United States Environmental Protection 1=1 m m Agency EPA/690/R-13/01 OF Final 4-04-2013 Provisional Peer-Reviewed Toxicity Values for Technical Grade Dinitrotoluene (CASRN 25321-14-6) Superfund Health Risk Technical Support Center National Center for Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- AUTHORS, CONTRIBUTORS, AND REVIEWERS CHEMICAL MANAGER Zhongyu (June) Yan, PhD National Center for Environmental Assessment, Cincinnati, OH Q. Jay Zhao, PhD, DABT National Center for Environmental Assessment, Cincinnati, OH PRIMARY INTERNAL REVIEWERS Ghazi Dannan, PhD National Center for Environmental Assessment, Washington, DC Paul G. Reinhart, PhD, DABT National Center for Environmental Assessment, Research Triangle Park, NC This document was externally peer reviewed under contract to Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421-3136 Questions regarding the contents of this document may be directed to the U.S. EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). l Technical Grade Dinitrotoluene ------- TABLE OF CONTENTS COMMONLY USED ABBREVIATIONS iii BACKGROUND 1 DISCLAIMERS 1 QUESTIONS REGARDING PPRTVs 1 INTRODUCTION 2 REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) 7 HUMAN STUDIES 17 Oral Exposures 17 Inhalation Exposures 17 ANIMAL STUDIES 23 Oral Exposures 23 Inhalation Exposures 33 Other Data (Other Examinations) 34 DERIVATION OF PROVISIONAL REFERENCE DOSES 37 DERIVATION OF ORAL REFERENCE DOSES 38 Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 38 Derivation of Chronic Provisional RfD (Chronic p-RfD) 38 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 38 CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 38 MODE-OF-ACTION DISCI SSION 40 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 41 Derivation of Provisional Oral Slope Factor (p-OSF) 41 Derivation of Provisional Inhalation Unit Risk (p-IUR) 41 APPENDIX A. PROVISIONAL SCREENING VALUES 42 APPENDIX B. DATA TABLES 51 APPENDIX C. BMD MODELING RESULTS 75 APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE SCREENING p-OSF 84 APPENDIX E. REFERENCES 107 li Technical Grade Dinitrotoluene ------- COMMONLY USED ABBREVIATIONS BMC benchmark concentration BMCL benchmark concentration lower bound 95% confidence interval BMD benchmark dose BMDL benchmark dose lower bound 95% confidence interval HEC human equivalent concentration HED human equivalent dose IUR inhalation unit risk LOAEL lowest-observed-adverse-effect level LOAELadj LOAEL adjusted to continuous exposure duration LOAELhec LOAEL adjusted for dosimetric differences across species to a human NOAEL no-ob served-adverse-effect level NOAELadj NOAEL adjusted to continuous exposure duration NOAELhec NOAEL adjusted for dosimetric differences across species to a human NOEL no-ob served-effect level OSF oral slope factor p-IUR provisional inhalation unit risk POD point of departure p-OSF provisional oral slope factor p-RfC provisional reference concentration (inhalation) p-RfD provisional reference dose (oral) RfC reference concentration (inhalation) RfD reference dose (oral) UF uncertainty factor UFa animal-to-human uncertainty factor UFC composite uncertainty factor UFd incomplete-to-complete database uncertainty factor UFh interhuman uncertainty factor UFl LOAEL-to-NOAEL uncertainty factor UFS subchronic-to-chronic uncertainty factor WOE weight of evidence 111 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR TECHNICAL GRADE DINITROTOLUENE (CASRN 25321-14-6) BACKGROUND A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant scientific literature using established Agency guidance on human health toxicity value derivations. All PPRTV assessments receive internal review by a standing panel of National Center for Environment Assessment (NCEA) scientists and an independent external peer review by three scientific experts. The purpose of this document is to provide support for the hazard and dose-response assessment pertaining to chronic and subchronic exposures to substances of concern, to present the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to characterize the overall confidence in these conclusions and toxicity values. It is not intended to be a comprehensive treatise on the chemical or toxicological nature of this substance. The PPRTV review process provides needed toxicity values in a quick turnaround timeframe while maintaining scientific quality. PPRTV assessments are updated approximately on a 5-year cycle for new data or methodologies that might impact the toxicity values or characterization of potential for adverse human health effects and are revised as appropriate. It is important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current information available. When a final Integrated Risk Information System (IRIS) assessment is made publicly available on the Internet (http://www.epa.eov/iris). the respective PPRTVs are removed from the database. DISCLAIMERS The PPRTV document provides toxicity values and information about the adverse effects of the chemical and the evidence on which the value is based, including the strengths and limitations of the data. All users are advised to review the information provided in this document to ensure that the PPRTV used is appropriate for the types of exposures and circumstances at the site in question and the risk management decision that would be supported by the risk assessment. Other U.S. Environmental Protection Agency (EPA) programs or external parties who may choose to use PPRTVs are advised that Superfund resources will not generally be used to respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program. QUESTIONS REGARDING PPRTVs Questions regarding the contents and appropriate use of this PPRTV assessment should be directed to the EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). 1 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 INTRODUCTION Technical grade dinitrotoluene (tgDNT), CAS No. 25321-14-6, is a mixture of dinitrotoluene (DNT) isomers with the molecular formula C7H6N2O4 (MA1. 2011). tgDNT comprises predominantly 2,4-DNT and 2,6-DNT (approximated as 76% and 19%, respectively). The remaining 5% is a combination of the four other DNT isomers: 2,3-, 2,5-, 3,4-, and 3,5-DNT. tgDNT may also contain trace amounts of trinitrotoluene, cresols, mononitrobenzene, and mononitrotoluenes and is used in the production of toluene diisocyanate, which is used in the preparation of polyurethane products and in the manufacture of explosives (NLM. 2011). Table 1 provides physicochemical properties of tgDNT. Table 1. Physicochemical Properties of tgDNT (CASRN 25321-14-6)a Property (unit) Value Boiling point (°C) 250 Melting point (°C) ND Density (g/cm3 at 71°C) 1.32 Vapor pressure (mm Hg at 25°C) 3.97 x 1(T4 pH (unitless) ND Solubility in water (mg/L at 22°C) 2.7 x 102 Relative vapor density (air =1) 6.27 Molecular weight (g/mol) 182.134 aNLM ("20ID. ND = no data. IRIS has developed assessments for 2,4-DNT (approximately 98% 2,4-DNT and 2% 2,6-DNT; U.S. EPA. 1993) and for a 2,4-/2,6-DNT mixture (various compositions of DNTs; U.S. EPA. 1990). There is also a PPRTV assessment for 2,6-DNT (approximately 99% 2,6-DNT; U.S. EPA, 2013). Table 2 provides a summary of available toxicity values from the U.S. Environmental Protection Agency (U.S. EPA) and other agencies/organizations for tgDNT, the 2,4-DNT and 2,6-DNT isomers, and the 2,4-/2,6-DNT mixture. For the purpose of this PPRTV, only the toxicity of tgDNT (approximated as 76% 2,4-DNT and 19% 2,6-DNT) is evaluated. 2 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2), 2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a Source/ Parameterb'c tgDNT Value (approximately 76% 2,4-DNT and 19% 2,6-DNT) 2,4-DNT Value (approximately 98% 2,4-DNT and 2% 2,6-DNT) 2,6-DNT Value (approximately 99% 2,6-DNT) 2,4-/2,6-DNT Mixture Value (various compositions of DNTs) Notes Reference Date Accessed Cancer IRIS/OSF NV NV NV 6.8 x 10 1 per mg/kg-d IRIS entry is for 2,4-/2,6-DNT mixture with no CASRN; principal study used rats dosed with a mixture of 98% 2,4-DNT and 2% 2,6-DNT to determine OSF U.S. EPA (1990) 9-13-2012 IRIS/drinking water unit risk NV NV NV 1.9 x 10~5 per Hg/L IRIS entry is for 2,4-/2,6-DNT mixture with no CASRN; principal study used rats dosed with a mixture of 98% 2,4-DNT and 2% 2,6-DNT to determine OSF U.S. EPA (1990) 9-13-2012 HEAST NV NV NV NV None U.S. EPA (2003) 9-13-2012 IARC/cancer WOE NV NV NV NV Group 2B—Possibly carcinogenic to humans for 2,4- and 2,6-DNT IARC (1996) 9-13-2012 NTP NV NV NV NV None NTP (2011) 9-13-2012 Cal EPA/unit risk NV 8.9 x 1(T5 per Hg/m3 NV NV Data source was RCHAS-S Cal EPA (2009) 9-13-2012 Cal EPA/OSF NV 3.1 x 10 1 per mg/kg-d NV NV Data source was RCHAS-S Cal EPA (2009) 9-13-2012 ACGIH (cited in NLM. 2011) NV NV NV NV Group A3—Confirmed animal carcinogen with unknown relevance to humans for tgDNT, 2,4- and 2,6-DNT NLM (2011) 9-13-2012 Drinking Water/ cancer risk health advisory 5 x 1CT3 mg/L 5 x 10~3 mg/L 5 x 10~3 mg/L NV None U.S. EPA (2011a) 9-13-2012 3 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2), 2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a Source/ Parameterb'c tgDNT Value (approximately 76% 2,4-DNT and 19% 2,6-DNT) 2,4-DNT Value (approximately 98% 2,4-DNT and 2% 2,6-DNT) 2,6-DNT Value (approximately 99% 2,6-DNT) 2,4-/2,6-DNT Mixture Value (various compositions of DNTs) Notes Reference Date Accessed Health effect assessment 2.3 x lCT1 per mg/kg-dd and 2.1 x lO^per mg/kg-de 6.8 x 10 1 per mg/kg-df NV NV dBased on a 104-wk study in rats with increased incidence of liver tumors in males; "Based on a 104-wk study in rats with increased incidence of liver tumors in females; fBased on a 2-yr study in rats with increased incidence of combined mammary/hepatic tumors; U.S. EPA (1987) 2-6-2013 PPRTV NV NV 1.5 x 10° per mg/kg-d NV Based on a BMDL10Hed of 0.25 from a 52-wk study in rats with increased incidence of liver tumors in males U.S. EPA (2013) 4-3-2013 Noncancer ACGIH/TLV 0.2 mg/m3 NV NV NV NA NLM (2011) 9-13-2012 ATSDR/acute oral MRL NV 5 x 10~2 mg/kg-d NV NV Toxicological profile for 2,4-DNT; based on neurotoxicity in dogs ATSDR (1998) 11-21-2012 ATSDR/chronic or intermediate- duration oral MRL NV 2 x 10~3 mg/kg-d8 4 x 1() 3 mg/kg-dh NV 8Chronic oral MRL for 2,4-DNT; based on neurotoxicity, Heinz bodies, and biliary tract hyperplasia in dogs; hIntermediate-duration oral MRLfor 2,6-DNTbased on hematological effects of splenic extramedullary erythropoiesis and lymphoid depletion in dogs ATSDR (1998) 11-21-2012 Cal EPA/REL NV NV NV NV NA Cal EPA (2012a. b) 8-1-2012 4 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2), 2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a Source/ Parameterb'c tgDNT Value (approximately 76% 2,4-DNT and 19% 2,6-DNT) 2,4-DNT Value (approximately 98% 2,4-DNT and 2% 2,6-DNT) 2,6-DNT Value (approximately 99% 2,6-DNT) 2,4-/2,6-DNT Mixture Value (various compositions of DNTs) Notes Reference Date Accessed Drinking water NV 2 x 1(T3 mg/kg-d (1-d Health advisory) 1 x 1CT1 mg/L (Drinking water equivalent level) 1 x 10° mg/L (1- and 10-d Health advisory for a 10-kg child) 1 x 10 3 mg/kg-d (1-d Health advisory) 4 x 10 2 mg/L (Drinking water equivalent level) 4 x 1CT1 and 4 x l(T2 mg/L (1- and 10-d Health advisory for a 10-kg child) NV NA U.S. EPA (201 la) 2-6-2013 NIOSH/REL 1.5 mg/m3 NV NV NV TWA for 10-hr workday; document specifies CASRN for tgDNT but notes that various isomers of DNT exist NIOSH 9-13-2012 (2007) OSH A/PEL 1.5 mg/m3 NV NV NV TWA for 8-hr workday OSHA 9-13-2012 (2006) IRIS/Oral RfD NV 2 x 1(T3 mg/kg-d NV NV Based on a 2-yr study in dogs dosed with 98 2,4-DNT and 2% 2,6-DNT; critical effect of CNS neurotoxicity, Heinz bodies in erythrocytes, and hyperplasia of biliary tract U.S. EPA 9-13-2012 (1993) IRIS/Inhalation RfC NV NV NV NV None U.S. EPA (1990) 9-13-2012 HEAST/ subchronic Oral RfD NV 2 x icr3 mg/kg-d NV NV Based on a 2-yr study in dogs dosed with a mixture of 98% 2,4-DNT and 2% 2,6-DNT; critical effect of CNS neurotoxicity, Heinz bodies in erythrocytes, and hyperplasia of biliary tract U.S. EPA (2003) 9-13-2012 5 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2), 2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a Source/ Parameterb'c tgDNT Value (approximately 76% 2,4-DNT and 19% 2,6-DNT) 2,4-DNT Value (approximately 98% 2,4-DNT and 2% 2,6-DNT) 2,6-DNT Value (approximately 99% 2,6-DNT) 2,4-/2,6-DNT Mixture Value (various compositions of DNTs) Notes Reference Date Accessed Health effects assessment NV NV NV NV NA U.S. EPA (1987) 2-6-2013 PPRTV NV NV 3 x 10 3 mg/kg-d (screening subchronic p-RfD) 3 x 1() 4 mg/kg-d (screening chronic p-RfD) NV Based on a LOAELhed of 3 mg/kg-d for splenic extramedullary hematopoiesis in male and female dogs in a 13-wk oral study U.S. EPA 4-3-2013 (2013) CARA HEEP NV NV NV NV None U.S. EPA (1994) 9-13-2012 WHO NV NV NV NV None WHO (2012) 8-1-2012 aNo information was available from any source for 2,3-, 2,5-, 3,4-, and 3,5-DNT. bSources: Integrated Risk Information System (IRIS); Health Effects Assessment Summary Tables (HEAST); International Agency for Research on Cancer (IARC); National Toxicology Program (NTP); California Environmental Protection Agency (Cal EPA); American Conference of Governmental Industrial Hygienists (ACGIH); Agency for Toxic Substances and Disease Registry (ATSDR); National Institute for Occupational Safety and Health (NIOSH); Occupational Safety and Health Administration (OSHA); Chemical Assessments and Related Activities (CARA); Health and Environmental Effects Profile (HEEP); World Health Organization (WHO). Parameters: weight of evidence (WOE); reference dose (RfD); reference concentration (RfC); oral slope factor (OSF); minimum risk level (MRL); time-weighted average (TWA); reference exposure level (REL); permissible exposure limit (PEL); Reproductive and Cancer Hazard Assessment Section (RCHAS). d ''See notes column for corresponding information. NA = not applicable; NV = not available. 6 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Literature searches were conducted on sources published from 1900 through July 10, 2012 for studies relevant to the derivation of provisional toxicity values for tgDNT. The following databases were searched by chemical name, synonyms, or CAS No.: ACGIH, ANEUPL, AT SDR, BIO SIS, Cal EPA, CCRIS, CD AT, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HERO, HMTC, HSDB, IARC, INCHEM IPCS, IP A, ITER, IUCLID, LactMed, NIOSH, NTIS, NTP, OSHA, OPP/RED, PESTAB, PPBIB, PPRTV, PubMed (toxicology subset), RISKLINE, RTECS, TOXLINE, TRI, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, and U.S. EPA TSCATS/TSCATS2. The following databases were searched for relevant health information: ACGIH, AT SDR, Cal EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, U.S. EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS. REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER) Table 3 provides an overview of the relevant database for tgDNT and includes potentially relevant repeated long-term-, subchronic-, and chronic-duration studies. The phrase "statistical significance," used throughout the document, indicates ap-value of <0.05. 7 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Human 1. Oral Acuteb ND Short-term0 ND Long-termd ND Chronic" ND 2. Inhalation Acuteb ND Short-term0 ND Long-termd 154/0 workers, occupational survey, 12 mo, mixed isomers of DNT (unknown composition of DNT) NV Complaints such as unpleasant taste, weakness, headache, loss of appetite, dizziness, nausea, insomnia, pain in extremities, vomiting, and numbness and tinging; clinical signs included low-grade anemia, and cyanosis NV DU NV McGee et al. (1942) PR 8 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Long-termd 714 workers (sex not reported), occupational survey, 3 yr, mixed isomers of DNT (unknown composition of DNT) <1 mg/m3 Fewer complaints of unpleasant taste, weakness, headache, loss of appetite, dizziness, nausea, insomnia, pain in extremities, vomiting, and numbness and tinging; reduced incidences of low-grade anemia, and cyanosis compared to McGee et al. (1942) studv NV DU NV McGee et al. A follow-up study to McGee et al. (1942) PR (1947) 30/0 workers, occupational survey, exposure duration (varies), mixed isomers of DNT (unknown composition of DNT) and coexposed to toluene diamine (TDA) undetectable- 0.23 mg/m3 (personal sample); undetectable- 042 mg/m3 (area sample) Significant reduction in sperm count NC NC NC Alirenliolz (1980) NPR 9 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Long-termd 50/0 workers (nonsemen study) and 41/0 workers (semen study); occupational survey, exposure duration (varies), mixed isomers of DNT (approximately 80% 2,4-DNT and 20% 2,6-DNT) and coexposed to TDA 0.00929-0.318 mg/m3 (time-weighted average, TWA) No significant difference in serum enzymes, sperm volume, sperm counts and morphological changes in workers in exposed group, and spontaneous abortions in their wives 0.0739 mg/m3 (mean, TWA) DU NV Alirenliolz and Mever (1982) NPR 203/0 workers, occupational survey, >6 mo, coexposure to DNT and TDA, mixed isomers of DNT (unknown composition of DNT) <1.5 mg/m3 (Permissible Exposure Limit, PEL) No significant differences in work history, medical history, physical examination, reproductive history, fertility, laboratory serum (follicle- stimulating hormone), mean sperm count, and sperm morphology in exposed group NV DU NV Hamill et al. (1982) Investigated only male reproductive and fertility endpoints PR 208/10 workers, occupational study, no specified exposure duration, DNT/TDA, mixed isomers of DNT (unknown composition of DNT) NV No significant associations found for male reproductive toxicity NV DU NV Levine (1983) Exposure-related information on fertility among female employees was insufficient to analyze NPR Reproductive study 10 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Long-termd 586 workers (sex not reported), retrospective survey, DNT/TDA mixed isomers of DNT (unknown composition of DNT) No significant difference was found between the fertility of workers exposed to DNT in the three U.S. chemical plants and the fertility of unexposed workers NV DU NV Levine et al. PR (1985) 156/0 and 301/0 (two cohorts from two different plants), occupational cohort study, exposure at least 30 d during 1950s for the first cohort; exposure for >30 d during the 1940s and 1950s for the second cohort. 