cclcccc(N)clNO 1 0 1 NDr HjN NH2 FYUDUZRLZITSTF- UHFFFAOYSA-N Nclcccc(CO)clN 1 0 1 NDr U \ /° H2N \ OH nh2 KKTUQAY CCLMNO A-UHFFFAOYSA-N Nc lcccc(C(=0)0)c IN 1 0 1 NDr 78 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod X o V VYFFPFYOFVMNGG- UHFFFAOYSA-N Cclcccc(NO)clN 1 0 1 NDr H0—<\ —nh, ' NH, GVEXOXFTENPDOH- UHFFFAOYSA-N Cc 1 c(0)ccc(N)c IN 0 1 1 NDr nh2 HO ^ NHj SRFOBSMZHWJDJN- UHFFFAOYSA-N Cc 1 cc(0)cc(N)c IN 0 1 1 NDr NH- 6:"-. DCYIPFHDKQUXBG- UHFFFAOYSA-N Cc lcccc(N)c 1N0S(=0)( =0)0 1 0 0 NDr X o fX^i h; DEYTUXOGDHQHRY -UHFFF AOY S A-N CC(=0)Nclcccc(C0)cl N 1 0 0 NDr o NH HO GMTRKMQJURSNIX- UHFFFAOYSA-N CC(=0)Nc 1 cccc(C)c IN 0 1 0 0 NDr 79 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod HO. JsN. ji .OH NH, HRGIXVSYYVWOGJ- UHFFFAOYSA-N Nclc(CO)cccclNO 1 0 0 NDr °H j HH nh2 / OH LFOLOGIZBFISKF- UHFFFAOYSA-N Nclcccc(CO)clNO 1 0 0 NDr NHj YJDFBYAXTGPZSK- UHFFFAOYSA-N Cclcccc(N0S(=0)(=0)0 )clN 1 0 0 NDr H HO YRXPMBZXLPQXIS - UHFFFAOYSA-N CC(=0)Nc 1 c(N)cccc 1C 0 1 0 0 NDr OH /X 1 ^NH \ ll j ZPZCAEZFJLLBMW - UHFFFAOYSA-N CC(=0)Nclc(C)cccclN 0 1 0 0 NDr A. XT FUVMDOMRPMZMO A-UHFFFAOYSA-N CC(=0)Nc lccc(0)c(C)c IN 0 1 0 NDr NH, 80 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod ill 7 H H I HB CUEGKUEMKCKO -UHFFFAOYSA-N CC(=0)Nc lc(N)ccc(0)c 1C 0 1 0 NDr ^CPr™ NH, PNMPOSDTYOCRAS- UHFFFAOYSA-N CC(=0)Nc lc(C)cc(0)cc IN 0 1 0 NDr OH 0 ^ n H ji ^ nh2 ZEBMHLAASWFAGS -UHFFFAOYSA-N CC(=0)Nc lcc(0)cc(C)c IN 0 1 0 NDr ^ ^h2 ARCDFSYMTMSQRI- UHFFFAOYSA-N CC 1=CC(=0)C=C(N)C 1 =0 0 0 1 NDr X o / X v/ CH CCIQDILKNGQPCA- UHFFFAOYSA-N N=C 1 C(N)=CC(=0)C=C ICO 0 0 1 NDr FTKYIFRLYMBQDC- UHFFFAOYSA-N CC1=C(N)C(=N)CC(SC C(NC(=0)CCC(N)C(=0) 0)C(=0)N CC(=0)0)C 1 =0 0 0 1 NDr 81 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod nh2 NAJRDVTXZXCQAM -UHFFFAOYSA-N Cclcccc(N=0)clN 0 0 1 NDr ^ ir\Jl OH NRERCLGIEBGCTA- UHFFFAOYSA-N Cc 1 cccc(N)c IN (O)S CC( NC(=0)CCC(N)C(=0)0 )C(=0)NCC(=0)0 0 0 1 NDr nh2 PMJTXIMMLKKLKL- UHFFFAOYSA-N Cc 1 cccc(N)c 1N=0 0 0 1 NDr PYGSXASUOAHHOW -UHFFFAOYSA-N Cclcccc(N(0)SCC(NC(= 0)CCC(N)C(=0)0)C(= 0)NCC(=0)0)c IN 0 0 1 NDr h2n ^ RJICMTPCHYXVJA- UHFFFAOYSA-N CC1=C(N)C(=N)C=CC1 =0 0 0 1 NDr Oa h2n NH2 UOGKMJUEKOCDAX -UHFFFAOYSA-N Nclcccc(C=0)clN 0 0 1 NDr 82 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod WDQVHPTNDVFMR -UHFFFAOYSA-N CC 1=CC(=0)C=C(N)C 1 =N 0 0 1 NDr yjri W WYXB GROOLIEEU -UHFFFAOYSA-N