76% 2,4-DNT, 19% 2,6-DNT, and 5% other isomers for the first cohort; 98% 2,4-DNT and 1% 2,6-DNT for the second cohort NV For total workers in both plants, no significant increases in death from any specific types of cancer were observed. Excess mortality from ischemic heart disease (IHD) at both plants with standardized mortality ratios (SMRs) of 13 land 143 (95% confidence intervals [CI]s: 65-234, and 107-187, respectively) NV NU NA Levine et al. (1986) Additional analyses revealed al5-yr latent period and suggested a relationship between duration and intensity of exposure PR 11 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry3 Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Long-termd 4989 exposed (M)/5636 unexposed workers (M), occupational cardiovascular mortality study, participants exposed for >5 mo, (unknown composition of DNT) NV No significant association for an increased risk of either IHD or cerebrovascular disease NV NU NA Stavner et al. (1992) PR 4989 exposed (M)/7436 unexposed (M), occupational carcinogenicity study, participants exposed for >5 mo, (unknown composition of DNT) NV Excess of hepatobiliary cancer in exposed workers; standardized mortality ratio (SMR) of 2.67 (95% confidence interval [CI]: 0.98-5.83) when compared with U.S. population; standard rate ratio (SRR) of 3.88 (95% CI: 1.04-14.41) when compared with unexposed group NV NU NV Stavner et al. (1993) Failed to demonstrate an exposure- response relationship between duration of exposure and hepatobiliary cancer mortality; limited by small number of participants with long exposure durations. PR The study subjects were selected from the second plant examined by Levi tie et al. (1986) Chronic6 500 workers (sex not reported), occupational carcinogenicity study, exposed for 7 to 37 yr, 30% tgDNT, which consisted of 75% 2,4-DNT and 20% 2,6-DNT. NV High exposure to DNT related to urothelial cancer NV NU NV Bruiting et al. PR Retrospective study (1999) 12 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Chronic" 180 workers exposed for 7 to 37 yr (sex not reported), 30% tgDNT, which consisted of 75% 2,4-DNT and 20% 2,6-DNT NV A straight dose-dependent pathological (tubular and/or glomerular damage) pattern; nephrotoxic effect toward the proximal tubule under the exposure conditions NV NU NV Bruning et al. PR (2001) Three case studies(sex not reported); >7 yr, exposure to DNT and possible exposure to nitrobenzene, mixed isomers of DNT (unknown composition of DNT) High exposure to DNT associated with human urothelial cancer NV NU NV Hartli et al. (2005) PR Animal 1. Oral Subchronic 10/10, albino Fischer 344 (F344), rat, diet, 4 wk M: 0,31.9,61.9, or 134 F: 0, 32.0, 63.6, or 120 Increases in hematological parameters, including methemoglobin (MetHb), reticulocytes, and Heinz bodies in males NDr NV 31.9 CUT (1983) NPR 13 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Chronic 10/10 (Study initial 130/130), F344, rat, diet, 26 wk M: 0,3.47,13.6, or 34.6 F: 0,3.22,13.9, or 34.9 Increased absolute and relative liver weights and increased hepatotoxicity in males 3.47 2.16 based on increased incidence of hepatocyte necrosis in males 13.6 CUT f1982a) NPR, PS 10/10, F344, rat, diet, 52 wk M: 0,3.47, 13.9, or 34.9 F: 0, 3.46, 13.9, or 35.1 Increased absolute and relative liver weight and hepatotoxicity in males NDr NU 3.47 CUT f 1982a) NPR 20/20, F344, rat, diet, 55 wk M: 34.9 F: 35.1 Hepatotoxicity in both males and females NDr NV NDr CUT f 1982a) All surviving high dose rats were terminated at 55 wk due to severe toxicity. NPR 20/20, F344, rat, diet, 78 wk M: 0, 3.49, or 14.0 F: 0, 3.45, or 14.0 Increased relative liver weight and hepatotoxicity in females NDr NU 3.45 CUT f 1982a) NPR 75-87/84-87, F344, rat, diet, 104 wk M: 0,3.51, or 14.0 F: 0,3.46, or 14.0 Increased absolute and relative liver weights and hepatotoxicity in females NDr 0.363 based on increased incidence of hepatocyte necrosis in males 3.46 CUT f1982a) NPR, PS 14 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL BMDL/ BMCL LOAEL Reference Comments Notes Chronic 28/0, F344, rat, diet, 52 wk 0,35 Decreased body weight, increased absolute and relative liver weights, accompanied by pathological findings in the liver and bile duct NDr NU 35 Leonard et al. PR (1987) Developmental Female, 22 control, 13, 7, 13,7, 13, 6 for treatment groups, respectively, F344 rat, gavage, GDs 7-20 0, 14,35,37.5, 75, 100, or 150 Increased maternal relative liver weight (maternal effects); increased resorption at 150 mg/kg-d (fetal effects) Maternal: 37.5 Fetal: 100 NU Maternal:75 Fetal: 150 Price et al. (1985) CUT (1982b) PR NPR Reproductive ND Carcinogenic 28/0, F344 rat, diet, 52 wk ADD: 0,35 HED: 0, 9.3 47% increase in incidence of hepatocellular carcinomas compared with controls NV NV NV Leonard et al. (1987) PR 75-87/84-87, F344, rat, diet, 104 wk ADD: 0,3.51, or 14 (M); 0, 3.46, or 14.03 (F) HED: 0,0.922, or 3.45 (M); 0, 0.851, or 3.23 (F) Dose-dependent increase in hepatocellular carcinomas and neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas NV BMDLiohed: 0.224 NV CUT (1982a) NPR, PS 15 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6) Number of Male/Female, Strain, Species, Study Type, BMDL/ Category Study Duration Dosimetry" Critical Effects NOAEL BMCL LOAEL Reference Comments Notes 2. Inhalation Subchronic ND Chronic ND Developmental ND Reproductive ND Carcinogenic ND ""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects. Values are also presented as a human equivalent dose (HED in mg/kg-d) for oral carcinogenic effects. All long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly) exposure. ''Acute = exposure for <24 hr (U.S. EPA. 2002). °Short-term = repeated exposure for >24 hr <30 d (U.S. EPA. 2002). 'Long-term = repeated exposure for >30 d <10% lifespan (based on 70-yr typical lifespan) (U.S. EPA. 2002). "Chronic = repeated exposure for >10% lifespan (U.S. EPA. 2002). Significant (increase/decrease/difference) means either statistically or biologically significant (increase/decrease/difference) in this document. GD = gestation day, ND = no data, NA = not applicable, NV = not available, NDr = not determinable, NC = not calculated, NPR = not peer-reviewed, PR = peer-reviewed, PS = principal study. 16 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 HUMAN STUDIES Oral Exposures No studies were identified. Inhalation Exposures Relevant data are available from epidemiological studies on the effects in humans of inhalation exposure to 2,4/2,6-DNT mixtures of various compositions. These effects have been evaluated in several occupational studies of workers in DNT manufacturing plants in which exposures were identified by the study authors to be predominantly via the inhalation route with contributions from the dermal route. No details on the exposure concentrations to DNT are given in these studies, and, therefore, they can only be used as qualitative descriptions of symptoms reported upon exposure. Also, in some of the studies, the isomer composition of the DNT mixture was not specified. The identified studies from occupational exposure to DNT have examined the clinical long-term effects (McGee et al.. 1947; McGee et al.. 1942). the potential for adverse reproductive effects (Levine et al, 1985; Levins, 1983; Ahrenholz and Meyer, 1982; Ham ill et al.. 1982; Ahrenholz, 1980). adverse effects on the cardiovascular system and carcinogenic risk (Harth et al.. 2005; Brunine et al.. 2001; Bruning et al. 1999; Stavner et al.. 1993; Stavner et al.. 1992; Levine et al.. 1986). Epidemiological Studies of General Toxicity McGee et al. (1942) and McGee et al. (1947) In an epidemiological study performed by McGee et al. (1942). 154 male workers in a military plant that manufactured powder containing a DNT mixture (primarily 2,4-DNT) were observed for 12 months. In the 12-month period, 96 individuals reported complaints of unpleasant taste (62%), 78 reported weakness (51%), 76 reported headache (49%), 72 reported loss of appetite (47%), 68 reported dizziness (44%), 57 reported nausea (37%), 57 reported insomnia (37%), 40 reported pain in extremities (26%), 35 reported vomiting (23%), and 29 reported numbness and tinging (19%). Additionally, 84 individuals exhibited clinical signs of sickness, which included pallor (36%), cyanosis (34%), and anemia (23%). These symptoms are consistent with methemoglobinemia and disappeared 2 to 3 days after exposure to the powder was terminated. The study authors also reported two instances of acute toxic hepatitis with jaundice. After an effort was initiated to reduce the exposure between 1942 and 1945, a follow-up study by the same investigators in the same plant (McGee et al.. 1947) evaluated 714 workers "3 (sex not reported) who were exposed to less than 1 mg/m DNT. Each of the individuals received medical examinations at intervals of 2 to 4 weeks. From these examinations, the study authors reported that signs and symptoms of illness were noticeably decreased when compared to the signs and symptoms experienced by the 154 workers from the 12-month study in 1942. Only 13.2%) and 8.7% of men from the follow up study reported weakness and headaches, respectively, while around 50% of the men from the 1942 study reported the same effects. The reports of loss of appetite, nausea and vomiting, vertigo, pain, or tingling/numbing in the extremities were also reduced in this follow-up study as compared to the initial study. Pallor was rarely observed in the follow-up study, no hepatitis was observed, and a marked reduction in cyanosis (8.7%) and anemia (10.2%) was reported as well. The study authors (McGee et al.. 1947; McGee et al.. 1942) did not provide detailed DNT compositions or exposure data, and no unexposed control groups were used as a basis for comparison. 17 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Epidemiological Studies of Reproductive Effects Ahrenholz (1980) and Ahrenholz and Meyer (1982) Ahrenholz (1980) reported on the potential for reproductive effects in male workers exposed to mixed isomers of DNT (composition of DNT was not reported, and exposure duration varied) and toluene diamine (TDA) in a TDA plant at Olin Chemical Company in Brandenburg, KY. TDA was produced through catalytic hydrogenation of DNT. The study authors conducted environmental and medical surveys in September 1979, and a follow-up investigation was conducted in January 1980. During both the initial and follow-up surveys, personal air samples were taken by mounting sample collection media in the operators' breathing zones. Area air sampling was also conducted. Medical evaluations consisting of a detailed questionnaire were used to acquire information on a range of potentially toxic effects. In addition, tobacco and alcohol consumption and medical history were recorded. A reproductive history was also elicited. The wives of workers were given a different, more detailed reproductive questionnaire in an attempt to validate the information given by the workers themselves. A physical examination with a special emphasis on the male reproductive system and secondary sex characteristics was performed. Blood specimens were obtained for analyses of blood urea nitrogen (BUN), creatinine, bilirubin, alkaline phosphatase, serum glutamic oxalic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum testosterone, serum luteinizing hormone, and serum follicle stimulating hormone. A semen specimen was also collected and analyzed for volume, sperm count, and morphologic pattern. The male workers that participated in the surveys were divided into three exposure groups: (1) exposed, (2) intermediate exposed (contact with DNT for one to several years on an intermittent basis but with no exposure for the last 2 years), and (3) unexposed (no exposure during their employment). The exposure groupings were determined by reviewing job descriptions and by discussing exposures with the individuals, the company, and the local union representatives. The study group consisted of 30 male workers (9 from the exposed group, 12 from the intermediate exposed group, and 9 from the unexposed group). However, only seven total personal samples and three area samples were collected with the concentration ranging from not 3 3 detectable to 0.23 mg/m (personal samples) and from not detectable to 0.42 mg/m (area samples). The author stated that the concentrations of DNT in the plant were below the OSHA "3 standard PEL of 1.5 mg/m over an 8-hour workday. Serum examinations of renal and hepatic profiles indicated no significant difference between any of the tested groups. A slight increase in miscarriages among the wives of the exposed workers was found but could not be conclusively related to the exposures. There were no significant differences in congenital defects and total pregnancies between the groups. However, sperm counts in the exposed group were significantly lower compared to the unexposed group. No significant differences were reported between unexposed and intermediate exposed groups or between intermediate exposed and exposed groups. The study author concluded that the results were strongly suggestive of a reproductive problem but more workers needed to be evaluated. Ahrenholz and colleagues (Ahrenholz and Meyer, 1982) also conducted a similar study at Olin Chemical Company, in Moundsville, WV where male workers were exposed to both DNT and TDA. DNT was reported in an approximate 80:20 ratio of 2,4-DNT and 2,6-DNT. Fifty males (in the nonsemen portion of the study) and 41 of these 50 workers (in the semen portion of the study) were divided into three groups based on the same criteria in Ahrenholz (1980). The exposure duration ranged from 3 to 27.5 years. Due to laboratory error in preparation of the sampling media, personal exposure data were determined to be invalid. Seven DNT area 18 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 3 3 3 samples ranged in concentration from 0.026 mg/m (0.00929 mg/m , TWA) to 0.89 mg/m (0.318 mg/m3, TWA) with a mean of 0.207 mg/m3 (0.0739 mg/m3, TWA). The study authors reported no significant difference between the exposed and unexposed groups in serum enzymes, sperm volume, sperm counts, and morphological changes. The questionnaire data on the employees' reproductive history did not show statistically significant differences in the number of spontaneous abortions in wives of workers employed in the DNT area when compared with non-DNT exposed workers in the plant. A NOAEL of 0.0739 mg/m is identified based on no significant differences in serum enzymes, sperm volume, sperm counts and morphological changes in workers in the exposed group, and spontaneous abortions in their wives. The studies conducted by Ahrenholz (1980) and Ahrenholz and Mever (1982) were limited by small sample size, limited exposure data, problematic grouping, coexposure with TDA and other unknown chemicals, and unknown composition of DNT (Ahrenholz. 1980). Hamill et at. (1982) Hamill et al. (1982) conducted a study to determine the reproductive effects of occupational DNT and/or TDA exposure among 203 male workers from a chemical complex in Lake Charles, LA. Of the 203 employees in the cohort, 84 were exposed to DNT and/or TDA (exposure level within the OSHA PEL of 1.5 mg/m within an 8-hour workday, detailed exposure data and composition of DNT were not reported), and 119 were not exposed. Each worker was subjected to a semen analysis for sperm count and morphology, blood testing (for serum follicle-stimulating hormone [FSH] measurement), a urogenital examination, an estimation of testicular volume, and an interview. Information was also acquired on reproductive history, medical and surgical history, past and present work history, assessment of exposure, exposure to other chemicals with potential reproductive toxicity, smoking habits, alcohol consumption, and recent medication. Data were collected between January and June 1981. Based on exposure history (intensity, frequency, and how recent exposure to DNT and/or TDA occurred), the participants were classified into four groups: (1) none to minimal, (2) low to high, (3) low to moderate, and (4) high. The duration was at least 6 months. No significant differences were observed between the exposure groups with respect to work history, medical history, or physical examination characteristics. Additionally, there were no significant differences in reproductive histories, or decreases in fertility related to DNT and/or TDA exposure. Finally, no significant differences were discovered in the laboratory findings including serum FSH, mean sperm count, and sperm morphology. The study authors concluded that both TDA and DNT did not present a detectable reproductive hazard to the workers. However, the study is limited due to lack of detailed exposure data, exposure to mixed chemicals, and unknown composition of DNT. Levine (1983) Levine (1983) investigated the effect of DNT exposure on the fertility of workers occupationally exposed to a DNT mixture (composition not further specified; exposure duration not reported) in a U.S. toluene diisocyanate (TDI) plant. DNT and TDA are intermediates in the manufacturing of TDI. DNT was used to manufacture TDA in the plants and data were collected in 1981. Due to the job rotation, it was assumed that there was a coexposure to both DNT and TDA. A total of 208 male (166 white, 42 nonwhite) and 10 married female workers were interviewed. Exposure-related fertility among female employees was insufficient to analyze. The fertility analysis for male employees revealed that their wives exhibited no evidence of an 19 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 abnormal aggregation of miscarriages, stillbirths, neonatal deaths, or birth defects. This study revealed little to suggest that occupational exposure to DNT may have affected reproduction adversely and is limited due to lack of comparison with unexposed male employees, lack of data regarding quantitative exposure, exposure to mixed chemicals and other unknown chemicals, and unknown composition of DNT. Levine et al (1985) Levine et al. (1985) investigated the effect of DNT exposure on the fertility of workers occupationally exposed to a DNT mixture (composition not further specified) in three U.S. chemical plants. The plants manufactured TDA, DNT, and/or TDI. Data were collected between 1979 and 1981, several years after the exposure period (between 1973 and 1976). A total of 586 workers (144 from Plant A, 207 from Plant B, and 235 from Plant C; sex not reported) were interviewed. No significant difference was found between the fertility of workers who were exposed to DNT in the three U.S. chemical plants and the fertility of unexposed workers. The study is limited due to the lack of quantitative exposure data provided, exposure to mixed and other unknown chemicals, and unknown composition of DNT. Epidemiological Studies of Carcinogenicity or Cardiovascular Diseases Levine et al (1986) Levine et al. (1986) evaluated workers at two army ammunition plants to assess the relationship between exposure to DNT and carcinogenicity. In the first plant, located in Joliet, IL, tgDNT (approximately 76% 2,4-DNT, 19% 2,6-DNT, and 5% other isomers) was manufactured and purified to at least 98% 2,4-DNT and about 1% 2,6-DNT. From the first plant, a total of 156 men, who worked in the DNT production line for at least 30 days during the 1950s, participated in the study. At the second plant, located in Radford, VA, the purified DNT (98%) 2,4-DNT and approximately 1% 2,6-DNT) was used in certain single-based propellant formulations. This cohort consisted of 301 men who had worked for 30 days or more during the 1940s and 1950s in specific jobs that had potential for DNT exposure. Workers from both plants were presumed white males and considered exposed to DNT via the inhalation and dermal routes. The exposure levels from the first plant were judged by the study authors to be high. Jobs at the second plant were categorized by plant technical personnel according to opportunity for exposure: high, moderate, low or none. Cohort mortality was followed from enrollment through the end of 1980. Numbers of observed and expected deaths were recorded for each underlying cause and the standardized mortality ratio (SMR) was computed using mortality rates of U.S. white males as the standard. Of the 457 men in both plants, 164 had died compared to 127 expected deaths using mortality rates of U.S. white males as the standard. The combined SMR of 129 (p = 0.001) for all causes of death was significantly high, and it increased to an SMR of 140 (p = 0.00007) after 15 years elapsed since entry into the study. This increase in overall mortality was attributed to increased death from disease of the circulatory system (SMR: 140,/? = 0.002) or due to death from accidents, poisonings, and violence (SMR: 191, p = 0.0007). Death as a result of malignant neoplasms was less than the expected mortality rate (SMR: 87); however, this decrease was not significant. No significant increases in death from any specific types of