Cclc(0)cc(SCC(NC(=0) CCC(N)C(=0)0)C(=0) NCC(=0)0)c(N)clN 0 0 1 NDr 2,4-Toluenediamine (CASRN 95-80-7) KN. .NK xc DPKOCFTZJRJTQL- UHFFFAOYSA-N Cc lcc(0)c(N)cc IN 1 1 1 1 RBQWGHBZCHFUQU -UHFFFAOYSA-N CC(=0)Nc lccc(C)c(N)c 1 1 1 1 1 nh2 A A H \ UAZGSMMESOKKQZ -UHFFFAOYSA-N CC(=0)Nc lcc(N)ccc 1C 1 1 1 NDr o r2 ^ Jr" HN-—\ / HQ BATES GGSVKZESGJ- UHFFFAOYSA-N CC(=0)Nclcc(N)c(C)cc 10 1 1 0 1 83 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod —M 1 ^OH HH2 FADNCTWKDWKIX -UHFFFAOYSA-N Nc 1 ccc(CO)c(N)c 1 1 0 1 NDr (/ \\ [sih V=^ OH H2N JCSKFCJYMXDFAD- UHFFFAOYSA-N Cclccc(NO)cclN 1 0 1 NDr OH ^N\ 1 KARRBUHHWCMGH B -UHFFF AOY S A-N Cclccc(N)cclNO 1 0 1 NDr /¦ \ ,0 JT\-J H.N / \ VmV oh NH, LDQMZKBIBRAZEA- UHFFFAOYSA-N Nc lccc(C(=0)0)c(N)c 1 1 0 1 NDr NHj 0 / \ Jl \W-OH hn—\ / LZEMQCKKBQRKFM -UHFFFAOYSA-N CC(=0)Nc lcc(N)c(0)cc 1C 1 1 0 NDr CEKKEYTVRSICNQ- UHFFFAOYSA-N Cclcc(0)c(N)c(SCC(NC (=0)CCC (N)C(=0)0)C(=0)NCC( =0)0)clN 1 0 0 NDr 84 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod h2n DYLOOKKMCKMEC T -UHFFFAOY S A-N Cclccc(N=0)cclN 1 0 0 NDr IODXTXYTNSSKSC- UHFFFAOYSA-N Cclccc(N)cclN(0)SCC( NC(=0)CCC(N)C(=0)0 )C(=0)NCC(=0)0 1 0 0 NDr p H2N\ _ II y^N LLROHMIZNDXUDK- UHFFFAOYSA-N Cclccc(N)cclN=0 1 0 0 NDr RGMVILWFQAJIRO- UHFFFAOYSA-N CC1=CC(=0)C(N)(SCC( NC(=0)CCC(N)C(=0)0 )C(=0)NCC(=0)0)CC 1 =N 1 0 0 NDr JT\^ ^-v / o h2N Nv Nh^ VMFJRVFZHAPENO- UHFFFAOYSA-N Nc lccc(C=0)c(N)c 1 1 0 0 NDr YIJGNYPGRJIBNG- UHFFFAOYSA-N CC 1=CC(=0)C(N)=CC 1 =N 1 0 0 NDr 85 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod r ^ ^C YLDTVWMQWTYLO W-UHFFFAOYSA-N Cclccc(N(0)SCC(NC(= 0)CCC(N)C(=0)0)C(= 0)NCC(=0)0)cc IN 1 0 0 NDr _/ DGBUOAIABOATGK- UHFFFAOYSA-N CC(=0)Nc lcc(N)ccc 1C 0 1 0 0 NDr 1 OH HJLRPSJGXCSTOA- UHFFFAOYSA-N CC(=0)Nc lccc(C)c(NO) cl 1 0 0 NDr NH? —~NH' NNY GTEAP VB YVOR -UHFFF AOY S A-N Cclcc(0S(=0)(=0)0)c( N)cclN 0 0 1 NDr -NH Pi '\^=J V -0H ty/ NTVDFUPWAZHKRU -UHFFF AOY S A-N Cc lccc(N0S(=0)(=0)0) cclN 0 0 1 NDr OHWHMZFQZAEYN R-UHFFF AOY S A-N Nclcc(NO)ccclCO 0 0 1 NDr 86 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod I f o X PQHCWNHKWOJEJH- UHFFFAOYSA-N CC(=0)Nc 1 cc(NO)ccc 1 C 0 0 1 NDr ft o ULBRTOCNNKAALW -UHFFFAOYSA-N CC(=0)Nc lccc(CO)c(N) cl 0 0 1 NDr fj 2 NH L WQJKPERQJHMQFH- UHFFFAOYSA-N Nclccc(CO)c(NO)cl 0 0 1 NDr OH HO / \_J/ nh2 h2n XKZAGZAKKCRYJI- UHFFFAOYSA-N Nc lcc(N)c(CO)cc 10 0 0 1 NDr tvi \ \\ / —OH ^ £/ YEDHXAKCFRBZAC- UHFFFAOYSA-N