cancer were observed. Increased mortality from disease of the circulatory system was determined to be primarily based on an increase in mortality from ischemic heart disease (IHD) (SMRs of 131 and 143 for the first and the second plant, respectively; 95% confidence intervals of 65-234 and 107-187 for the first 20 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 and the second plant, respectively). Deaths from IHD remained high even when compared with expected numbers derived using mortality rates of the counties in which the plants were located. The relationship between mortality from IHD to duration and intensity of DNT exposure was analyzed at 15 years from entry into the study onward. The results are suggestive of a dose- and duration-response relationship for DNT and mortality from IHD. The study authors suggested that the increase in mortality from heart disease was a result of damage to the coronary arteries from exposure to DNT. The study is limited due to lack of exposure data, exposure to other unknown chemicals, and lack of unexposed controls. Stavner et al. (1992) Stayner et al. (1992) performed a retrospective cohort study using current and former white male workers from a propellant production facility in Radford, VA. The study aimed to determine the possible relationship between exposure to DNT (composition of DNT is not reported) and the risk of death as a result of cardiovascular disease including ischemic heart IHD and cerebrovascular disease. A total of 4989 workers with probable exposure to DNT and 5636 unexposed workers were selected for the study (workers' operations were rated concerning the probability of exposure to DNT). All exposed and unexposed workers who participated in the study had been employed for at least 5 months at the study plant between January 1949 and January 1980. The difference in mortality between the cohorts (exposed and unexposed groups) and the U.S. population was evaluated using SMRs. SMRs were also used to evaluate specific causes of death in addition to standardized rate ratios (SRRs), which are ratios of observed deaths in the exposed groups to the observed deaths in the unexposed groups. Death from all causes (including cardiovascular and noncardiovascular causes) was similar for DNT-exposed (SMR: 1.00) and unexposed (SMR: 0.99) groups when compared to the U.S. population. Mortality from cerebrovascular disease in the DNT-exposed group was less than that in the unexposed group (SMR: 0.95, SRR: 0.89). IHD mortality in the DNT-exposed group was similar to that of the unexposed group (SMR: 0.98, SRR: 0.99). Hypertension without heart disease (SMR: 1.17) and other myocardial degeneration (SMR: 1.41) were slightly elevated in the DNT-exposed group. The study authors concluded that DNT exposure did not appear to be associated with an increased risk of either IHD or cerebrovascular disease. The study authors also concluded that potential biases related to the company's medical screening program for workers exposed to DNT may have limited the ability to detect these effects. However, the study authors did not provide exposure or DNT composition data, and the definition of DNT-exposed groups was not clear. Stavner et al. (1993) Stavner et al. (1993) investigated the relationship between workers exposed to DNT and cancer of the liver and biliary tract. The cohort was selected from the second plant examined by Levine et al. (19861 but with more subjects who were exposed to DNT. A total of 4989 male workers exposed to DNT and 7436 unexposed male workers were included in this investigation. All the enrolled workers had worked at least 5 months at the study facility between January 1949 and January 1980. The vital status (i.e., whether dead or alive) of the workers at the end of 1982 was collected and used to develop SMRs and SRRs to analyze the various relationships between DNT exposure and mortality. 21 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Mortality for the study cohort was compared to the expected mortality for the U.S. population. Death as a result of cancer was less than expected for the DNT exposed and unexposed groups (SMRs of 0.84 and 0.78, respectively). An increase in hepatobiliary cancers (defined as biliary, liver, and gall bladder cancers combined) was observed in the DNT-exposed cohort as compared to the U.S. population (SMR: 2.67, 95% CI = 0.98-5.83) (but this was identified as borderline statistically significant \p = 0.052]). When compared to unexposed workers of the same facility, however, the SRR for hepatobiliary cancers in the DNT-exposed group was significantly increased (SRR: 3.88, 95% CI = 1.04-14.41,/? = 0.04). No exposure-response relationship was detected between the duration of exposure to DNT and hepatobiliary cancer mortality. According to the investigators, the study had several limitations, mainly that it was originally designed to evaluate the risk associated with exposure to nitroglycerin (which was also manufactured at the plant) rather than DNT. Other limitations included the small number of hepatobiliary cancer cases (six), the small number of workers with a long exposure period to DNT, and the lack of quantitative DNT exposure data. Worker exposure was classified qualitatively, and those workers that were "probably" exposed were included in the exposed group. Individuals in this group may have had minimal contact with DNT, as the group was defined only by having contact with materials containing DNT as opposed to being exposed to DNT directly. Therefore, inclusion of these individuals could have introduced bias by preventing the detection of the true level of excess risk for hepatobiliary cancer following exposure to DNT. Another limitation identified by the study authors was the possibility of exposure to chemicals other than DNT. Nevertheless, the study authors concluded that the excess in hepatobiliary cancer mortality observed among DNT-exposed workers in this study added some support to the hypothesis that occupational exposure to DNT may be carcinogenic. The authors noted that this study investigated more subjects, and hence, it has more statistical power to detect an excess of hepatobiliary cancer than the Levine et al. (1986) study. Bruningetal. (1999) Bruning et al. (1999) performed a retrospective survey on underground miners who were formerly exposed to an explosive (Donarit) containing 30% tgDNT, which consisted of approximately 75% 2,4-DNT and 20% 2,6-DNT (the remaining 5% was unknown). The cohort was selected from a mining area in Mansfeld, which is located in the former German Democratic Republic (GDR). Health records were used to identify miners with former exposures to DNT (n = 500) and their incidence of urogenital malignant diseases. Of these 500 subjects (sex not reported), a group of 340 miners with available information on malignant urogenital tract disease was asked to participate. Among the 340 miners, 183 gave their consent and were subjected to a standard medical examination and a retrospective occupational exposure assessment. Additional information was obtained on occupational histories, including exposures to any type of hazardous chemicals, smoking histories, history of former kidney and renal diseases, as well as history of cancers within families. The study authors reported that the miners had been exposed through two routes: inhalation of the smoke after explosions and skin contact with DNT-containing explosive sticks. The exposures were ranked into low, medium, high, and very high exposure categories. Between 1984 and 1997, 14 cases of renal cell cancer and 6 cases of urothelial cancer were identified in the group of 500 underground miners with former exposure to DNT. Exposure duration ranged from 7-37 years, and latency periods ranged from 21-46 years. The incidences of urothelial and 22 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 renal cancer in this group were 4.5 and 14.3 times higher than anticipated, respectively, on the basis of the cancer registers of the GDR. The exposure categorization of the 14 renal cell cancer cases revealed a distribution (i.e., number of workers in low, medium, high and very high exposure categories) similar to that of the 183 exposed miners without cancer. However, the six cases of urothelial cancer were predominantly confined to the high exposure category. The study authors concluded that high exposure to DNT might be associated with urothelial tumor formation. The study was limited due to the lack of quantitative exposure data and coexposure to other unknown chemicals. Bruninz et al (2001) In another study, Brunine et al. (2001) investigated signs of subclinical renal damage in the same subjects that were reported in Brunine et al. (19991 consisting of a group of 161 no cancer miners and 19 cases with renal (n= 14) or urothelial cancer (n = 5), all of whom had been exposed to explosives containing tgDNT [the same exposure duration and DNT composition information as presented in Burning et al. (1999)1. The exposures were categorized semiquantitatively, according to the type and duration of contact with DNT, into low, medium, high, and very high. Evaluation of urinary protein excretion patterns indicated that there was a straight dose-dependent relationship in the pathology of tubular and/or glomerular damage, indicating that DNT-induced damage is directed toward the renal tubular system. In addition, there was a dose-dependent increase in the biomarkers of alphal-microglobulin and glutathione S-transferase alpha, indicating a nephrotoxic effect toward the proximal tubule under the exposure conditions. By contrast, there was no similar change in glutathione S-transferase pi, indicating no nephrotoxicity to the distal tubule. The study was limited due to the lack of exposure data and coexposure to other unknown chemicals. Earth et al. (2005) In this case report, Harth et al. (2005) reported a cluster of three cases of urothelial cancer (sex not reported) among a group of about approximately 60 workers who were exposed to DNTs during manufacture of a DNT explosive (Donarit) at a factory in the former GDR. The cases occurred within a period of 12 years (1990-2002) leading to a 15.9-fold higher incidence of cancer of the urinary bladder than that of the federal state where the chemical factory was located, even though no adjustments were made for age and smoking. From 1970 until 1974, the production of DNT and nitrobenzene was located in one building, raising the possibility of coexposure to nitrobenzene during this period. The exposure durations for the three cases were longer than 7 years. The observation of the cluster of urothelial cancer in people highly exposed to DNTs underlines the possibility of human carcinogenicity of DNTs, with the human urothelium as a relevant target tissue. The study was limited due to the lack of exposure data, unknown composition of DNT, and coexposure to other unknown chemicals. ANIMAL STUDIES Oral Exposures The effects of oral exposure of animals to tgDNT have been evaluated in one subchronic (CUT. 1983). two chronic, (Leonard et al.. 1987; CUT. 1982a) one developmental (Price et al.. 1985; CUT. 1982b). and two carcinogenicity studies (Leonard et al.. 1987; CUT. 1982a). 23 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Subchronic Studies cut a983) In an unpublished, nonpeer-reviewed study, CUT (1983) administered tgDNT (consisting of 76.49% 2,4-DNT, 18.83% 2,6-DNT, 2.43% 3,4-DNT, 1.54% 2,3-DNT, 0.65% 2,5-DNT, and 0.040%) 3,5-DNT) via diet to groups of 10 albino F344 rats/sex/group for 4 weeks. The nominal doses were 0, 37.5, 75, or 150 mg/kg-day per group. Based on weekly tgDNT consumption provided by the study authors, the adjusted daily doses (ADDs, calculated based on TWA doses) are 0, 31.9, 61.9, or 134 mg/kg-day for males and 0, 32.0, 63.6, or 120 mg/kg-day for females. Fresh diets were prepared weekly and adjusted based on body weight and food consumption data. No certificate of good laboratory practice (GLP) was included in the study report. The study authors observed animals for signs of morbidity and mortality twice daily and recorded signs of gross toxicity and/or pharmacologic effects, food consumption, and individual body weights weekly. Blood samples were collected from nonfasted females and males on Days 27 and 28, respectively. Blood samples were analyzed for methemoglobin (MetHb), reticulocytes, and Heinz bodies. After sacrifice, gross pathological examinations were performed on the lungs, liver, spleen, kidneys, ovaries, and vagina. Organ weights were not measured. No treatment-related mortalities occurred during the study period. The only clinical signs noted by the study authors were alopecia around the right eye of two females in the low-dose group and urine stains on the fur of four high-dose females at Week 3 and two high-dose females at Week 4. At Weeks 3 and 4, body weights of females treated with 120 mg/kg-day were significantly decreased by 17% and 21%, respectively. All the high-dose treated males experienced a significant reduction in mean body weight in a time-dependent manner compared with controls (10%, 26%, 36%, and 38% reductions compared to controls at Weeks 1, 2, 3, and 4, respectively). In addition, body weights of the mid-dose males were significantly decreased by 11% and 17% compared with controls at Weeks 3 and 4, respectively (see Table B. 1). Food consumption was reduced in all treatment groups compared with controls in a dose-dependent manner (data not shown). Therefore, the decreased body weights were likely related to the reduction in food consumption in addition to tgDNT treatment. Dose-dependent, significant increases in mean reticulocytes and Heinz bodies were reported in all treated animals (see Table B.2). MetHb was significantly increased in the low and high-dose females and in the high-dose males (see Table B.2). The study authors noted the following gross pathological observations in high-dose females and in low- and high-dose males: discoloration (yellow), mottled appearance, and/or rough or granular surface of the liver. Discoloration (green) of the kidneys was observed in high-dose males and females. Dark yellow vaginal stains were also noted in two high-dose females, and an ovarian cyst was found in one mid-dose female. Table B.3 presents the gross pathology results. Based on the significant hematological changes in male rats, a LOAEL of 31.9 mg/kg-day is identified. Data preclude identification of a NOAEL. Chronic Studies CUT (1982a) CUT (1982a) is selected as the principal study for deriving the screening subchronic and chronic p-RfDs. CUT (1982a) administered tgDNT (consisting of 76.5% 2,4-DNT, 24 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 18.8% 2,6-DNT, 2.4% 3,4-DNT, and <2.3% 2,3-, 2,5-, and 3,5-DNT) to groups of 130 F344 rats/sex/group (Charles River Breeding Laboratories, Wilmington, MA) via diet at target dose levels of 0, 3.5, or 14.0 mg/kg-day for 2 years, or 35 mg/kg-day for 55 weeks (sacrificed early due to treatment-related incidence of liver tumors). The control group rats received a basal diet only. The study authors conducted interim sacrifices at 26 weeks, 52 weeks, 55 weeks (for high-dose group only), 78 weeks, and 104 weeks at termination. For each dose group, a ADD was calculated based on tgDNT concentration in food, food consumption, and time periods (calculated based on TWA) provided by the study authors. The calculated ADDs are shown in each data summary table (see Tables B.4 to B.15). The test diets were prepared weekly based on measured body weight and food consumption data to ensure constant intake of tgDNT on a mg/kg body-weight basis. The study authors did not provide a statement confirming GLP status. Animals were examined twice daily for general physical appearance, mortality, and morbidity. The study authors recorded food consumption in each cage and individual body weights every week for the first 14 weeks, biweekly for the following 12 weeks, and every fourth week for the remainder of the test period. Mean daily tgDNT consumption was calculated for each dose level weekly from Weeks 1 through 14, biweekly through Week 26, and monthly for the remainder of the treatment period. Hematology, clinical chemistry, and urinalyses were performed on 10 animals/sex/group at Weeks 26 and 52, and on 20 animals/sex/group at Weeks 78 and 104. An additional 20 animals from the high-dose group were tested for hematology and clinical chemistry at Week 55. The measured hematological parameters included hematocrit, hemoglobin (Hb), and MetHb; red blood cell (RBC), reticulocyte, Heinz body, total white blood cell (WBC), and differential leukocyte counts; and mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). Serum samples were also analyzed for clinical chemistry, including alkaline phosphatase (ALP), BUN, and SGPT. Urinalysis measured appearance, pH, specific gravity, glucose (GLU), ketone, total protein (TPR), occult blood, and sediment. Ophthalmologic examinations were performed on both eyes of each animal using an indirect ophthalmoscope 1 or 2 days prior to sacrifice at 26, 52, 78, and 104 weeks. Ten rats/sex/group were sacrificed at Weeks 26 and 52. At Week 55, all surviving high-dose rats were sacrificed due to severe toxicity (high incidence of tumors). At Week 78, 20 rats/sex/ group were sacrificed, and at Week 104, all surviving rats were sacrificed. The study authors performed examinations for gross signs of toxicity and incidence and recorded the location of tumors at identical intervals. At necropsy, the following organs were excised and weighed: brain, heart, liver, kidneys, lungs, and testes with epididymides. Ovaries were not weighed until after fixation. The following tissues were examined histopathologically from animals in the control and high-dose groups at Weeks 26 and 52, in the control and mid-dose groups at Weeks 78 and 104, and in the high-dose groups at Week 55: brain (cerebellum, cerebrum, brain stem), eyes, testes with epididymides, thoracic spinal cord, pituitary, thyroid, parathyroid, adrenal, heart, aorta, lungs, spleen, liver, kidneys, stomach, small intestine (duodenum, jejunum, ileum), large intestine (upper and lower colon, rectum), pancreas, ovary with oviduct, uterus, prostate, thymus, esophagus, trachea, nasal turbinate, adipose tissue, submaxillary salivary gland, lymph nodes (mesenteric and thoracic), urinary bladder, thigh skeletal muscle with sciatic nerve, bone marrow (sternum), skin (flank), mammary gland, and 25 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 unusual lesions. Histopathological examination was also performed on the livers from the low-dose groups at Weeks 26, 52, 78, and 104 and from the mid-dose group at Weeks 26 and 52. The study authors reported a decrease in survival in male rats in the mid-dose group at termination and in the high-dose group at Week 55. All surviving rats in the high dose group were sacrificed at Week 55 due to severe liver toxicity and tumor formation noted at the Week 52 interim sacrifice. Survival in the remaining dose groups was similar to controls. The study authors reported treatment-related hunched, thin, and/or bloated appearance in all animals. The study summaries are presented below in the order of the sacrifice. The study authors reported that the results of urinalysis were unremarkable throughout the 2-year period. 26-week study The ADDs at this interim sacrifice were calculated to be as follows: 0, 3.47, 13.6, or 34.6 mg/kg-day for males, and 0, 3.22, 13.9, or 34.9 mg/kg-day for females. At Week 26, 10 animals/sex/group were sacrificed. Hematology, clinical chemistry, urinalyses, gross pathology, organ/body-weight ratios, and histopathology evaluations were conducted. Selected hematology results are provided in Table B.5. There was a 31% increase in WBC count in the mid-dose males and dose-related increases in MetHb levels (by up to 400%), reticulocyte count (by up to 83%), as well as a decrease in RBC count in high-dose males (8%). The hematology results for females are not summarized in this document because no biological significance was found. RBC and reticulocyte counts were not significantly changed, while MetHb was significantly decreased in all treatment groups. Clinical chemistry results indicated that BUN levels in high-dose males and females were significantly increased by 17% and 27%, respectively (see Table B.6). Both males and females experienced dose-related decreases in body weight compared to control animals (see Tables B.4 and B.7). However, the decreased body weight may be partially due to the reduction of food intake (see Table B.4). The study authors reported significant dose-dependent increases in both absolute (12% and 50%>) and relative (25% and 95%) liver weight in the mid- and high-dose males, respectively (see Table B.8). A similar liver weight increase was also reported in tgDNT-treated females (see Table B.9). In addition, there were dose-dependent increases in relative kidney weights with significant changes observed in the high dose group (38% in males and 24% in females; see Tables B.8 and B.9). Other observed significant organ-weight changes included increased relative heart, relative lung weight in high-dose males and females, and increased relative testis weight in high-dose males. Gross pathology findings revealed tgDNT-treatment-related gross alterations in the livers. Histopathology findings indicated hepatotoxicity in the mid- and high-dose males and females (see Table B. 10). Indicators of hep atotoxi city consisted of minimally to moderately severe nonsuppurative pericholangitis in high-dose males and in mid- and high-dose females; necrosis of hepatocytes in mid- and high-dose males and females; vacuolated hepatocytes in high-dose males and females; and slightly to moderately severe biliary hyperplasia, periportal fibrosis, and necrosis of bile duct epithelium in high-dose males. Other signs of histopathology were found in the heart, spleen, and kidney (incidence data are not displayed in this document). These findings included an increase in the incidence and severity of chronic myocarditis in high-dose males. Considering 4/10 control males also had minimal chronic myocarditis, the study authors concluded that this probably represents a treatment-related exacerbation of spontaneous disease. Also, an increase in spleen hemosiderin and extramedullary hematopoiesis was observed in the high-dose males and females. In the kidney, 26 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 slightly increased incidence of chronic interstitial nephritis was noted in the high-dose males. In addition, there was increased incidence and amount of tubular pigment in the mid- and high-dose males and females. Although moderately severe testicular degeneration was observed in high-dose males, the study authors suggested that the unilateral change may represent a spontaneous lesion. Two high-dose males had hepatocellular carcinomas, which were not observed in the control group (see Table B.14), suggesting these tumors may have been induced by tgDNT. Based on increased absolute and relative liver weight and hepatotoxicity in mid-dose males, a LOAEL of 13.6 mg/kg-day and aNOAEL of 3.47 mg/kg-day are identified. 52-week study The ADDs at this interim sacrifice were calculated as follows: 0, 3.47, 13.9, or 34.9 mg/kg-day for males, and 0, 3.46, 13.9, or 35.1 mg/kg-day for females. At Week 52, 10 animals/sex/group were sacrificed. Hematology, clinical chemistry, urinalyses, gross pathology, organ/body-weight ratio, and histopathology evaluations were conducted. Compared to controls, there were dose-related increases in MetHb levels (not significant) and reticulocyte count (significant in high-dose group), and decreases in RBC count (significant in the mid-and high-dose groups) and Hb (significant in the high-dose group; see Table B.5). Also, WBC counts in the high-dose group were increased. In female rats, no treatment-related hematological alterations were observed. Body-weight changes were similar to the findings at Week 26 (see Table B.7). Significant increases in both absolute and relative liver weights were reported in the low-dose males and mid- and high-dose males and females. There were dose dependent increases in male (significant in the mid- and high-dose groups) and female relative kidney weight (significant in the high-dose group) and in male relative heart weight (significant in the high-dose group) (see Tables B.8 and B.9). Gross pathology findings revealed distinct gross alterations of the livers, namely, focal discolorations in the mid- and high-dose males and females. Also, liver nodular lesions were noted in 8/10 males and 4/10 females in the high-dose group. The liver histopathology findings included hyperbasophilia, megalocytosis of hepatocytes, and vacuolation and necrosis of individual hepatocytes (see Table B. 11). Other treatment-related lesions in the high-dose males consisted of exacerbation of chronic interstitial nephritis and renal tubular pigment, increased incidence and severity of testicular degeneration, and increased proliferation of hematopoietic cells in the splenic red pulp and sternal marrow, suggesting an increased RBC turnover rate. Also, the study authors noted that the cardiomyopathy observed in the high-dose males at 26 weeks was not obvious in this group at 52 weeks. A dose-dependent increase in hepatocellular carcinomas (3/10 in mid-dose and 10/10 in high-dose) was observed in males (see Table B. 14). Hepatocellular carcinomas were observed in 4/10 females in the high-dose group. Neoplastic nodules were noted in the livers of 4/10 mid-dose and 3/10 high-dose males, as well as 8/10 high-dose females. Cholangiocarcinomas were observed in 2/10 high-dose males and 2/10 high-dose females. One of 10 mid-dose males had biliary hyperplasia with atypia of the bile duct epithelium, which the study authors believed to be a precursor lesion of cholangiocarcinoma. 27 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Based on increased absolute and relative liver weight and hepatotoxicity in low-dose males, a LOAEL of 3.47 mg/kg-day is identified. Data preclude identification of aNOAEL. 55-week study The ADDs at this interim sacrifice were calculated as follows: 34.9 mg/kg-day for males and 35.1 mg/kg-day for females (only high-dose group). At Week 55, all surviving high-dose animals were sacrificed. Hematology, clinical chemistry, urinalyses, and histopathology evaluations were conducted on 20 rats/sex, but the results on hematology and clinical chemistry were only reported on 10 rats/sex. Because there were no significant hematological findings in the treated females, Table B.5 displays male results only. Tables B.6, B.8, and B.9 present clinical chemistry and organ/body-weight ratios. Histopathological findings (not displayed in tables) in high-dose animals indicated that hepatocellular carcinomas occurred in 20/20 males and in 11/20 females. Two male rats had hepatocellular-cholangiocarcinomas and 3/20 males had cholangiocarcinomas. Biliary hyperplasia with atypia of bile duct epithelium was also observed in 2/20 males. Due to lack of a control group, data preclude identification of a NOAEL or a LOAEL. 78-week study The ADDs at this interim sacrifice were calculated as follows: 0, 3.49, or 14.0 mg/kg-day for males, and 0, 3.45, or 14.0 mg/kg-day for females. At Week 78, 20 animals/sex from each dose level were sacrificed (only control, low-dose, and mid-dose groups were available; the high-dose males and females were all sacrificed at Week 55). Hematology, clinical chemistry, urinalyses, gross pathology, organ/body-weight ratio, and histopathology evaluations were conducted. Among the hematology findings were decreased hematocrit and RBC counts in the mid-dose males and dose-dependent increases of 13% and 112% in reticulocyte counts in the low-and mid-dose males, respectively (see Table B.5). In treated female rats, significantly increased WBC counts were observed in mid- and high-dose groups, and no other significant hematological changes were observed. Clinical chemistry indicated significantly higher mean SGPT values in mid-dose males (93% higher than controls) (see Table B.6). Body-weight changes in the mid-dose males and females were similar to those observed in the 26-week treatment. Absolute and relative liver weights were significantly increased in the low- and mid-dose males and in the mid-dose females (all the high-dose rats were sacrificed at Week 55; see Tables B.8 and B.9). In low- and mid-dose males, the study authors also reported significantly dose-dependent increases in the relative weights of testes (27% and 37%, respectively), lungs (14% and 30%, respectively), and kidneys (12% and 55%, respectively; see Table B.8). Also, the relative kidney weight was increased by 32% and the absolute brain weight was decreased by 11% in the mid-dose females see Table B.9). Hepatotoxicity including cystic degeneration, necrosis of individual hepatocytes and fatty metamorphosis were observed in low- and mid-dose males and females (see Table B. 12). An increase in the severity of chronic interstitial nephritis was observed in mid-dose males and females. Treatment-related testicular pathology observed at the Week 52 interim sacrifice could not be determined because all control and mid-dose males had interstitial cell testicular tumors. Hepatocellular carcinomas were observed in 19/20 mid-dose males (see Table B.14); neoplastic nodules were observed as follows: 1/20 in low-dose males, 11/20 in mid-dose males, 2/20 in low-dose females, and 10/20 in mid-dose females. Four cholangiocarcinomas were recognized in mid-dose males in addition to 2/20 incidence of biliary hyperplasia with atypia of bile ductal epithelium in the same 28 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 group. In this interim sacrifice, the study authors observed increases in relatively common benign neoplasms, such as mammary fibroadenomas, subcutaneous fibromas, pituitary chromophobe adenomas, and pulmonary carcinomas. Based on increased relative liver weight and hepatotoxicity in low-dose females, a LOAEL of 3.45 mg/kg-day is identified. Data preclude identification of a NOAEL. 104-week study The ADDs doses at this terminal sacrifice were calculated as follows: 0, 3.51, or 14 mg/kg-day for males, and 0, 3.46, or 14 mg/kg-day for females. At Week 104, all surviving animals were sacrificed. Hematological results indicated that in males there were dose-dependent increases in MetHb (not significant); a significant increase in reticulocyte counts in the mid-dose group; and significant decreases in hematocrit, Hb, and RBC counts in the mid-dose group (see Table B.5). No remarkable hematological changes were observed in treated female rats (data not shown). Evaluation of clinical chemistry revealed significantly increased SGPT and BUN in the mid-dose males (see Table B.6). Similar body weight changes were observed in mid-dose males and females as those observed in the 52 or 78-week treatments (see Table B.7). Dose-dependent, significantly increased organ weights in the low- and mid-dose groups included relative heart weights in males and females, both absolute and relative liver weights in males and females, relative testis weight in males, and relative kidney and ovary weights in females (see Table B.8 and Table B.9). Gross pathology findings revealed apparent treatment-related liver lesions and nodular and/or mass formation in low- and mid-dose males and females. Histopathology indicated that hepatotoxicity occurred in the livers of treated animals and consisted of fatty metamorphosis, necrosis, cystic degeneration, and megalocytosis in low- and mid-dose males and females (see Table B. 13). An increase in the severity of chronic interstitial nephritis occurred in the kidneys of mid-dose males and females. The study authors did not observe treatment-related histopathological changes in the ovaries or in the hearts from either sexes; therefore, the biological significance of the increased relative heart and ovary weights in the low- and mid-dose groups is unclear. Testicular pathological changes were not determined as observed in Week 78. Therefore, the biological significance of the increased relative testes weight is also unclear. There is a clear dose-dependent increased incidence of hepatocellular carcinomas and neoplastic nodules in both males and females (see Table B. 15). "Neoplastic nodule" is a term used in rodent liver pathology. Because neoplastic nodules are believed to progress to hepatocellular carcinomas (Bannasch et al.. 1982; Bannasch.. 1976). this endpoint is included in the dose-response analysis. Hepatocholangiocarcinomas were observed in 1/68 mid-dose female, and cholangiocarcinomas were observed in 2/23 mid-dose males. In addition, 2/23 mid-dose males had biliary hyperplasia with atypia of bile duct epithelium. Parathyroid hyperplasia was also observed in mid-dose males and females, and 2/23 mid-dose males had parathyroid adenomas. Increased relatively common benign neoplasms were observed in both males and females (see Table B.15). Based on increased absolute and relative liver weight and hepatotoxicity in low-dose females, a LOAEL of 3.46 mg/kg-day was identified. Data preclude identification of a NOAEL. 29 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Leonard et al. (1987) Leonard et al. (1987) administered 0 or 35 mg/kg-day tgDNT (final composition of 76.5% 2,4-DNT, 18.8% 2,6-DNT, 2.43% 3,4-DNT, 1.54% 2,3-DNT, 0.69% 2,5-DNT, and 0.04%) 3,5-DNT) via diet to groups of 28 male F344/CrlBR rats (Charles River Breeding Laboratories, Kingston, NY) for 1 year. New food batches were prepared monthly and the tgDNT concentration was adjusted in each batch based on food consumption and average body weight in order to maintain target dose levels. It is unclear if this study was conducted under GLP. Rats were housed four per cage, and average dietary consumption for each cage was determined weekly. Body weights were measured every 2 weeks throughout the study. Four animals in each group were sacrificed after 6 and 26 weeks of feeding. At the end of the 52-week treatment period, all surviving animals were sacrificed and necropsied, the liver and lungs were weighed, histopathological examination was performed, and hepatic microsomal epoxide hydrolase (EH) and cytosolic DT-diaphorase (DTD) activities were measured. Serum enzyme activities (SGPT) and glutamyl transferase (GGT) were also determined. Other clinical chemistry, hematology, and pathology examinations besides liver and lung were not conducted. The study authors reported that body weight was significantly reduced 11% and 26% in rats treated with tgDNT compared with controls at 26 and 52 weeks, respectively (see Table B. 16). Relative liver weight was significantly increased at both time periods (see Table B. 16) as well. At the end of 52 weeks, absolute liver weight was 89% more and relative liver weight was 155% more than that of controls (see Table B.16). After 52 weeks of treatment, the study authors noted nonneoplastic lesions consisting of hepatocytic degeneration and vacuolation in the majority of animals, as well as acidophilic and basophilic cell foci in over 90% of the animals. Bile duct hyperplasia and a highly variable incidence of cholangiofibrosis were also noted. Based on significant decreases in body weight, and increases in absolute liver and relative liver weight accompanied by pathological findings in the liver and bile duct, a LOAEL of 35 mg/kg-day is identified from this study. Data preclude identification of a NOAEL. Table B. 17 summarizes neoplastic lesions of the liver. Neoplastic nodules were found in the livers in 53% of animals treated with tgDNT. Hepatocellular carcinomas were seen in 47% of the tgDNT-treated animals; all lesions had a typical trabecular pattern. Cholangiocarcinomas were also reported in 11% of the treated animals. The study authors concluded that tgDNT is a potent, complete hepatocarcinogen in male F344 rats. The study is limited by the use of only one dose of tgDNT, which precludes examining dose-response relationships. In addition, the study authors did not provide quantitative data for nonneoplastic lesions and only reported general findings. Developmental Studies Price et al. (1985) and CUT (1982b) Research Triangle Institute (RTI) performed a study investigating the potential developmental toxicity of tgDNT following maternal gestational exposure in F344 rats. The study was performed in 1980, and a Final Report was submitted under the Toxic Substances Control Act (TSCA) to EPA's Office of Toxic Substances by CUT (1982b). Additionally, Price and colleagues, the study authors from RTI, reported on the maternal and fetal toxicity in a 30 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 peer-reviewed, published study (Price et ai. 1985). The tgDNT used in the study contained the following composition of isomers: 76% 2,4-DNT; 19% 2,6-DNT; 2.4% 3,4-DNT; 1.5% 2,3-DNT; <1%; 2,5-DNT; and <1%; 3,5-DNT. The rats were administered the tgDNT by gavage, with laboratory-grade corn oil used as the vehicle. The study authors administered tgDNT at doses of 0 (vehicle control), 35, 75, or 150 mg/kg-day (first breeding round) to groups of 13 pregnant F344 rats on gestation days (GDs) 7 through 20. Due to high mortality rates observed in the 150 mg/kg-day dose group, the doses used in the second and third breeding rounds were reduced to 14, 37.5, and 100 mg/kg-day. Table B.18 shows the numbers of females in each dose group. The study authors examined dams daily for clinical signs of toxicity and recorded body weights on GDs 0 and 7-20. On GD 20, 13, 7, 13, 7, 13, and 6 dams from each dose group (14, 35, 37.5, 75, 100, and 150 mg/kg-day, respectively) and 22 dams from the control group were sacrificed and evaluated for implantations, resorptions, and dead or live fetuses. In addition, body weight, liver weight, spleen weight, number of corpora lutea, and gravid uterine weight for each dam were recorded. The pregnancy rates in mated dams from the control and low- through high-dose groups were 20 (91%), 10 (77%), 7 (100%), 12 (92%), 6 (86%), 12 (92%), and 5 (83%>), respectively. No histopathological examinations were conducted on the dams. Live fetuses were examined for uterine position, body weight, crown-rump length, placenta weight, sex, and gross morphological abnormalities. Maternal and fetal blood samples from the 100-mg/kg-day treatment group were analyzed for MetHb content. In addition, blood samples from dams and one male and one female fetus per litter from the 100-mg/kg-day treatment group were evaluated for RBC count, WBC count, hematocrit, MCV, RBC distribution width (RDW), and platelet count. The study authors also examined 50% of the fetuses in each litter for visceral and skeletal malformations, malformations of the head, and liver and spleen weights. A high mortality rate was observed in dams from the first breeding date exposed to 150 mg/kg-day tgDNT, as 46.2% of rats (6/13) in this treatment group died between GDs 11 and 18. Therefore, in the second and third breedings, the study authors reduced the tgDNT doses. Treatment with tgDNT at 14, 35, and 100 mg/kg-day also resulted in mortality rates of 4.5%) (1/22), 7.7%) (1/13), and 4.3% (1/23), respectively, through GD 20 (see Table B.18). No deaths occurred in females treated with the vehicle control (corn oil). The study authors stated that the cause of death of one rat/group from the 14-, 35-, and 100-mg/kg/day-DNT groups was initially suspected to be related to gavage error. Gavage error was also suspected to be the cause of death in 2/6 rats from the highest dose group (150 mg/kg); however, the study authors concluded that the cause of death in the remaining 4 rats appeared to be treatment related as death was preceded by clinical signs of toxicity. Clinical signs related to tgDNT treatment included rough coat, lethargy, and hind-limb weakness, and were observed in 7/13 females in the 150-mg/kg-day dose group beginning on the fifth to eighth day of dosing (GDs 11-14) and continuing until death (GDs 12-18) or scheduled sacrifice (GD 20). On GD 20, significant increases in MetHb, reticulocyte count, MCV (RBC size), RDW, and platelet count were observed in dams treated with 100-mg/kg-day tgDNT. Significant decreases in RBC count and hematocrit were also observed in this group (see Table B.19). Because the study authors did not analyze low-dose blood samples, it is not clear if hematological parameters were adversely affected at lower doses. 