Cclccc(N)cclNOS(=0)( =0)0 0 0 1 NDr X ° y° j* .L IFQWB WVICSDD SO- UHFFFAOYSA-N C 1=C(C(=CC=C 1NC(C) =0)C(=0)0)N 0 0 0 1 87 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod h2n oh RMHSOFVBSGKWJZ- UHFFFAOYSA-N C1 (=C(C=CC(=C 10)N) C)N 0 0 0 1 Nt-t P H°\ / ^ Vy N- N. / UZDRYZZYGHVZBF- UHFFFAOYSA-N C 1=CC(=C(C(=C 1 C)N) 0)NC(C)=0 0 0 0 1 -xrv 0 UZGYKGBZSGOY CM -UHFFF AOY S A-N C 1=CC(=C(C=C 1NC(C) =0)NC(C)=0)C(=0)0 0 0 0 1 XAHUNQAOCGD SB W-UHFFFAOYSA-N C1 (=CC=C(C=C 1NC(C) =0)NC(C)=0)C 0 0 0 1 NH2 h2m ^ OH YKMOHRBCUQRNQ M-UHFFF AOY S A-N C1=C(C=C(C(=C10)C) N)N 0 0 0 1 HO fV r 0 / 0 ¥ IFQWB WVICSDD SO- UHFFFAOYSA-N C 1=C(C(=CC=C 1NC(C) =0)C(=0)0)N 0 0 0 1 88 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod 2,5-Toluenediamine (CASRN 95-70-5) GWFPMSIIVJMYRZ- UHFFFAOYSA-N CC(=0)Nc 1 ccc(N)cc 1C 1 1 1 NDr A A OH h2n—// \\ N/ L JHMYKU SHZQTMV -UHFFF AOY S A-N Cclcc(N)ccclNO 1 0 1 NDr nh2 OH NKN CGBHPGCHY CQ -UHFFF AOY S A-N Nc 1 ccc(N)c(CO)c 1 1 0 1 NDr QNLWDEIYRQGAEE- UHFFFAOYSA-N Cclcc(NO)ccclN 1 0 1 NDr -VCC QXWUFIZOBXUMSM -UHFFF AOY S A-N CC(=0)Nc lccc(N)c(C)c 1 1 1 1 NDr OH UONVFNLDGRWLKF -UHFFF AOY S A-N Nc 1 ccc(N)c(C(=0)0)c 1 1 0 1 NDr 89 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod y-Y »- AHAAQFCJXQOJQM- UHFFFAOYSA-N Cc lcc(N)ccc 1N0S(=0)( =0)0 1 0 0 NDr BAYXOGMUGKSOIY -UHFFF AOY S A-N Cc lcc(N)c(0)cc IN 1 0 0 NDr GDRJGNLVIHNOTC- UHFFFAOYSA-N CC(=0)Nc lcc(0)c(N)cc 1C 1 0 0 NDr -—^ j Vy—NH, HN- \ / HO JCEYYZACUKLBJT- UHFFFAOYSA-N CC(=0)Nc lcc(C)c(N)cc 10 1 0 0 NDr 0H HO JI TJHj LPVMVIHWFLVFLL- UHFFFAOYSA-N Nclcc(CO)c(N)cclO 1 0 0 NDr ,0 w 11 f\\ 0 JKNEPY QGJF QCX- UHFFFAOYSA-N CC(=0)Nc 1 ccc(NO)cc 1 C 1 0 0 NDr 90 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod -°x fTS^"' H0' § PJLKOWKKMQTFPM -UHFFFAOYSA-N Cclcc(N0S(=0)(=0)0)c cclN 1 0 0 NDr OH HN— PMH SKY OPGGHU CD -UHFFFAOYSA-N Nclccc(NO)cclCO 1 0 0 NDr ,0 PH QWRHGJVFZGEQMN -UHFFFAOYSA-N CC(=0)Nc lccc(NO)c(C) cl 1 0 0 NDr OH QWUDZFZVWQFZNJ- UHFFFAOYSA-N CC(=0)Nc lccc(N)c(CO) cl 1 0 0 NDr WJQKIKWBEAJBTM- UHFFFAOYSA-N CC(=0)Nc 1 ccc(N)cc 1C 0 1 0 0 NDr OH YFLDRIRBIYOFLP- UHFFFAOYSA-N Nc lccc(NO)c(CO)c 1 1 0 0 NDr ho v y 91 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod CJGYEDFJVQTRDO- UHFFFAOYSA-N Cc 1 c(N)ccc(N)c 1S CC(N C(=0)CCC(N)C(=0)0)C (=0)NCC(=0)0 0 0 1 NDr y={_ GAANDMBIVOKAES- UHFFFAOYSA-N Cclcc(N)c(SCC(NC(=0) CCC(N)C(=0)0)C(=0) NCC(=0)0)cclN 