31 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 A significant dose-related decrease in absolute maternal weight gain (maternal weight gain during treatment minus gravid uterine weight) was observed in the 14, 100, and 150-mg/kg-day tgDNT dose groups (see Table B.20). However, no decreases in absolute maternal weight gain was observed at 35, 37.5, and 75 mg/kg-day, suggesting the reduction of weight gain at 14 mg/kg-day was not treatment related, at least at dose levels <100 mg/kg-day. Significant dose-related increases in relative maternal liver weight were observed in the 75- (11%), 100- (12%), and 150-mg/kg-day (17%) treatment groups (see Table B.20). A significant increase in maternal relative spleen weight was observed at all doses of tgDNT >35 mg/kg-day (see Table B.20). Due to lack of histopathological data, the biological significance of the increased spleen weight is not clear. There was an increased resorption rate (46.0%) in the high-dose group compared to the control (16.8%); however, this change was not significant. No other significant effects on measures related to reproduction (incidence of live or dead fetuses per dam, see Table B.20) or fetal morphology (see Table B.21) were observed. The study authors, therefore, concluded that tgDNT was not observed to be teratogenic in F344 rats even at dose levels that produced significant maternal toxicity. In litters with live fetuses, no significant difference was observed in the proportion of male fetuses per litter, average fetal body weight per litter, average fetal crown-rump length per litter, or average placental weight per litter (see Table B.22). Changes in liver/body and spleen/body-weight ratios were observed in some treatment groups, but no dose-response relationship was apparent (see Table B.22). Fetuses from the 100-mg/kg-day group exposed to tgDNT exhibited decreased reticulocyte count, decreased RBC count, and increased MCV (see Table B. 19). Although the decreased RBC count and increased MCV were significant, the P/o RBC count decrease (2.15 x io6 compared to 2.17 x io6) and 2% RBC size increase (160.61 |im3 compared to 156.54 |im3) were not considered biologically significant. The Final Report submissions (CUT. 1982b) include the teratological study [the same information included in Price et al. (1985)1 as well as a postnatal developmental evaluation. In the postnatal developmental evaluation, the remaining female rats that were not sacrificed on GD 20, including 8, 5, 15, 6, 9, and 1 pregnant females from the low- through high-dose groups, respectively, and 15 controls were observed through parturition, death, or GD 24, whichever came first. One female in the 14-mg/kg-day group died on GD 22, and 1 female in the 150-mg/kg-day group died on GD 23 (the females from this group were not available for postnatal developmental evaluation). Twelve females failed to deliver by GD 24 and were sacrificed; 11 of these females were determined not to be pregnant. In total, pups were observed from 5-14 litters per treatment group from birth (postnatal day [PND] 0) to PND 60. Body weight and crown-rump length of each live pup were recorded on PND 0, then litters were culled to no more than eight live pups (four male and four female, as possible). The study authors recorded body weight daily and noted age of appearance of physical landmarks (pinna detachment, pilation, incisor eruption, eye opening, testes descent, vaginal opening), neurobehavioral landmarks (surface righting, cliff avoidance, auditory startle, wire grasping, and mid-air righting), and open field behavior on PND 30. A limited number of pups were sacrificed from each treatment group on PNDs 0, 10, 25, and 50; the remaining pups were sacrificed on PND 60. Body weight, liver weight, and spleen weight were recorded at each sacrifice date; testis weights were recorded from the sacrifice at PND 60. All dams were sacrificed on PND 30, 32 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 and maternal body weight, liver weight, and spleen weight at sacrifice were recorded. Blood samples were collected from dams at PND 30 and pups on PNDs 0, 10, 25, and 50 from the 75- and 100-mg/kg-day groups and the corresponding controls. The study author (CUT. 1982b) reported that dams in the 100-mg/kg-day group exhibited decreased body weight on PND 15, and dams in the 75-mg/kg-day group had reduced reticulocyte counts on PND 30; no other signs of maternal toxicity were observed. The study authors state that significant differences in some observations from vehicle controls were observed in the litters, but these differences were not dose-related, including elevated litter size on PND 0 in the 75-mg/kg-day group; elevated female crown-rump length on PND 0 in the 14- and 37.5-mg/kg-day groups; increased male body weight on PND 0 in the 14-mg/kg-day group; increased reticulocyte count in the 75-mg/kg-day group or decreased reticulocyte count in the 100-mg/kg-day group on PND 50; and either early or delayed appearance of eye opening in the 14-mg/kg-day group or the 35- and 75-mg/kg-day groups, respectively. There was a dose-dependent increase in relative liver weight for pups in all treatment groups on PND 0, but no difference was observed on PND 60 in any group, indicating that tgDNT toxicity was reversed by PND 60. The study authors stated that a dose-related decrease in rearing behavior in the open field was observed at 100 mg/kg-day in female pups, suggestive of sex-specific neuromotor deficits (see Table B.23). The Price et al. (1985) study stated that tgDNT was not found to be teratogenic following oral administration and concluded that there was no evidence for selective sensitivity of the developing conceptus to tgDNT because prenatal viability was reduced only at the dose near the maternal LD50. The CUT (1982b) studies concluded that while various dosages of tgDNT could produce facilitation or retardation of growth or development, dose-response relationships for these changes do not exist. Based on significantly increased relative liver weight in pregnant F344 dams at GD 20, a LOAEL of 75 mg/kg-day and a NOAEL of 37.5 mg/kg-day are identified for maternal toxicity. Considering a 29.2% increase in resorption rate accompanied by an increase in dead fetuses and a decrease in live fetuses in the high-dose group, a LOAEL of 150 mg/kg-day and a NOAEL of 100 mg/kg-day are established for developmental toxicity. Further, a LOAEL of 100 mg/kg-day and a NOAEL of 75 mg/kg-day were identified for postnatal toxicity based on decreased rearing behavior. Reproductive Studies No studies were identified. Carcinogenicity Studies Carcinogenicity studies by CUT (1982a) and Leonard et al. (1987) were summarized in the Chronic Studies section. The carcinogenicity study by CUT (1982a) is selected as the principal study for deriving the screening provisional oral slope factor (p-OSF). Inhalation Exposures No studies were identified. 33 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Other Data (Other Examinations) Mutagenicity or genotoxicity of tgDNT has been evaluated in in vitro and in vivo test systems, and Table 4 summarizes the study results. Mixed results were reported from both in vitro and in vivo test systems. Mutagenicity tests were positive in Salmonella typhimurium Ames assays, with and without metabolic activation, while they were negative in mammalian cell systems (e.g., HGPRT gene mutations in Chinese hamster ovary [CHO] cells and TK mutations in mouse lymphoma cells). Similar to the in vitro test systems, mixed results were also seen in in vivo studies. While unscheduled DNA synthesis showed a positive response in most rat hepatocytes (Mirsalis et at.. 1989; Hamilton and Mirsalis. 1987; Ashbv et at.. 1985; Mirsalis and Butterworth. 1982; Mirsalis et at.. 1982) and in rat lymphocytes (Ktigerman et at.. 1982). a mouse bone marrow micronucleus test (Ashbv et at.. 1985) and dominant lethal assay (Spares and Lock, 1980) indicated negative responses. 34 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 4. Summary of tgDNT Genotoxicity Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation Genotoxicity studies in prokaryotic organisms Reverse mutation S. typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 1000 |ig/platc + (TA98, TA1538) + (TA98, TA1538) NA Couch et al. (1981) Forward mutation S. typhimurium strain TM 677 500 (ig/mL + + NA Couch et al. (19811 Genotoxicity studies in mammalian cells—in vitro HGPRT Mutation Chinese hamster ovary (CHO) <2mM NA Abernethv and Couch (19821 TK+/- P388 mouse lymphoma cells 1.6-1000 (ig/mL — — NA Styles and Cross (19831 Unscheduled DNA synthesis (UDS) Primary hepatocyte cultures from adult male F344 rats 0, 1 x 1CT4, or 1 x l(T5 M ND Evaluated Bermudez et al. (19791 Genotoxicity studies in mammals—in vivo Sister chromatid exchange (SCE) Rat lymphocyte culture from male F344 rats 0 or 100 mg/kg (oral) + ND NA Kligerman et al. (19821 UDS Hepatocytes/Male Alderley Park rats 0, 100, or 200 mg/kg (oral) + ND NA Aslibv et al. (19851 UDS Hepatocytes/Male F344 rats 0, 25, 100, 150, or 200 mg/kg (oral) + ND NA Ashbv et al. (1985) UDS Hepatocytes/Male F344 rats 0 or 100 mg/kg (oral) + ND Mixture of DNTs, with dissimilar composition as tgDNT Hamilton and Mirsalis (1987) UDS Hepatocytes/Male and female F344 rats <200 mg/kg (oral) + ND NA Mirsalis and Buttcrworth (1982) UDS Hepatocytes/Male germ-free (axenic) F344 rats or Charles River Altered Schaedler Flora rats (CRASF; similar to normal gut microflora) 0 or 100 mg/kg (oral) Axenix - CRASF + ND Results indicate that gut flora is necessary for tgDNT to induce UDS Mirsalis et al. (1982) UDS Hepatocytes/Male F344 rats 0, 35, 125, or 250 mg/kg (oral) + (at doses >125 mg/kg) ND NA Mirsalis et al. (19891 35 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 4. Summary of tgDNT Genotoxicity Endpoint Test System Dose Concentration3 Resultsb Comments References Without Activation With Activation Mouse biochemical or visible specific locus test T stock male and C57BL/6J female mice; C57BL/6 J male and C57BLBL/6J female mice 0 or 100 mg/kg ND Recessive spot test; different matings were tested; treated on a day designated as 1/4 of pregnancy Soares and Lock (1980) Micronucleus test Bone marrow/Male (CBA x BalbQFl mice 0, 200, 400 mg/kg (IP) ND NA Ashbv et al. (1985) Dominant lethal Male DBA/2J mice 0 or 250 mg/kg (IP or oral) - ND Treated on two consecutive days Soares and Lock (1980) aLowest effective dose for positive results or highest dose tested for negative results. b+ = positive; IP= intraperitoneal injection; NA = not applicable; ND = no data. 36 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 DERIVATION OF PROVISIONAL REFERENCE DOSES Tables 5 and 6 present a summary of noncancer reference values and cancer values, respectively. Table 5. Summary of Noncancer Reference Values for Technical Grade Dinitrotoluene (CASRN 25321-14-6) Toxicity Type (Units) Species/Sex Critical Effect p-Reference Value POD Method PODhed UFC Principal Study Screening Subchronic p-RfD (mg/kg-d) F344 Rat/M Increased hepatocyte necrosis at week 26 5 x 1(T3 BMDLjo 0.52 100 CUT f1982a) Screening Chronic p-RfD (mg/kg-d) F344 Rat/M Increased hepatocyte necrosis at week 104 9 x 1(T4 BMDL10 0.087 100 CUT CI 982a) Subchronic p-RfC (mg/m3) NDr Chronic p-RfC (mg/m3) NDr NDr = not determined. Table 6. Summary of Cancer Values for Technical Grade Dinitrotoluene (CASRN 25321-14-6) Toxicity Type Species/Sex Tumor Type Cancer value Principal Study Screening p-OSF F344 Rat/M Combined tumor incidence for hepatocellular carcinomas, liver neoplastic nodules, mammary fibroadenomas and subcutaneous fibromas 4.5 x 10"1 (mg/kg-d)-1 CUT f 1982a) p-IUR NDr NDr = not determined. 37 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 DERIVATION OF ORAL REFERENCE DOSES Derivation of Subchronic Provisional RfD (Subchronic p-RfD) A subchronic p-RfD cannot be derived for tgDNT because the only potential principal study by CUT (CUT. 1983) is limited and is not suitable to derive a subchronic p-RfD (see Appendix A for details). However, Appendix A provides a "screening level" value for subchronic oral exposure based on a comprehensive unpublished study (CUT, 1982a). Derivation of Chronic Provisional RfD (Chronic p-RfD) A chronic p-RfD cannot be derived for tgDNT because no peer-reviewed studies are suitable to derive a chronic p-RfD (see Appendix A for details). However, Appendix A provides a "screening level" value for chronic oral exposure based on a comprehensive unpublished chronic study (CUT. 1982a). DERIVATION OF INHALATION REFERENCE CONCENTRATIONS No studies were identified that could be used to derive provisional inhalation RfCs for tgDNT. Available epidemiological studies consist primarily of occupational studies in which workers were exposed to a tgDNT mixture, and/or other known and unknown chemicals. In addition, none of the epidemiological studies provided quality exposure information. No animal inhalation studies for tgDNT were identified. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR Table 7 identifies the cancer weight-of-evidence (WOE) descriptor for tgDNT. 38 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table 7. Cancer WOE Descriptor for tgDNT Possible WOE Descriptor Designation Route of Entry (Oral, Inhalation, or Both) Comments "Carcinogenic to Humans " NA Inhalation Relevant human cancer studies on tgDNT are available on the effects of human inhalation/dermal exposure to mixed DNT. "Likely to Be Carcinogenic to Humans" Selected Both3 In the cancer studv bv CUT (1982a). oral exposure to tgDNT caused an increased incidence of hepatocellular tumors, liver neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas in both male rats and an increased incidence of hepatocellular tumors, liver neoplastic nodules, and subcutaneous fibromas in female F344 rats. In another cancer studv bv Leonard et al. (1987), tsDNT caused an increased incidence of hepatocellular carcinomas and liver neoplastic nodules in F344 male rats after 1 year of oral exposure. "Suggestive Evidence of Carcinogenic Potential" NA NA The evidence from animal and human data is more than suggestive of carcinogenicity, which raises a concern for carcinogenic effects and is judged sufficient for a stronger conclusion. "Inadequate Information to Assess Carcinogenic Potential" NA NA There is evidence to assess the carcinogenic potential of tgDNT. "Not Likely to Be Carcinogenic to Humans " NA NA Evidence of the carcinogenic potential of tgDNT is available in animals and humans. atgDNT is considered "Likely to be Carcinogenic to Humans" by all routes of exposure based on Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005). which indicates that for tumors occurring at a site other than the initial point of contact, the cancer WOE descriptor may apply to all routes of exposure that have not been adequately tested at sufficient doses. NA = not applicable. Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005). the WOE descriptor for tgDNT is "Likely to Be Carcinogenic to Humans'' by all routes of exposure (see Table 7). This descriptor is based on (1) suggestive evidence of carcinogenicity in humans and (2) strong evidence in animals by oral exposure. There is some evidence for an increased risk of certain types of cancer in occupational populations exposed to tgDNT. An association between tgDNT exposure and an increased risk of hepatobiliary cancer was found in a retrospective mortality study involving workers at a U.S. Army munitions facility (Stavner et al.. 1993). A study of underground mining workers exposed to tgDNT as an explosive (Burning et ai. 1999) also indicated that tgDNT might be associated with urothelial tumor formation. The workers in this study were believed to be 39 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 exposed to tgDNT via dermal or inhalation exposure. However, these studies were limited by a variety of factors, including inadequate exposure information (i.e., concentration and duration), and therefore, do not permit a definitive conclusion on the carcinogenicity of tgDNT in humans. Results from experimental animal studies showed that tgDNT increased the incidence of multiple tumor types in F344 rats in two separate studies (Leonard et al .. 1987; CUT. 1982a). Significant increases in hepatocellular neoplastic nodules and carcinomas (males and females), subcutaneous fibromas (males and females), and mammary fibroadenomas (males only) were observed in F344 rats in chronic-duration dietary exposure bioassays (CUT, 1982a), and increases in hepatocellular carcinoma were also observed in male F344 rats in a 1-year dietary study (Leonard et at., 1987). In addition, tgDNT caused hepatocellular tumors in rats as early as 26 weeks; therefore, the positive tumor results observed in these studies can be considered an early onset of carcinogenicity. As stated in the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), examples for a chemical to be considered "Likely to Be Carcinogenic to Humans" are (1) "an agent that has tested positive in animal experiments in more than one species, sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans;" (2) "a positive tumor study that raises additional biological concerns beyond that of a statistically significant result, for example, a high degree of malignancy, or an early age at onset." Based on these examples from the cancer guidelines and the carcinogenicity data from available human and animal studies, the WOE descriptor of "Likely to Be Carcinogenic to Humans'' is appropriate for tgDNT. The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) indicate that for tumors occurring at a site other than the initial point of contact, the cancer WOE descriptor may apply to all routes of exposure that have not been adequately tested at sufficient doses. An exception occurs when there are convincing toxicokinetic data that absorption does not occur by other routes. Information available on the carcinogenic effects of tgDNT demonstrates that tumors occur in tissues remote from the site of absorption. tgDNT has been shown to be a hepatocarcinogen in rats in two bioassays of various experimental designs by oral exposure. Increased hepatocellular carcinoma in munition workers and urothelial cancer in mining workers are presumed to have been exposed predominantly through the inhalation route with a contribution from the dermal route. Information on the carcinogenic effects of tgDNT via the dermal route in humans and animals is limited or absent. There are no toxicokinetic data indicating absorption does not occur by other routes. Therefore, based on the observation of liver tumors in animals following oral exposure and in humans following occupational inhalation and dermal exposure, it is assumed that an internal effective dose will be achieved regardless of the route of exposure. Thus, tgDNT is considered "Likely to be Carcinogenic to Humans" by all routes of exposure. MODE-OF-ACTION DISCUSSION The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) define mode-of-action "as a sequence of key events and processes, starting with the interaction of an agent with a cell, proceeding through operational and anatomical changes, and resulting in cancer formation". Examples of possible modes of carcinogenic action for any given chemical include "mutagenicity, mitogenesis, inhibition of cell death, cytotoxicity with reparative cell proliferation, and immune suppression". 40 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 The potential mode of action for tgDNT is unclear. Table 4 summarizes the studies examining genotoxicity (e.g., clastogenicity, mutagenicity) of tgDNT. tgDNT was shown to be positive for mutagenicity in S. typhimurium strains (Couch et ai. 1981) but was not mutagenic in mammalian cell systems [e.g., HGPRT mutation in CHO cells and TK mutation in mouse lymphoma cells (Styles and Cross. 1983; Abernethv and Couch. 1982). While most assays of unscheduled DNA synthesis in rat hepatocytes showed a positive response following oral dosing (Mirsalis et at.. 1989; Hamilton and Mirsalis. 1987; Ashbv et ai. 1985; Mirsalis and Butterworth. 1982; Mirsalis et ai. 1982). mouse bone marrow micronuclei (Ashbv et ai. 1985) and dominant lethal assays (Spares and Lock. 1980) were negative. DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of Provisional Oral Slope Factor (p-OSF) A p-OSF cannot be derived for tgDNT because no peer-reviewed studies are suitable to derive a p-OSF. However, Appendix A provides a "screening level" value for a p-OSF based on a comprehensive unpublished carcinogenicity study (CUT, 1982a). Derivation of Provisional Inhalation Unit Risk (p-IUR) No human or animal studies examining the carcinogenicity of tgDNT following inhalation exposure were identified. Therefore, derivation of a p-IUR is precluded. 41 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 APPENDIX A. PROVISIONAL SCREENING VALUES For the reasons noted in the main document, subchronic and chronic p-RfDs for tgDNT could not be derived. However, information is available for this chemical which, although insufficient to support derivation of a provisional toxicity value, under current guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk Technical Support Center summarizes available information in an appendix and develops a "screening value". Appendices receive the same level of internal and external scientific peer review as the main documents to ensure their appropriateness within the limitations detailed in the document. Users of screening toxicity values in an appendix to a PPRTV assessment should understand that there is considerably more uncertainty associated with the derivation of an appendix screening toxicity value than for a value presented in the body of the assessment. Questions or concerns about the appropriate use of screening values should be directed to the Superfund Health Risk Technical Support Center. DERIVATION OF SCREENING SUBCHRONIC PROVISIONAL RfD (SCREENING SUBCHRONIC p-RfD) No human oral studies were identified. The database for oral tgDNT toxicity in animals includes one subchronic-duration study (CUT. 1983). two chronic-duration studies (Leonard et at., 1987; CUT, 1982a), and one developmental study (Price et ai, 1985; CUT, 1982b). The chronic-duration F344 rat study by CUT (1982a) is composed of five interim evaluations at Weeks 26, 52, 55, 78, and 104. C " ' : I ?83) (4 weeks exposure duration) reported significant treatment-related hematological effects. As a result, a LOAEL of 31.9 mg/kg-day is identified based on the significant hematological changes in male rats. However, this study was not selected as the principal study because it only focused on hematological and gross pathological examinations, and no clinical chemistry, organ weight, and histopathology endpoints were examined. The 26-week interim study within the chronic-duration study (CUT, 1982a) provided a comprehensive toxicity evaluation and is the closest exposure duration to the standard 13 weeks of a subchronic-duration study. Thus, in the absence of a comprehensive evaluation following a shorter exposure duration, the 26-week study from CUT (1982a) is selected as the principal study in lieu of the 4-week study by CUT (1983) and is protective of subchronic-duration exposure. In this 26-week study, hematology, clinical chemistry, urinalyses, gross pathology, and histopathology were all conducted to evaluate tgDNT toxicity. The study authors reported toxicity of tgDNT on organ weight (e.g., liver and kidneys) and hematological (e.g., MetHb levels, reticulocyte count, MCV, and RBC count), and histopathological end points (e.g., liver and spleen). A NOAEL of 3.47 mg/kg-day and a LOAEL of 13.6 mg/kg-day were identified based on increased absolute and relative liver weight and hepatotoxicity in male rats. For comparison purposes, Table A.l summarizes all the potential critical effects from the 26-week study. All the endpoints shown in the table were modeled with benchmark dose software (BMDS) (version 2.2.2), and the estimated BMDLi0s are also summarized in the table. Among all the candidate endpoints for potential critical effect, the increased incidence of hepatocyte necrosis in male rats resulted in the lowest BMDLio of 2.16 mg/kg-day, which is followed by a BMDLio of 2.27 mg/kg-day for periportal hyperbasophilic hepatocytes in females. Therefore, increased hepatocyte necrosis is considered the critical effect, and using the BMDLio for this endpoint as the point of departure (POD) would protect all the sensitive effects observed in rats after 26 weeks of oral exposure. In addition to the subchronic-duration studies mentioned above, there is a developmental study (Price et at.. 1985) that is also considered as part of the 42 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 database for derivation of a screening subchronic p-RfD. In this study, a NOAEL of 37.5 mg/kg-day and LOAEL of 75 mg/kg-day were established based on maternal toxicity (i.e., increased relative liver weight). A NOAEL of 100 mg/kg-day and a LOAEL of 150 mg/kg-day were established for developmental toxicity based on increased resorption rate accompanied by an increase in dead fetuses and a decrease in live fetuses. Among all available subchronic data (including subchronic-duration and developmental studies), the BMDLio of 2.16 mg/kg-day for increased incidence of hepatocyte necrosis in male rats is the most sensitive and is considered protective for all potential tgDNT-induced effects including developmental toxicity. Therefore, the BMDLio of 2.16 mg/kg-day is selected as the POD for derivation of the screening subchronic p-RfD. The NOAEL of 3.47 mg/kg-day for increased absolute and relative liver weights in males in the same 26-week interim sacrifice is considered supportive. Table A.l. Potential Critical Effects in Male and Female F344 Rats After Dietary Exposure to tgDNT for 26 Weeks NOAEL LOAEL BMDL10 POD End points (mg/kg-d) (mg/kg-d) (mg/kg-d) (mg/kg-d) Males Relative liver weight 3.47 13.6 No fit 3.47 Relative kidney weight 13.6 34.6 No fit 13.6 Hepatocyte necrosis 3.47 13.6 2.16 2.16 Females Relative liver weight 3.22 13.9 5.60 5.60 Relative kidney weight 13.9 34.9 No fit 13.9 Periportal hyperbasophilic hepatocytes 3.22 13.9 2.27 2.27 In Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011b). the Agency endorses a hierarchy of approaches to derive human equivalent oral exposures from data from laboratory animal species, with the preferred approach being physiologically based toxicokinetic modeling. Other approaches may include using some chemical-specific information, without a complete physiologically based toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of human equivalent oral exposures, EPA endorses body-weight scaling to the 3/4 power (i.e., BW3 4) as a default to extrapolate toxicologically equivalent doses of orally administered agents from all laboratory animals to humans for the purpose of deriving a RfD under certain exposure conditions. More specifically, the use of BW3 4 scaling for deriving a RfD is recommended when the observed effects are associated with the parent compound or a stable metabolite but not for portal-of-entry effects or developmental endpoints. A validated human physiologically based pharmacokinetic (PBPK) model for tgDNT is not available for use in extrapolating doses from animals to humans. In addition, the selected POD of 2.16 mg/kg-day is based on increased incidence of hepatocyte necrosis, which is not a portal-of-entry or developmental effect. Therefore, scaling by BW3 4 is relevant for deriving human equivalent doses (HEDs) for this effect. 43 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Following U.S. EPA (2011b) guidance, the POD for the 26-week rat study is converted to a HED through the application of a dosimetric adjustment factor (DAF1) derived as follows: DAF = (BWa1/4 - BWh1/4) where DAF = dosimetric adjustment factor BWa = animal body weight BWh = human body weight Using a BWa of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA. |), the resulting DAF is 0.24. Applying this DAF to the BMDLio identified in the 26-week rat study yields a BMDLiqhed as follows: PODhed = BMDLio (mg/kg-day) x DAF = BMDLio (mg/kg-day) x 0.24 = 2.16 (mg/kg-day) x 0.24 = 0.52 mg/kg-day Screening Subchronic p-RfD = PODhed ^ UFc = 0.52 mg/kg-day -M00 = 5 x 10~3 mg/kg-day Table A.2. Uncertainty Factors for the Screening Subchronic p-RfD for tgDNT UF Value Justification ufa 3 For the POD based on an increased incidence of heratocvte necrosis (CUT. 1982aN). an UF, of 3 dO0 5) has been applied to account for uncertainty in characterizing the toxicodynamic differences between rats and humans following oral tgDNT exposure. The toxicokinetic uncertainty has been accounted for by calculation of a HED through application of a DAF as outlined in the Recommended Use of Body Weisht3'4 as the Default Method in Derivation of the Oral Reference Dose ('U.S. EPA. 20 lib). ufd 3 An UFn of 3 has been applied because there is one developmental toxicity studv (Price et al.. 1985^ in addition to subchronic- and chronic-duration studies, but there are no two-generation reproductive toxicity studies. UFh 10 An UFh of 10 has been applied for inter-individual variability to account for human-to-human variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and toxicodynamics of tgDNT in humans. ufl 1 An UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL10. UFS 1 An UFS of 1 has been applied because a subchronic-duration study was selected as the principal study. UFC 100 :As described in detail in Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011b'). rate-related processes scale across species in a manner related to both the direct (BW11) and allometric scaling (BW3/4) aspects such that BW3 4 BW11 = BW ' converted to a DAF = BWa1/4 - BWhI/4. 44 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 DERIVATION OF SCREENING CHRONIC PROVISIONAL RfD (SCREENING CHRONIC p-RfD) The chronic-duration study database for tgDNT includes two studies (Leonard et al.. 1987; CUT. 1982a). Leonard et al. (1987) conducted a 1-year study with two doses of tgDNT (0 and 35 mg/kg-day) administered to male F344 rats. The study authors reported significantly decreased body weight, increased absolute and relative liver weights, and liver pathological changes. A LOAEL of 35 mg/kg-day is established based on the observed effects; no NOAEL could be identified. The chronic-duration study by CUT (1982a) is a comprehensive study composed of three interim chronic-duration evaluations (i.e., 52-,78-, and 104-week interim sacrifices). Table A.3 summarizes all potential critical effects and corresponding NOAELs and LOAELs for each interim study. All the endpoints shown in this table were modeled with BMDS (version 2.2.2), and the estimated BMDLs are also summarized. As shown in Table A.3, the interim sacrifices consistently indicated that the liver and kidneys are the target organs of tgDNT, and the liver is a more sensitive target organ than the kidneys. During all three interim sacrifices, the responses observed in male rats were consistently more sensitive than in the female rats (e.g., relatively lower PODs for hepatocyte necrosis ranged from 0.059 to 0.5 mg/kg-day in males vs. 1.31 to 3.64 [LOAEL] in females). Among the responses observed in male rats, hepatocyte necrosis is consistently shown as the most sensitive response. Therefore, male rat hepatocyte necrosis data have been further evaluated (see Table A.4) to find the most sensitive POD. Although, the necrosis incidences in male rats at 52 weeks appeared to be more sensitive than the 78- and 104-week groups, the results might be questionable because (1) the 52-week high-dose group showed a much lower response (50%) compared to 70% and 90% incidence at the low- and mid-doses, respectively; and (2) the 78- and 104-week studies, which had a longer exposure duration and larger sample sizes, showed less response (35—60%) at low- and mid-doses compared to those (70% and 90%, respectively) at 52-week sacrifice. At 104 weeks, male hepatocyte necrosis incidence at the low-dose (54%) was higher than that at the mid dose (48%); therefore, BMD modeling cannot adequately model the full data set (see Appendix C for details). In order to provide a best estimate for this data set, the mid-dose data point (48%>) was dropped and two data points (control and low dose) were modeled with BMDS (version 2.2.2), with an estimated BMDLio of 0.363 mg/kg-day (see Appendix C for details). As part of the database to derive a chronic RfD, the developmental study by Price et al. (1985) identified higher NOAELs for maternal toxicity (37.5 mg/kg-day) and developmental toxicity (100 mg/kg-day). Therefore, the BMDLio of 0.363 mg/kg-day for hepatocyte necrosis at 104 weeks in the CUT (1982a) study is the most sensitive and is considered protective for all the observed effects and is chosen as the POD for derivation of the screening chronic p-RfD. 45 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table A.3. Potential Critical Effects in Male and Female F344 Rats After Dietary Exposure to tgDNT for 52, 78, and 104 Weeks Endpoints NOAEL (mg/kg-d) LOAEL (mg/kg-d) BMDL10 (mg/kg-d) POD (mg/kg-d) POD Selection (mg/kg-d) 52-Week Males Relative liver weight NA 3.47 1.66 1.66 Relative kidney weight 3.47 13.9 No fit 3.47 Hepatocyte necrosis NA 3.47 0.059 0.059 0.059 Hyperbasophilic hepatocyte 3.47 13.9 No fit 3.47 Vacuolation NA 3.47 No fit 3.47 (LOAEL) 52-Week Females Relative liver weight 3.46 13.9 No fit 3.46 Relative kidney weight 13.9 35.1 No fit 13.9 Hepatocyte necrosis 13.9 35.1 3.64 3.64 Hepatocyte megalocytosis 13.9 35.1 6.86 6.86 Hyperbasophilic hepatocyte 3.46 13.9 2.20 2.20 2.20 78-Week Males Relative liver weight NA 3.49 1.34 1.34 Relative kidney weight NA 3.49 1.07 2.72 Hepatocyte necrosis NA 3.49 0.50 0.5 0.5 Cystic degeneration 3.49 14 No fit 3.49 78-Week Females Relative liver weight NA 3.45 2.15a 2.15 Relative kidney weight 3.45 14 3.56 3.67 Hepatocyte necrosis 3.45 14 1.31 1.31 1.31 104-Week Males Relative liver weight NA 3.51 1.36a 1.36 Relative kidney weight Original data illegible Hepatocyte necrosis NA 3.51 0.3633 0.363 0.363 Cystic degeneration 3.51 14.0 No fit 3.51 Hepatocyte megalocytosis NA 3.51 No fit 3.51 (LOAEL) 104-Week Females Relative liver weight NA 3.46 Control SD from original data is illegible 3.46 (LOAEL) 3.46 (LOAEL) Relative kidney weight 3.46 14.0 No fit 3.46 Hepatocyte necrosis NA 3.46 No fit 3.46 (LOAEL) Hepatocyte megalocytosis NA 3.46 2.09 2.09 aBMD modeling was performed with two data points (control and low dose). 46 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 A.4. Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 52, 78 and 104 Weeks" Week Control Low-dose Mid-dose High-dose 52 (n = 10) 0/10 7/10 (70°) 9/10 (90) 5/10 (50) 78 {n = 20) 0/20 7/20 (35) 12/20 (60) NAb 104 (n = 61, 70, and 23 for control, low-, and mid-dose groups, respectively) 0/61 38/70 (54) 11/23 (48) NAb "CUT (1982a). bAll the high-dose rats were sacrificed at Week 55. °Percent animals with necrosis Following U.S. EPA (201 lb) guidance, the POD for the rat 104-week study is converted to a HED through an application of a DAF derived as follows: DAF = (BWa1/4 - BWh1/4) where DAF = dosimetric adjustment factor BWa = animal body weight BWh = human body weight Using a BWa of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA. 1988). the resulting DAF is 0.24. Applying this DAF to the BMDLm identified in the rat 104-week study yields a BMDLiohed as follows: PODhed = BMDLio (mg/kg-day) x DAF = BMDLio (mg/kg-day) x 0.24 = 0.363 (mg/kg-day) x 0.24 = 0.087 mg/kg-day Screening Chronic p-RfD = PODhed ^ UFc = 0.087 mg/kg-day -M00 = 9 x 10~4 mg/kg-day 2As described in detail in Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011b'). rate-related processes scale across species in a manner related to both the direct (BW11) and allometric scaling (BW3/4) aspects such that BW3 4 BW11 = BW ' converted to a DAF = BWa1/4 - BWhI/4. 