0 0 1 NDr GFXJDKBBELRHKA- UHFFFAOYSA-N Cclcc(N)cc(SCC(NC(=0 )CCC(N)C(=0)0)C(=0) NCC(=0)0)clN 0 0 1 NDr GVAJQXAWDPTTRS- UHFFFAOYSA-N Cc 1 cc(N)ccc IN (O)S CC( NC(=0)CCC(N)C(=0)0 )C(=0)NCC(=0)0 0 0 1 NDr nh2 0 KIWGKZZMXPLHDG -UHFFF AOY S A-N Nc 1 ccc(N)c(C=0)c 1 0 0 1 NDr YvC / yT * LDEKAEXXYZEVAI- UHFFFAOYSA-N Cclcc(N(0)SCC(NC(=0 )CCC(N)C(=0)0)C(=0) NCC(=0)0)ccc IN 0 0 1 NDr 92 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod LFHSUMCUWFSVFL- UHFFFAOYSA-N CC1=CC(=N)C=CC 1 =N 0 0 1 NDr ¦£ z UFBHQACBYVXASE- UHFFFAOYSA-N Cclcc(N=0)ccclN 0 0 1 NDr h2n // \ // 2 \ ) N XIRVYFRBHGMGMO -UHFFF AOY S A-N Cc 1 cc(N)ccc 1N=0 0 0 1 NDr J V Z Y C WUKASEIL JBB - UHFFF AOYSA-N C1 (=CC(=CC=C 1NC(C) =0)NC(C)=0)C 0 0 0 1 2,6-Toluenediamine (CASRN 823-40-5) NH2 ( OH V CIEFZSDJGQKZNS- UHFFF AOYSA-N Nc lcccc(N)c 1 C(=0)0 1 0 1 NDr H*N j H NVKFKABKBGAWB K-UHFFF AOYSA-N Cclc(N)cccclNO 1 0 1 NDr 93 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod -- N NH, H 2 TZEOVCYRUCGICH- UHFFFAOYSA-N CC(=0)Nc 1 cccc(N)c 1C 1 1 1 1 nh2 ZJRAQHULMYRRQG- UHFFFAOYSA-N Nclcccc(N)clCO 1 0 1 NDr H,N (v ,) OH \\ // ' nh2 SHWMCLVULOXIMZ -UHFFF AOY S A-N Cc 1 c(N)ccc(0)c IN 0 1 1 1 Sb BIMQURICJYB VBU - UHFFF AOYSA-N Cclc(N)cccclNOS(=0)( =0)0 1 0 0 NDr /\—CH HO H DPFBFFAJBRRLGH- UHFFF AOYSA-N Nclcccc(NO)clCO 1 0 0 NDr 0 YY AYA, HO FHWODLDY COFLFK- UHFFF AOYSA-N CC(=0)Nc 1 cccc(N)c 1C 0 1 0 0 NDr 94 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod X o \ /I LNZDEXXJPTWLTL- UHFFFAOYSA-N CC(=0)Nc 1 cccc(NO)c 1 C 1 0 0 NDr H°. 0 tT 1 1 1a nh2 POQKQLONFCRNFP- UHFFFAOYSA-N CC(=0)Nc lc(0)ccc(N)c 1C 0 1 0 NDr x^k: FCSVJIUNOCBBTJ- UHFFFAOYSA-N CC1=C(N)C(=0)C(SCC( NC(=0)CCC(N)C(=0)0 )C(=0)NCC(=0)0)CC 1 =N 0 0 1 NDr KWPKZWQEAFVXH D-UHFFFAOYSA-N Cclc(N)cccclN(0)SCC( NC(=0)CCC(N)C(=0)0 )C(=0)NCC(=0)0 0 0 1 NDr ~R~ PXORAQVFLKLDLZ- UHFFFAOYSA-N CC 1=C(N)C(=0)C=CC 1 =N 0 0 1 NDr NH2 QWENKMCDOWMU AG-UHFFFAOYSA-N Nclcccc(N)clC=0 0 0 1 NDr 95 3,4-T oluenediamine ------- EPA/690/R-21/003F Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues across Software Tools and Observations from In Vivo Rodent Studies3 Structure InChI Key SMILES Meteor Nexusb TIMES In Vivoc TIMES In Vitro0 Observed In Vivod VOOYTMAMLLHB SZ -UHFFF AOY S A-N Cclc(N)cccclN=0 0 0 1 NDr YPGMZCDVNOMVO G-UHFFFAOYSA-N Cclc(N)c(0)cc(SCC(NC (=0)CCC(N)C(=0)0)C( =0)NCC(=0)0)c IN 0 0 1 NDr xxx H KSPKSSDXPVUERG- UHFFFAOYSA-N CC(=0)Nc