47 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table A.5. Uncertainty Factors for the Screening Chronic p-RfD for tgDNT UF Value Justification ufa 3 For the POD based on an increased incidence of heratocvte necrosis (CUT. 1982aN). a UF -, of 3 CIO05) has been applied to account for uncertainty in characterizing the toxicodynamic differences between rats and humans following oral tgDNT exposure. The toxicokinetic uncertainty has been accounted for by calculation of a HED through application of a dosimetric adjustment factor (DAF) as outlined in Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011W. ufd 3 An UFn of 3 has been applied because there is one developmental toxicity studv (Price et al.. 1985s) in addition to subchronic- and chronic-duration studies, but there are no two-generation reproductive toxicity studies. UFh 10 An UFh of 10 has been 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 tgDNT in humans. ufl 1 An UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDLi0. UFS 1 An UFS of 1 has been applied because a chronic-duration study was selected as the principal study. UFC 100 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of Screening Provisional Oral Slope Factor (p-OSF) There are two oral carcinogenicity studies in rats (Leonard et ai. 1987; CUT. 1982a). Leonard et al. (1987) indicated that exposure to tgDNT (35 mg/kg-day) for 1 year caused a 47% increase in the incidence of hepatocellular tumors in male rats compared to the control (only one treatment dose). In the study conducted by CUT (1982a). the incidence of hepatocellular carcinoma was observed beginning at Week 26, and the highest incidence occurred at Week 104. In addition, increased liver neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas were observed in males at Week 104. Appendix B summarizes these carcinogenicity results. The overall tumor response is relatively higher in male rats than in female rats at 104 weeks from the CUT (1982a) study, and, therefore. Table A.6 summarizes only the male incidence data. Table A.6 shows BMD dose-response modeling was conducted for various tumor types. All these tumor incidence data successfully fit to the Multistage-Cancer model in BMDS (version 2.2.21 see Appendix D for details), and Table A.7 summarizes the modeling results. Based on the BMD modeling results, the calculated cancer slope factors (oral slope factor or OSF) for hepatocellular carcinomas, liver neoplastic nodules, combined hepatocellular carcinoma and/or neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas are 0.047, 0.037, 0.060, 0.027, and 0.072 (mg/kg-day)-1, respectively. Because treatment with tgDNT produced multiple types of tumors in male rats in three different tissues in the CUT bioassay (CUT. 1982a). the overall oral cancer slope factor for tgDNT exposure was derived based on the male incidence data for combined hepatocellular carcinoma and/or neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas by assuming that different tumor types are independent from each other. The overall tumor incidence was fit with the MSCombo multiple tumor model (BMDS version 2.2.2; see Appendix D for details), and the estimated 48 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 BMDLio is 0.852 mg/kg-day. Similar to the screening subchronic and chronic p-RfDs, this BMDLio was further converted from an animal dose to an HED, and then used as the PODHed to derive the p-OSF for tgDNT. Table A.6. Incidences of Hepatocellular Carcinomas, Liver Neoplastic Nodules, Mammary Fibroadenomas, and Subcutaneous Fibromas in Male Rats at Week 104a Parameter Exposure Group, ADD mg/kg-db Control (0) Low-dose (3.51) High-dose (14.0) Number examined 61 70 23 Hepatocellular carcinoma 1(2) 9(13)* 21 (91)* Neoplastic nodules 9(15) 11(16) 15 (65)* Hepatocellular carcinomas and/or neoplastic nodules 10(16) 19 (27) 23 (100)* Mammary fibroadenomas 3(5) 7(10) 5 (22)* Subcutaneous fibromas 5(8) 14 (20) 14 (61)* "CUT (1982a). bValues expressed as number of animals (% of animals with lesion/effect); % calculated by EPA. * Statistically different from controls, p < 0.05. Table A.7. Goodness-of-Fit Statistics and BMDio and BMDLio Values for Dichotomous Model for Four Types of Tumors and Combined Tumors in Male F344 Rats Exposed to tgDNT Orally for 104 Weeks" Multistage-Cancer Model Goodness-of-fit p-V alueb BMD10 (mg/kg-d) BMDL10 (mg/kg-d) Cancer Slope Factor (mg/kg-d)1 (Animal Dose) Hepatocellular carcinoma 0.6332 3.04 2.15 0.047 Liver neoplastic nodules 0.5454 4.86 2.69 0.037 Hepatocellular carcinoma and/or neoplastic nodule(s) 0.2389 2.42 1.68 0.060 Mammary fibroadenomas 0.9081 7.37 3.73 0.027 Subcutaneous fibromas 0.4138 2.01 1.38 0.072 Combined tumors NA 1.20 0.852 aCIIT (1982a1. bValues >0.1 meet conventional goodness-of-fit criteria. 49 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 PODhed = BMDLio(mg/kg-day) x DAF = BMDLio (mg/kg-day) x (BWa1/4 - BWh1/4) = BMDLio (mg/kg-day) x (0.3331/4 - 701/4) = 0.852 (mg/kg-day) x 0.263 = 0.224 mg/kg-day Note: The BWa of 0.333 kg is the mean body weight from the low-dose male group at Week 104 (see Table B.7). Screening p-OSFHuman = BMR h- BMDLiohed (mg/kg-day) = 0.1-0.224 = 4.5 x 10-1 (mg/kg-day)-1 The p-OSF is 4.5 x 10_1 (mg/kg-day)-1 based on combined tumor incidences in male rats from CUT (1982a). 50 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 APPENDIX B. DATA TABLES Table B.l. Average Body Weights (g) of Male and Female F344 Rats After Dietary Exposure to tgDNT for 4 Weeks" Number of animals/group 10 10 10 10 Males Time period Exposure Group (ADD, mg/kg-d; 0 (Control) 31.9 61.9 134 Initial 176.5 ± 11.0 177.9 ± 11.1 (101) 177.1 ± 10.8(100) 177.9 ± 11.7(101) Week 1 207.1 ±7.4 205.9 ± 10.4(99) 201.8 ±9.4 (97) 186.6 ± 10.5* (90) Week 2 228.0 ±8.4 221.4 ± 11.8(97) 207.8 ±9.8* (91) 167.6 ± 10.7* (74) Week 3 236.7 ±9.6 231.4 ± 13.0(98) 209.6 ± 10.1* (89) 152.2 ± 10.2* (64) Week 4 251.8 ± 13.2 236.9 ± 13.2* (94) 208.8 ± 11.9* (83) 155.8 ± 10.9* (62) Females Time period Exposure Group (ADD, mg/kg-d)b 0 (Control) 32.0 63.6 120 Initial 125.8 ±3.2 127.7 ±3.4 (102) 128.4 ±3.7 (102) 126.3 ±6.7 (100) Week 1 136.2 ±2.7 138.0 ±5.5 (101) 137.2 ±4.3 (101) 139.1 ±7.5 (102) Week 2 146.0 ±3.2 147.3 ±5.8 (101) 142.3 ±4.8 (97) 135.6 ±9.5* (93) Week 3 153.8 ±6.0 151.9 ±7.7 (99) 149.4 ± 6.6 (97) 127.7 ± 11.0* (83) Week 4 158.4 ±4.3 155.1 ±7.5 (98) 149.4 ±7.4* (94) 124.5 ± 10.1* (79) "CUT fl.983). bValues expressed as mean ± SD (% of control); % calculated by EPA. * Statistically different from controls, p < 0,05. 51 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.2. Hematology Results for Male and Female F344 Rats After Dietary Exposure to tgDNT for 4 Weeks" Number of animals/group 10 10 10 10 Males Parameter Exposure Group (ADD, mg/kg-d)b 0 (Control) 31.9 61.9 134 MetHb (%) 1.12 ± 0.61 1.20 ±0.40 (107) 1.38 ±0.71 (123) 2.63 ± 1.03* (235) Reticulocytes (%) 1.11 ± 0.31 1.99 ±0.82* (179) 2.81 ± 1.55* (253) 6.84 ±3.05* (616) Heinz bodies (%) 0.00 ±0.00 0.04 ±0.05* 0.06 ±0.07* 5.25 ±3.28* Females Exposure Group (ADD, mg/kg-d)b 0 (Control) 32.0 63.6 120 MetHb (%) 0.57 ±0.48 1.46 ±0.76* (256) 1.04 ±0.77 (182) 1.99 ±0.88* (349) Reticulocytes (%) 1.45 ±0.27 3.27 ±0.94* (226) 2.95 ±0.81* (203) 4.05 ±0.91* (279) Heinz bodies (%)° 0.00 ±0.00 0.07 ±0.07* 0.09 ±0.06* 0.14 ± 0.11* aCIIT fl.983). bValues expressed as mean ± SD (% of control); % calculated by EPA. * Statistically different from controls, p < 0,05. 52 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.3. Gross Pathology for Male and Female F344 Rats After Dietary Exposure to tgDNT for 4 Weeks" Number of animals/group 10 10 10 10 Parameter Exposure Group (ADD, mg/kg-d)b 0 (Control) 31.9 61.9 134 Males Mottled lungs (dark-red or brown areas) 0 6 (60) 10 (100) 3 (30) Liver Surface rough or granular 0 5 (50) 10 (100) 0(0) Mottled appearance 0 5 (50) 0(0) 3 (30) Yellowish tinge 0 3 (30) 0(0) 6(60) Spleen Dark or black 0 1(10) 0(0) 10 (100) Surface rough or granular 0 1(10) 0(0) 0(0) Thickened 0 0(0) 0(0) 3 (30) Enlarged 0 0(0) 0(0) 1(10) Kidneys Greenish tinge 0 0(0) 0(0) 3 (30) Dark zone at cortico-medullary junction 0 1(10) 0(0) 1(10) Small white raised nodule on ventral surface 0 0(0) 0(0) 1(10) Parameter Exposure Group (ADD, mg/kg-d)b 0 (Control) 32.0 63.6 120 Females Mottled lungs 0 0(0) 0(0) 1(10) Liver Yellowish tinge 0 0(0) 0(0) 4 (40) Spleen Dark or black 0 0(0) 0(0) 5 (50) Thickened 0 0(0) 0(0) 7 (70) Enlarged 0 0(0) 0(0) 2 (20) Kidneys Greenish tinge 0 0(0) 0(0) 1(10) Water cyst in ovary 0 0(0) 1(10) 0(0) Dark yellow stains around vagina 0 0(0) 0(0) 2 (20) "CUT (19831. bValues expressed as number of animals affected (% affected); % calculated by EPA. 53 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.4. Selected Average Body Weights (g) and Food Consumption (g/week/rat) of Male and Female F344 Rats After Dietary Exposure to tgDNT for up to 2 Years" Time Period Exposure Group (ADD, mg/kg-d) Control (0) Low-dose (3.51) Mid-dose (14.0) High-dose (34.9) Average body weightb (Males) Week 0(« = 130) 154 ± 14.5 153 ± 14.2 (99) 151 ± 14.2 (98) 153 ± 14.6 (99) Week 26 (« = 130) 351 ±22 338 ±21.6 (96) 301 ± 19.2 (86) 265 ± 17.6 (75) Week 50 (« = 113-119)c 387 ±23.9 370 ± 20.3 (96) 316 ± 18.3 (82) 270 ± 18 (70) Week 78 (n = 95-106) 396 ±22.3 373 ±21.7 (94) 309± 25.8 (78) NA Week 102 (n = 25-75) 370 ±31 351 ±30.8 (95) 293 ± 39.3 (80) NA Average food consumption (Males) (g/week/rat) Week 0(n = 130) 0 0 0 0 Week 26 (n = 130) 108 111(103) 101 (94) 101 (94) Week 50 (n = 113-119)c 112 109 (97) 110(98) 114(102) Week 78 (n = 95-106) 107 107 (100) 106 (99) NA Week 102 (n = 25-75) 105 104 (99) 92 (88) NA Time Period Exposure Group (ADD, mg/kg-d) Control (0) Low-dose (3.46) Mid-dose (14.0) High-dose (35.1) Average body weightb (Females) Week 0(n = 130) 118 ±8.4 121 ±8.2 (103) 121 ±7.7 (103) 118 ±8.4 (100) Week 26 (n = 130) 198 ± 10.7 193 ±11.6 (97) 187 ± 11.2 (94) 172 ± 11.2 (87) Week 50 (n = 120)c 226 ± 14.5 220 ± 14.9 (97) 197 ± 13.5 (90) 180 ± 11.1 (80) Week 78 (n = 104-107) 272 ± 20 251 ±24.1 (92) 213 ± 12.8 (78) NA Week 104 (n = 61-69) 288 ± 29 267 ±25.1 (93) 213 ± 18.8 (74) NA Average food consumption (Females) (g/week/rat) Week 0(« = 130) 0 0 0 0 Week 26 (n = 130) 79 78 (99) 73 (92) 73 (92) Week 50 (n = 120)c 81 79 (98) 72 (89) 71 (88) Week 78 (n = 104-107) 87 85 (98) 80 (92) NA Week 104 (n = 61-69) 88 89 (101) 91(103) NA TUT fl.982a). ''Body-weight values expressed as mean ± SD (% of control); % calculated by EPA. °Body weight and food consumption data were collected on Week 50, not Week 52. 54 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.5. Selected Hematology Results for Male F344 Rats After Dietary Exposure to tgDNT for up to 2 Years3 Parameter Exposure Group (ADD, mg/kg-df Control (0) Low-dose (3.47) Mid-dose (13.6) High-dose (34.6) Week 26 (n = 10) Hb (g/dL) 17.12 ±0.42 17.36 ±0.48 (101) 17.26 ± 1.12(101) 16.48 ± 0.44 (96) Hematocrit (100%) 48.70 ±2.21 50.00 ±2.71 (103) 52.05 ±4.37 (107) 49.30 ±2.21 (101) RBC (x 106/mm3) 8.967 ±0.554 9.209 ±0.428 (103) 8.921 ±0.525 (99) 8.272 ± 0.392* (92) Reticulocyte (%) 1.97 ±0.80 2.23 ±0.70 (113) 2.83 ±0.85 (144) 3.60 ±0.84* (183) MetHb (%) 0.36 ±0.36 0.97 ±0.71 (269) 0.74 ± 0.55 (205) 1.80 ± 1.18* (500) WBC (x 103/mm3) 12.31 ± 1.89 14.60 ±3.15 (119) 16.13 ±2.11* (131) 15.04 ±2.73 (122) Parameter Exposure Group (ADD, mg/kg-dr Control (0) Low-dose (3.47) Mid-dose (13.9) High-dose (34.9) Week 52 (n = 10) Hb (g/dL) 16.05 ±0.59 16.11 ±0.61 (100) 16.29 ±0.66 (101) 14.07 ± 1.16* (88) Hematocrit (100%) 46.70 ± 1.70 46.45 ± 1.57 (99) 46.90 ± 1.91 (100) 47.30 ±2.89 (101) RBC (x 106/mm3) 8.507 ±0.778 8.572 ±0.570 (101) 7.585 ±0.647* (89) 5.546 ± 0.728* (65) Reticulocyte (%) 2.08 ±0.73 2.42 ±0.79 (116) 2.01 ±0.74 (97) 3.87 ± 1.90* (186) MetHb (%) 0.87 ±0.56 1.37 ±0.61 (157) 1.49 ±0.96 (171) 1.55 ±0.92 (178) WBC (x 103/mm3) 13.29 ±3.23 11.60 ±4.13 (87) 14.45 ±3.60 (109) 19.30 ± 3.47* (145) Parameter Exposure Group (ADD, mg/kg-dr NA NA NA High-dose (34.9) Week 55 (n = 10) Hb (g/dL) NA NA NA 12.82 ±0.78 Hematocrit (100%) NA NA NA 38.37 ±2.08 RBC (x 106/mm3) NA NA NA 4.129 ± 1.245 Reticulocyte (%) NA NA NA 1.61 ±0.77 MetHb (%) NA NA NA 1.42 ± 1.33 WBC (x 103/mm3) NA NA NA 4.33 ±0.69 55 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.5. Selected Hematology Results for Male F344 Rats After Dietary Exposure to tgDNT for up to 2 Years3 Parameter Exposure Group (ADD, mg/kg-df Control (0) Low-dose (3.49) Mid-dose (14.0) NA Week 78 (n = 20) Hb (g/dL) 16.81 ± 1.60 18.25 ±2.16 (109) 15.34 ±2.79 (91) NA Hematocrit (100%) 51.37 ± 3.55 55.60 ±5.58* (108) 44.80 ±7.11* (87) NA RBC (x 106/mm3) 9.303 ±0.897 9.608 ±0.859 (103) 7.998 ± 1.328* (86) NA Reticulocyte (%) 2.02 ± 1.04 2.28 ± 1.25* (113) 4.29 ±2.64* (212) NA MetHb (%) 1.77 ± 1.16 1.56 ±0.46 (88) 1.31 ± 1.01(74) NA WBC (x 103/mm3) 10.36 ±2.26 9.71 ±2.86 (94) 10.76 ± 3.89 ( 104) NA Parameter Exposure Group (ADD, mg/kg-dr Control (0) Low-dose (3.51) Mid-dose (14.0) NA Week 104 (n = 19-20) Hb (g/dL) 18.48 ±3.13 19.40 ±2.86 (105) 12.08 ±2.72* (65) NA Hematocrit (100%) 54.60 ±9.38 56.42 ±8.09 (103) 36.95 ±7.21* (68) NA RBC (x 106/mm3) 9.227 ± 1.602 9.543 ± 1.112 (103) 6.819 ± 1.807* (74) NA Reticulocyte (%) 3.53 ±3.53 2.99 ± 1.89 (85) 6.72 ±2.93* (190) NA MetHb (%) 1.16 ± 1.01 1.32 ±0.99 (114) 1.46 ±0.86 (126) NA WBC (x 103/mm3) 11.22 ±4.06 12.85 ±3.62 (115) 9.35 ± 10.16(83) NA "CUT (1982a). bValues expressed as mean ± SD (% of control); % calculated by EPA. * Statistically different from controls, p < 0.05. 56 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.6. Selected Clinical Chemistry Results for Male and Female F344 Rats After Dietary Exposure to tgDNT for up to 2 Years" Males Parameter Exposure Group (ADD, mg/kg-d)b Week 26 (n = 10) Control (0) Low-dose (3.47) Mid-dose (13.6) High-dose (34.6) SGPT (IU/L) 43.0 ± 13.6 24.7 ±9.3* (57) 25.8 ±4.9* (60) 39.1 ±8.3 (91) ALP (IU/L) 77.60 ± 7.90 67.70 ± 7.24* (87) 63.10 ±3.90* (81) 78.56 ±6.17 (101) BUN (mg/dL) 21.03 ±2.09 17.98 ± 1.37* (85) 18.66 ± 1.55 (89) 24.56 ±2.63* (117) Week 52 (n = 10) 0 (Control) Low-dose (3.47) Mid-dose (13.9) High-dose (34.9) SGPT (IU/L) 41.5 ±6.5 22.2 ±5.8 (53) 25.1 ±5.7 (60) 138.7 ±72.4* (334) ALP (IU/L) 68.70 ± 10.95 53.50 ±4.25 (78) 57.60 ± 8.67 (84) 134.20 ±65.45* (195) BUN (mg/dL) 18.00 ± 1.08 17.30 ±0.96 (96) 16.77 ± 1.87 (93) 29.91 ±5.83* (166) Week 55 (n = 10) NA NA NA High-dose (34.9) SGPT (IU/L) NA NA NA 136.8 ±96.1 ALP (IU/L) NA NA NA 161.55 ±66.89 BUN (mg/dL) NA NA NA 28.92 ±3.37 Week 78 (n = 20) Control (0) Low-dose (3.49) Mid-dose (14.0) NA SGPT (IU/L) 28.7 ±6.5 24.1 ±16.2 (84) 55.3 ± 56.3* (193) NA ALP (IU/L) 74.80 ± 10.35 64.95 ± 29.20 (87) 79.95 ±47.52 (107) NA BUN (mg/dL) 21.55 ± 1.73 17.09 ±2.22* (79) 25.14 ±6.52 (117) NA Week 104 (n = 19-20) 0 Low-dose (3.51) Mid-dose (14.0) NA SGPT (IU/L) 22.5 ±4.6 21.7 ±5.9 (96) 78.7 ±42.7* (350) NA ALP (IU/L) 65.10 ± 11.54 45.05 ± 9.59* (69) 89.58 ±51.09 (138) NA BUN (mg/dL) 27.54 ±7.53 19.33 ±3.08* (70) 77.49 ±47.03* (281) NA Females Parameter Exposure Group (ADD, mg/kg-d)b Week 26 (n = 10) Control (0) Low-dose (3.22) Mid-dose (13.9) High-dose (34.9) BUN (mg/dL) 18.82 ± 1.43 19.20 ±3.04 (102) 18.90 ± 1.49(100) 23.88 ±4.31*( 127) aCIIT (1982a). bValues expressed as mean ± SD (% of control); % calculated by EPA. * Statistically different from controls, p < 0.05. 57 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.7. Terminal Body Weights (g) of Male and Female F344 Rats After Dietary Exposure to tgDNT for 2 Years" Average body weight—interim and terminal sacrifices (males) Time Period Exposure Group (ADD, mg/kg-d)b Week 26 (n = 10) Control (0) Low-dose (3.47) Mid-dose (13.6) High-dose (34.6) 328 ± 23 311 ±14* (95) 297 ±31* (91) 253 ± 13* (77) Week 52 (n = 10) Control (0) Low-dose (3.47) Mid-dose (13.9) High-dose (34.9) 363 ± 22 335 ±21 (92) 287 ±21* (79) 245 ± 14* (67) Week 55 (n = 101) NA NA NA High-dose (34.9) NA NA NA 259 ± 18 Week 78 (« = 20) Control (0) Low-dose (3.49) Mid-dose (14.0) NA 374.30 ± 16.936 358.20 ± 15.531* (96) 286.00 ±20.261* (76) NA Week 104 (n = 58, 68, and 19 for 0, 3.5, and 14 mg/kg-d, respectively) 0 Low-dose (3.51) Mid-dose (14.0) NA 352.03 ±28.836 333.19 ±20.806* (95) 257.79 ±30.608* (73) NA Average body weight—interim and terminal sacrifices (females) Time Period Exposure Group (ADD, mg/kg-d)b Week 26 (n = 10) Control (0) Low-dose (3.22) Mid-dose (14.0) High-dose (34.9) 184 ±8 182 ± 12* (99) 182 ± 13* (99) 158 ± 6* (86) Week 52 (n = 10) Control (0) Low-dose (3.46) Mid-dose (13.9) High-dose (35.1) 214 ± 14 207 ± 15 (97) 190 ± 12* (89) 169 ±11* (79) Week 55 (n = 109) NA NA NA High-dose (35.1) NA NA NA 170 ± 13 Week 78 (n = 20) Control (0) Low-dose (3.45) Mid-dose (14.0) NA 259.65 ± 14.651 238.80 ± 11.551* (92) 201.35 ±11.815* (78) NA Week 104 (n = 55, 59, and 59 for 0, 3.5, and 14 mg/kg-d, respectively) Control (0) Low-dose (3.46) Mid-dose (14.0) NA 271.88 ±28.15 256.22 ± 22.627* (94) 198.84 ± 19.27* (73) NA "CUT Q982a). bValues expressed as mean ± SD (% of control); % calculated by EPA. * Statistically different from controls, p < 0.05. 58 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.8. Selected Organ Weights (g) of Male F344 Rats After Dietary Exposure to tgDNT for 2 Years" Time Period Exposure Group (ADD, mg/kg-d)b Week 26 (n = 10) Control (0) Low-dose (3.47) Mid-dose (13.6) High-dose (34.6) Absolute liver weight 8.78 ±0.87 8.80 ±0.56 (100) 9.87 ±0.80* (112) 13.19 ± 1.01* (150) Relative liver weight 2.68 ±0.23 2.83 ±0.07 (105) 3.35 ±0.36* (125) 5.23 ±0.48* (195) Absolute brain weight 1.97 ±0.05 1.96 ±0.06 (99) 1.95 ±0.05 (99) 1.95 ±0.04 (99) Relative heart weight 0.32 ±0.03 0.32 ±0.03 (100) 0.30 ±0.03 (94) 0.37 ±0.05* (116) Relative kidney weight 0.685 ± 0.04 0.687 ±0.025 (100) 0.736 ± 0.066 (107) 0.942 ±0.044* (138) Relative lung weight 0.415 ±0.049 0.407 ± 0.022 (98) 0.416 ±0.045 (100) 0.496 ±0.031* (120) Relative testis weight 1.32 ± 0.11 1. 35 ±0.07 (102) 1.34 ±0.08 (102) 1.67 ±0.18* (127) Week 52 (n = 10) Control (0) Low-dose (3.47) Mid-dose (13.9) High-dose (34.9) Absolute liver weight 9.11 ±0.66 10.15 ±0.54 (111) 15.46 ± 1.23* (170) 28.73 ±7.86* (315) Relative liver weight 2.51 ±0.12 3.00 ±0.20 (120) 5.41 ±0.54* (216) 11.81 ± 3.54* (471) Absolute brain weight 2.13 ±0.16 2.01 ±0.08 (94) 1.99 ±0.07 (93) 2.00 ±0.13 (94) Relative heart weight 0.296 ±0.02 0.335 ±0.05 (113) 0.355 ±0.035 (120) 0.455 ±0.25* (154) Relative kidney weight 0.704 ±0.083 0.718 ±0.066 (102) 0.909 ±0.035* (129) 1.160 ±0.190* (165) Relative lung weight 0.417 ±0.055 0.400 ± 0.054 (96) 0.476 ±0.060 (114) 0.591 ±0.130* (142) Relative testis weight 1.42 ±0.21 1.46 ±0.19 (103) 1.71 ±0.10 (120) 1.59 ±0.40 (112) Week 55 (n = 101) NA NA NA High-dose (34.9) Absolute liver weight NA NA NA 29.88 ±5.77 Relative liver weight NA NA NA 11.65 ±2.45 Absolute brain weight NA NA NA 1.97 ±0.08 Relative heart weight NA NA NA 0.39 ±0.04 Relative kidney weight NA NA NA 1.15 ±0.28 Relative lung weight NA NA NA 0.620 ±0.471 Relative testis weight NA NA NA 1.48 ±0.45 Week 78 (n = 20) Control (0) Low-dose (3.49) Mid-dose (14.0) NA Absolute liver weight 9.422 ±0.6001 11.363 ±0.9594* (121) 18.580 ±3.8101*° (197) NA Relative liver weight 2.518 ±0.1279 3.175 ±0.2682* (126) 6.259 ± 1.0671* (249) NA Absolute brain weight 2.1010 ±0.11026 2.1180 ±0.06661 (101) 2.0150 ±0.12089 (96) NA Relative heart weight 0.290 ±0.03 0.302 ±0.02 (104) 0.400 ±0.60* (138) NA 59 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.8. Selected Organ Weights (g) of Male F344 Rats After Dietary Exposure to tgDNT for 2 Years" Relative kidney weight 0.665 ± 0.0282 0.744 ±0.0419* (112) 1.029 ±0.1003* (155) NA Relative lung weight 0.365 ±0.262 0.416 ±0.819* (114) 0.476 ± 0.0372* (130) NA Relative testis weight 1.450 ±0.3114 1.845 ±0.2662* (127) 1.981 ±0.3586* (137) NA Week 104 (n = various) Control (0) Low-dose (3.51) Mid-dose (14.0) NA Absolute liver weight 10.615 ± 1.7036 12.126 ± 1.5875* (114) 17.349 ±2.112* (163) NA Relative liver weight 3.024 ±0.4842 3.644 ±0.4394* (121) 6.901 ± 1.1686* (228) NA Absolute brain weight 2.0791 ±0.08113 2.0926± 0.10566 (101) 2.0542 ±0.10089 (99) NA Relative heart weight 0.321 ±0.31 0.337 ±0.38* (105) 0.389 ±0.08* (121) NA Relative kidney weight c 0.642 ±0.0661 1.227 ±0.2115* NA Relative lung weight 0.490 ±0.116 0.519 ±0.112 (106) 0.588 ±0.170* (120) NA Relative testis weight 2.150 ±0.4924 2.581 ±0.5747* (120) 2.978 ± 1.316* (139) NA aCIIT (1982a). bValues expressed as mean ± SD (% of control); % calculated by EPA. Data illegible. * Statistically different from controls, p < 0.05. 