lccc(0)c(N)c 1C 0 1 0 1 o o Ar-y-A NAXDRCLBFGZQSR- UHFFFAOYSA-N C1 (=C(C=CC=C 1NC(C) =0)NC(C)=0)C 0 0 0 1 al/0 denotes whether a metabolite was identified/not identified by a software tool or experimental animal data captured in Table A-3. ' Meteor Nexus (Dimitrov et at. 2005: Mekenvan et al.. 20041. Tn Vivo/In Vitro Rat Tissue Metabolism Simulator (Dimitrov et al.. 2005: Mekenvan et al.. 20041. dMetabolites reported from in vivo animal studies for the TDA isomers. Experimental data for 2,3- and 3,4-TDA were not available. Refer to Table A-3 for additional details. InChI = IUPAC International Chemical Identifier; IUPAC = International Union of Pure and Applied Chemistry; NDr = not determined; SMILES = Simplified Molecular Input Line Entry System; TDA = toluenediamine; TIMES = Tissue Metabolism Simulator. 96 3,4-T oluenediamine ------- EPA/690/R-21/003F Figure D-l. Metabolic Tree for the 2,4-Toluenediamine (CASRN 95-80-7) Analogue. Diagram displays the relationship of the metabolites identified from Meteor Nexus (Dimitrov et aL 2005; Mekenyan et at.. 2004) to the parent compound and notes the corresponding pathway transformations (a-g). 97 3,4-T oluenediamine ------- EPA/690/R-21/003F APPENDIX E. REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists). (2020). 2020 TLVs and BEIs: Based on the documentation of the threshold limit values for chemical substances and physical agents & biological exposure indices. Cincinnati, OH. Air Products and Chemicals (Air Products and Chemicals Inc.). (1976). Toxicity data on toluenediamine with attachments prepared by Air Prods and Chems Inc. (EPA/OTS: Doc #878213786). Wilmington, DE: E.I. Dupont Denemours & Co. Inc. Allied Chemical (Allied Chemical Corporation). (1979a). Short-term mutagenicity and cell transformation screening of toluene diamine - Bacterial mutagenicity of four toluene diamine isomers with cover letter dated 062392. https://ntrl.ntis.gov/NTRL/dashboard/scarchRcsults/titleDctail/OTS0540103.xhtml Allied Chemical (Allied Chemical Corporation). (1979b). Short-term mutagenicity and cell transformation screening of toluene diamine - effect of 4 toluene diamine isomers on murine testicular dna synthesis with cover letter dated 062392. (OTS0540099. TSCATS/427279. Doc #86-920000969). Allied Chemical (Allied Chemical Corporation). (1983a). 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