60 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.9. Selected Organ Weights (g) of Female F344 Rats After Dietary Exposure to tgDNT for up to 2 Years" Parameter Exposure Group (ADD, mg/kg-d)b Week 26 (n = 10) Control (0) Low-dose (3.22) Mid-dose (13.9) High-dose (34.9) Absolute liver weight 4.81 ±0.17 4.98 ± 0.35 (103) 5.89 ±0.44* (118) 7.26 ±0.56* (151) Relative liver weight 2.62 ±0.10 2.74 ±0.08 (105) 3.24 ±0.11* (124) 4.60 ±0.34* (176) Absolute brain weight 1.85 ±0.07 1.84 ±0.06 (99) 1.80 ±0.03 (97) 1.80 ±0.04 (97) Relative heart weight 0.36 ±0.02 0.36 ±0.03 (100) 0.37 ±0.03 (103) 0.41 ±0.04* (114) Relative kidney weight 0.689 ±0.017 0.725 ± 0.027 (105) 0.738 ±0.027* (107) 0.853 ±0.051* (124) Relative lung weight 0.53 ±0.04 0.50 ±0.01 (94) 0.53 ±0.05 (100) 0.59 ±0.05* (111) Relative ovary weight 0.040 ± 0.008 0.041 ±0.007 (103) 0.043 ±0.007 (108) 0.048 ± 0.007 (120) Week 52 (n = 10) Control (0) Low-dose (3.46) Mid-dose (13.9) High-dose (35.1) Absolute liver weight 5.74 ±0.30 5.80 ±0.50 (101) 7.06 ±0.41* (123) 8.90 ±0.61* (155) Relative liver weight 2.69 ±0.18 2.82 ±0.27 (105) 3.73 ±0.14* (139) 5.28 ±0.48* (196) Absolute brain weight 1.94 ±0.16 1.85 ±0.06 (95) 1.83 ±0.08 (94) 1.85 ±0.07 (95) Relative heart weight 0.43 ±0.10 0.34 ±0.03* (79) 0.38 ±0.03 (88) 0.46 ±0.03 (107) Relative kidney weight 0.727 ± 0.074 0.720 ± 0.047 (99) 0.762 ±0.105 (105) 0.949 ±0.044* (131) Relative lung weight 0.538 ±0.113 0.480 ± 0.050 (89) 0.500 ±0.040 (93) 0.570 ± 0.020 (106) Relative ovary weight 0.045 ± 0.008 0.051 ±0.0125 (113) 0.0740 ± 0.0699 (164) 0.057 ±0.015 (127) Week 55 (n = various) NA NA NA High-dose (35.1) Absolute liver weight NA NA NA 9.92 ± 1.21 Relative liver weight NA NA NA 5.87 ±0.93 Absolute brain weight NA NA NA 1.84 ± 0.11 Relative heart weight NA NA NA 0.44 ±0.04 Relative kidney weight NA NA NA 0.964 ± 0.080 Relative lung weight NA NA NA 0.616 ±0.114 Relative ovary weight NA NA NA 0.058 ±0.032 Week 78 (n = 20 for control and low-dose, n = 19 for mid-dose) Control (0) Low-dose (3.45) Mid-dose (14.0) NA Absolute liver weight 6.639 ± 0.4462 6.731 ±0.7549 (101) 9.074 ± 0.4790* (137) NA Relative liver weight 2.560 ±0.155 2.826 ±0.334* (110) 4.543 ± 0.2875* (177) NA Absolute brain weight 2.1035 ±0.15936 2.0290 ±0.18547 (96) 1.8730 ±0.13483* (89) NA Relative heart weight 0.378 ± 0.0626 0.374 ± 0.0803 (99) 0.430 ±0.0619 (114) NA Relative kidney weight 0.718 ±0.0741 0.731 ±0.0906 (102) 0.951 ±0.2626* (132) NA 61 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.9. Selected Organ Weights (g) of Female F344 Rats After Dietary Exposure to tgDNT for up to 2 Years" Parameter Exposure Group (ADD, mg/kg-d)b Relative lung weight 0.475 ± 0.0598 0.491 ±0.0798 (103) 0.508 ±0.826 (107) NA Relative ovary weight 0.0852 ±0.17849 0.1293 ±0.17126 (152) 0.0573 ±0.01170 (67) NA Week 104 (n = various) Control (0) Low-dose (3.46) Mid-dose (14.0) NA Absolute liver weight 7.462 ± 1.0733 8.702 ± 1.1791* (117) 12.309 ± 1.7879* (165) NA Relative liver weight 2.783 ±c 3.486 ±0.4438 * (125) 6.159 ±0.8252* (221) NA Absolute brain weight 1.9211 ±0.18071 1.9129 ±0.08483 (100) 1.9085 ±0.11577 (99) NA Relative heart weight 0.342 ±0.0512 0.373 ± 0.0456* (109) 0.449 ±0.0501* (131) NA Relative kidney weight 0.732 ±0.0971 0.793 ± 0.0732* (108) 1.157 ±0.1445* (128) NA Relative lung weight 0.496 ±0.1963 0.488 ± 0.0934 (98) 0.647 ±0.3783*c (130) NA Relative ovary weight 0.0392 ±0.01892c 0.0460 ±0.01491* (117) 0.0823 ±0.1136* (210) NA TUT f 1982a). Values expressed as mean ± SD (% of control); % calculated by EPA. Data were illegible. * Statistically different from controls, p < 0.05. 62 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.10. Selected Hepatotoxicity Incidences for Male and Female F344 Rats After Dietary Exposure to tgDNT for 26 Weeks" Parameter Exposure Group (ADD, mg/kg-d)b Males (10/group) Control (0) Low-dose (3.47) Mid-dose (13.6) High-dose (34.6) Fatty metamorphosis 0 4 (40) 4(40) 0 Hepatocyte necrosis 0 0 7 (70)* 9 (90)* Megalocytosis 0 0 0 4 (40) Periportal hyperbasophilic hepatocytes 0 0 8(80)* 0 Vacuolation 0 1(10) 0 4(40) Parameter Exposure Group (ADD, mg/kg-d)b Females (10/group) Control (0) Low-dose (3.22) Mid-dose (13.9) High-dose (34.9) Fatty metamorphosis 0 0 0 0 Hepatocyte necrosis 0 0 2 (20) 4 (40) Megalocytosis 0 0 0 0 Periportal hyperbasophilic hepatocytes 1(10) 2 (20) 6(60) 10 (100)* Vacuolation 0 0 2(20) aCIIT (1982a). bValues expressed as number of animals with lesions (% of animals with lesion/effect); % calculated by EPA. * Statistically different from controls, p < 0,05. 63 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.ll. Selected Hepatotoxicity Incidences for Male and Female F344 Rats After Dietary Exposure to tgDNT for 52 Weeks" Parameter Exposure Group (ADD, mg/kg-d)b Males (10/group) Control (0) Low-dose (3.47) Mid-dose (13.9) High-dose (34.9) Hepatocyte necrosis 0 7(70)* 9(90)* 5 (50)* Megalocytosis 0 1(10) 7 (70)* 3 (30) Periportal hyperbasophilic hepatocytes 0 2 (20) 9(90)* 6 (60)* Vacuolation 0 7 (70)* 7 (70)* 6 (60)* Parameter Exposure Group (ADD, mg/kg-d)b Females (10/group) Control (0) Low-dose (3.46) Mid-dose (13.9) High-dose (35.1) Hepatocyte necrosis 0 0 2 (20)* 7 (70)* Megalocytosis 0 0 2 (20) 4 (40) Periportal hyperbasophilic hepatocytes 0 1(10) 6 (60)* 5 (50)* Vacuolation 0 0 0 1(10) "CUT f1982a). bValues expressed as number of animals with lesions (% of animals with lesion/effect); % calculated by EPA. * Statistically different from controls, p < 0,05. 64 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.12. Selected Hepatotoxicity Incidences at Week 78a Fatty Metamorphosis Incidence Males (20/group) Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) Low-dose (3.49) Mid-dose (14.0) Minimal 0 7(35) 1(5) Slight 0 6(30) 13 (65) Moderate 0 0 3(15) Moderately severe 0 0 0 Females (20/group) Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) Low-dose (3.45) Mid-dose (14.0) Minimal 0 0 5(25) Slight 0 1(5) 11 (55) Moderate 0 0 0 Moderately severe 0 0 1(5) Other Hepatotoxicity Incidence Males (20/group) Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) Low-dose (3.49) Mid-dose (14.0) Cystic degeneration 0 0 8 (40)* Necrosis of individual hepatocytes 0 7 (35)* 12 (60)* Females (20/group) Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) Low-dose (3.45) Mid-dose (14.0) Necrosis of individual hepatocytes 0 3(15) 20 (100)* Megalocytosis of hepatocytes 0 0 2(10) aCIIT (1982a). bValues expressed as number of animals with lesions, (% of animals with lesion/effect), % calculated by EPA. * Statistically different from controls, p < 0,05. 65 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.13. Selected Hepatotoxicity Incidences at Week 104a Fatty Metamorphosis Incidence Males Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) (n = 61) Low-dose (3.51) (n = 70) Mid-dose (14.0) (n = 23) Minimal 8(13) 17 (24) 3(13) Slight 1(2) 33 (47) 5(22) Moderate 0(0) 1(1) 5(22) Moderately severe 0(0) 0(0) 0(0) Females Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) (n = 57) Low-dose (3.46) (n = 61) Mid-dose (14.0) (n = 68) Minimal 2(4) 6(10) 1(1) Slight 3(5) 6(10) 24 (35) Moderate 2(4) 1(2) 38 (56) Moderately severe 0(0) 0(0) 2(3) Other Hepatotoxicity Incidence Males Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) (n = 61) Low-dose (3.51) (n = 70) Mid-dose (14.0) (n = 23) Cystic degeneration 2(3) 2(3) 16 (70)* Necrosis of individual hepatocytes 0(0) 38 (54)* 11 (48)* Megalocytosis of hepatocytes 0(0) 34 (49)* 8 (35)* Females Exposure Group (ADD, mg/kg-d)b Grading of Finding Control (0) (n = 57) Low-dose (3.46) (n = 61) Mid-dose (14.0) (n = 68) Number examined 57 61 68 Cystic degeneration 3(5) 0(0) 6(9) Necrosis of individual hepatocytes 1(2) 18 (30)* 22 (32)* Megalocytosis of hepatocytes 1(2) 22 (36)* 37 (54)* "CUT (1982a). bValues expressed as number of animals with lesions, (% of animals with lesion/effect), % calculated by EPA. * Statistically different from controls, p < 0,05. 66 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.14. Incidences of Hepatocellular Carcinomas in Male F344/CrlBR Rats at Weeks 26, 52, and 78a Parameter Exposure Group (ADD, mg/kg-d)b 26 Week (n = 10) Control (0) Low-dose (3.47 mg/kg-d) Mid-dose (13.6 mg/kg-d) High-dose (34.6 mg/kg-d) Hepatocellular carcinoma 0 0 0 2 (20) 52 Week (n = 10) Control (0) Low-dose (3.47 mg/kg-d) Mid-dose (13.9 mg/kg-d) High-dose (34.9 mg/kg-d Hepatocellular carcinoma 0 0 3 (30) 10 (100)* 78 Week (n = 20) Control (0) Low-dose (3.49 mg/kg-d) Mid-dose (14.0 mg/kg-d) NA Hepatocellular carcinoma 0 0 19 (95)* NA aCIIT (1982a). bValues expressed as number of animals (% of animals with lesion/effect); % calculated by EPA. * Statistically different from controls, p < 0,05. 67 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.15. Incidences of Hepatocellular Carcinomas, Liver Neoplastic Nodules, and Other Tumors in Male and Female F344 Rats at Week-104a Parameter Exposure Group (ADD, mg/kg-d)b Males Control (0) Low-dose (3.51 mg/kg-d) Mid-dose (14.0 mg/kg-d) Number examined 61 70 23 Hepatocellular carcinoma 1(2) 9 (13)* 21 (91) * Neoplastic nodule(s) 9(15) 11(16) 15 (65)* Hepatocellular carcinoma and /or neoplastic nodule(s) 10 (16) 19 (27) 23 (100)* Mammary fibroadenomas 3(5) 7(10) 5 (22)* Subcutaneous fibromas 5(8) 14 (20) 14(61)* Parameter Exposure Group, (ADD, mg/kg-d)b Females Control (0) Low-dose (3.46 mg/kg-d) Mid-dose (14.0 mg/kg-d) Number examined 57 61 68 Hepatocellular carcinoma 0 0 40 (59)* Neoplastic nodule(s) 5(9) 12 (20) 53 (78)* Hepatocellular carcinoma and /or neoplastic nodule(s) 5(9) 12 (20) 66 (97)* Mammary fibroadenomas 15 (26) 12 (20) 24 (35) Subcutaneous fibromas 0 2(3) 7(10)* TUT (1982a). Values expressed as number of animals (% of animals with lesion/effect); % calculated by EPA. * Statistically different from controls, p < 0.05. 68 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.16. Body and Liver Weights of Male F344 Rats After Dietary Exposure to tgDNT for 1 Year3 Parameter Exposure Group (Adjusted Daily Dose, mg/kg-d)b 0 (Control) 35 26 Week Number of animals 4 4 Body weight (g) 395 ±2 353 ± 8 (89)* Liver/body weight 2.43 ±0.07 2.82 ±0.07 (116)* 52 Weeks Number of animals 20 19 Terminal body weight (g) 434 ±3 321 ±4 (74)* Liver weight (g) 10.30 ±0.16 19.49 ±0.35 (189)* Liver/body weight (relative liver weight) 2.38 ±0.04 6.08 ±0.10 (255)* "Leonard et al. (1987). Values expressed as mean ± SEM (% of control); % calculated by EPA. * Statistically different from controls, p < 0.05. Table B.17. Incidence of Hepatic Neoplastic Lesions and Hepatic Metastases in Male F344 Rats After Dietary Exposure to tgDNT for 1 Year" Exposure Group (Adjusted Daily Dose, mg/kg-d)b Parameter 0 (Control) 35 Total number of animals 20 19 Neoplastic nodules 0 10 (53)* Hepatocellular carcinoma Trabecular 0 9 (47)* Adenocarcinoma 0 0 Cholangiocarcinoma 0 2(11) "Leonard et al. (1987). bValues expressed as number of animals (% with lesion). * Statistically different from controls, p < 0.05. 69 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.18. Distribution of Experimental Subjects Across Dose Groups and Breeding Dates in the Developmental Study of tgDNT in F344 Rats3 Parameter Exposure Group (Adjusted Daily Dose, mg/kg-d)b Vehicle Control Technical DNT 14 35 37.5 75 100 150 Mortality for all treated females Total females treated 37 22 13 22 13 23 13 No. deaths (GDs 0-20) (%) 0(0) 1 (4.5) 1 (7.7) 0(0) 0(0) 1 (4.3) 6 (46.2) Assignment of surviving females for maternal and developmental evaluation First breeding 9 0 7 0 7 0 6 Second breeding 7 6 0 6 0 6 0 Third breeding 6 7 0 7 0 7 0 Total No. assigned 22 (91) 13 7 13 7 13 6 No. of Pregnancy (% pregnantd) 20 (91) 10 (77) 7 (100) 12 (92) 6 (86) 12 (92) 5(83) "Price et al. (1985). bValues expressed as number of animals (% of animals death). °Number of dams scarified at GD 20. dPercent of pregnant dams. 70 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.19. Hematology Parameters in F344 Dams and Fetuses on GD 20 Following Maternal Exposure to tgDNT from GDs 7-20a Parameter Exposure Group (Adjusted Daily Dose, mg/kg-d)b Dams Fetuses Vehicle Control tgDNT (100) Vehicle Control tgDNT (100) % MetHb 3.8 ±0.5 (16) 7.7 ±0.6** (11) 13.4 ± 1.6(14) 8.9 ± 1.4(10) % Reticulocytes 2.32 ±0.57 (12) 6.31 ±1.51* (11) 99.67 ± 0.06 (23) 98.98 ±0.22* (21) WBC (x 103/mm3) 5.34 ±0.32 (5) 6.06 ± 0.64 (5) 0.93 ±0.19 (8) 0.83 ±0.12 (8) RBC (x 1 oW) 6.24 ± 0.20 (5) 4.88 ±0.18* (5) 2.17 ±0.08 (10) 2.15 ±0.06** (9) Hematocrit (%) 32.5 ± 1.00 (5) 26.0 ±0.98* (5) 34.08 ± 1.24(10) 32.17 ± 1.97 (9) MCV (nm3) 52.14 ±0.29 (5) 54.54 ±0.55* (5) 156.54 ±0.88 (10) 160.61 ±1.17* (9) RDW 7.96 ± 0.24 (5) 9.68 ±0.41* (5) 14.71 ±0.12 (10) 14.71 ±0.19 (9) Platelets (x 103/mm3) 1063.00 ±20.92 (4) 1625.00 ± 97.50* (5) 406.86 ± 27.90 (7) 412.2 ±35.76 (5) "Price et al. (1985). bData are presented as x ± SE. Number of individual maternal or fetal blood samples evaluated is shown in parentheses, except for fetal MetHb, which represents the number of litters evaluated after pooling individual fetal blood within each litter. *p < 0.05 t-test (two-tailed). **p < 0.01 /-test (two-tailed). 71 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.20. Summary of Select Organ Weights and Production Endpoints in Pregnant F344 Dams Exposed to tgDNT from GDs 7-20a Parameter Exposure Group (Adjusted Daily Dose, mg/kg-d)b Vehicle Control tgDNT 14 35 37.5 75 100 150 Number sacrificed 20 10 7 12 6 12 5 Weight gain, GDs 0-20 (g)c 61.83 ±3.61f 64.94 ±4.63 (105) 66.09 ±6.01 (107) 55.81 ±5.76 (90) 64.75 ± 6.78 (105) 52.67 ± 4.54 (85) 8.08 ±20.13 (13)** Gravid uterine weight (g) 37.70 ±3.22 47.75 ±2.39 (127) 39.39 ±3.25(104) 33.64 ±5.16 (89) 41.69 ±5.90 (111) 37.43 ± 3.84 (99) 22.09 ± 9.82 (59) Absolute weight gain (g)d 24.14 ±2.09§§ff 17.19 ±3.88 (71)* 26.70 ±4.31 (111) 22.17 ±2.10 (92) 23.06 ±2.31 (95) 15.24 ± 1.94 (63)** 14.01 ± 13.38 (58)** Liver weight (% body weight) 4.09±0.08§§ff 3.91 ±0.10 (96)* 4.12 ±0.09 (101) 3.96 ±0.09 (97) 4.55 ±0.10 (111)** 4.58 ±0.08 (112)** 4.79 ±0.36 (117) Spleen weight (% body weight) 0.197 ±0.003ff 0.185 ±0.007 (94)* 0.223 ±0.011 (113)* 0.215 ±0.006 (109)* 0.246 ±0.010 (125)** 0.320 ± 0.027 (162)** 0.284 ±0.059 (144)* % Resorptions6 16.8 ±5.4 2.3 ± 1.5 4.1 ± 4.1 14.6 ±5.2 11.0 ±9.3 12.7 ±5.4 46.0 ±22.3 % Dead fetuses6 0.0 2.4 ± 1.6 0.0 0.0 1.3 ± 1.3 0.0 3.6 ±3.6 % Live fetuses6 83.2 ±5.4 95.4 ± 1.9 95.9 ±4.1 85 ±5.2 87.7 ±9.1 87.3 ± 5.4 50.4 ±20.6 "Price et al. (1985). bData are presented as x ± SE (% compared to control). Number using dam or average litter value as the experimental unit. Includes gravid uterine weight. dWeight gain during gestation minus gravid uterine weight. "Expressed as the percentage of total implants per dam (compared to controls). *p < 0.05 Mann-Whitney U test (two-tailed). **p < 0.01 Mann-Whitney U test (two-tailed), tp < 0.05 Kruskal-Wallis one-way ANOVA. < 0.01 Kruskal-Wallis one-way ANOVA. §§p < 0.01 Jonckheere's test. 72 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.21. Teratological Defects in F344 Rat Fetuses Following Maternal Exposure to tgDNT or Vehicle Control on GDs 7-20a Exposure Group (Adjusted Daily Dose, mg/kg-d^ b tgDNT Parameter Vehicle Control 14 35 37.5 75 100 150 Number of live litters examined 20 10 7 12 6 12 3 No. of live fetuses examined0 146 92 63 77 50 88 22 Gross malformations Anophthalmia (bilateral or right side) 0 1 2 0 0 0 0 Agnathia 0 1 0 0 0 0 0 Umbilical hernia 0 1 0 0 0 0 0 Visceral malformations Hydronephrosis (bilateral) 0 1 0 0 0 0 0 Skeletal malformations Abnormal skull fusion 4 2 0 1 0 3 0 Fused thoracic arches 0 1 0 0 0 1 0 Thoracic centra off center 0 0 0 0 0 1 0 Lumbar centra off center 0 0 0 0 0 1 0 Ribs fused to each other 0 1 0 0 0 0 0 Short rib 0 1 0 0 0 1 0 Variations 0 0 0 0 0 0 0 Hematoma (back) 0 0 0 2 0 0 0 Misaligned sternebrae 3 4 0 0 0 6 0 Doubled centra 2 1 8 1 3 1 0 Clubbed limb without bone change 2 0 0 0 0 0 0 "Price et al. (1985). bData are expressed as the number of fetuses exhibiting each type of defect. Thus, a single fetus may be represented more than once in this table. °Only 50% of the fetuses were examined for visceral malformations and internal malformations of the head. 73 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table B.22. Status of Live Fetuses from F344 Rats Following Maternal Exposure to tgDNT or Vehicle Control on GDs 7-20a Parameter Exposure Group (Adjusted Daily Dose, mg/kg-d)b Vehicle Control tgDNT 14 35 37.5 75 100 150 No. litters with live fetuses 20 10 7 12 6 12 3 Live litter size 7.3 ±0.7 9.2 ±0.7 9.0 ±0.9 6.4 ± 1.1 8.3 ± 1.3 7.3 ±0.9 7.3 ± 1.7 Male/live x 100 (%) 48.8 ±5.0 53.0 ±6.3 46.4 ±4.5 44.4 ±8.0 52.7 ±4.9 43.0 ±5.9 57.4 ±22.8 Body weight (g) 3.21 ±0.05 3.39 ±0.07 (106) 3.29 ±0.07 (100) 3.34 ±0.06 (104) 3.29 ±0.13 (102) 3.17 ±0.08 (98) 3.14 ± 0.18 (98) Crown-rump length (cm) 3.55 ±0.03 3.51 ±0.07 (99) 3.57 ±0.07 (101) 3.58 ±0.07 (101) 3.53 ±0.05 (99) 3.46 ±0.07 (97) 3.53 ±0.15 (99) Liver weight (% body weight) 8.09 ± O.lltt 7.38 ±0.12 (91)** 8.35 ±0.14 (103)* 7.82 ±0.10 (96) 8.44 ±0.29 (104) 8.12 ±0.08 (100) 8.50 ±0.30 (105) Spleen weight (% body weight) 0.097 ± 0.0005ff 0.081 ± 0.008 (83) 0.131 ±0.006 (135)** 0.084 ± 0.004 (87) 0.119 ± 0.003 (123)* 0.085 ± 0.004 (88) 0.128 ± 0.012(132) Placental weight (g) 0.494 ± 0.022 0.539 ± 0.054 (109) 0.440 ± 0.009 (89) 0.536 ± 0.046 (108) 0.453 ± 0.018 (92) 0.51 ± 0.028 (103) 0.458 ± 0.057 (93) "Price et al. (1985). bData are presented as x ± SE. Number using dam or average litter value as the experimental unit. *p < 0.05 Mann-Whitney U test (two-tailed). **p < 0.01 Mann-Whitney U test (two-tailed), "ft/? < 0.01 Kruskal-Wallis one-way ANOVA. Table B.23. Rearing Behavior in Postnatal F344 Rat Female Pups when Dams were Exposed to tgDNT from GDs 7-20a End point Exposure Group (Adjusted Daily Dose, mg/kg-d)b tgDNT 0 (control) 14 35 37.5 75 100 PND 30 Rearing behavior 20.2 ±2.2 14.5 ±2.4 24.6 ±2.2 17.0 ±0.8 16.7 ± 1.5 12.7 ± 1.8* aCIIT ("1982b1. bData are presented as x ± SE. * Statistically different from controls, p < 0.05. 74 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 APPENDIX C. BMD MODELING RESULTS MODELING PROCEDURE FOR DICHOTOMOUS DATA The BMD modeling of dichotomous data was conducted with EPA's BMDS (version 2.2.2). For these data, all of the dichotomous models (i.e., Gamma, Multistage, Logistic, Log-logistic, Probit, Log-probit, and Weibull) available within the software were fit using a default benchmark response (BMR) of 10% extra risk. Adequacy of model fit was judged based on the % goodness-of-fitp-value (p > 0.1), magnitude of scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. Among all models providing adequate fit, the lowest BMDL was selected if the BMDLs estimated from different models varied greater than 3-fold; otherwise, the BMDL from the model with the lowest Akaike Information Criterion (AIC) was selected as a potential POD from which to derive the RfD. In addition, in the absence of a mechanistic understanding of the biological response to a toxic agent, data from exposures much higher than the study LOAEL do not provide reliable information regarding the shape of the response at low doses. Such exposures, however, can have a strong effect on the shape of the fitted model in the low-dose region of the dose-response curve. Thus, if lack of fit is due to characteristics of the dose-response data for high doses, then the Benchmark Dose Technical Guidance document allows for data to be adjusted by eliminating the high-dose group (U.S. EPA. 2012). Because the focus of BMD analysis is on the low-dose region of the response curve, elimination of the high-dose group is deemed reasonable. MODELING PROCEDURE FOR CONTINUOUS DATA The BMD modeling of continuous data was conducted with EPA's BMDS (version 2.2.2). For these data, all continuous models available within the software were fit using a default BMR of 1 standard deviation relative risk. For liver-, body-, and kidney-weight changes, a BMR of 10% relative risk was also used. An adequate fit was judged based on the X goodness-of-fit p-v alue (p> 0.1), magnitude of the scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In addition to these three criteria forjudging adequacy of model fit, a determination was made as to whether the variance across dose groups was homogeneous. If a homogeneous variance model was deemed appropriate based on the statistical test provided by BMDS (i.e., Test 2), the final BMD results were estimated from a homogeneous variance model. If the test for homogeneity of variance was rejected (p < 0.1), the model was run again while modeling the variance as a power function of the mean to account for this nonhomogeneous variance. If this nonhomogeneous variance model did not adequately fit the data (i.e., Test 3; p-v alue < 0.1), the data set was considered unsuitable for BMD modeling. Among all models providing adequate fit, the lowest BMDL was selected if the BMDLs estimated from different models varied greater than 3-fold; otherwise, the BMDL from the model with the lowest AIC was selected as a potential POD from which to derive the RfD. INCREASED INCIDENCE OF HEPATOCYTE NECROSIS IN MALE RATS TREATED WITH tgDNT FOR 26 WEEKS The procedure outlined above was applied to the data for increased hepatocyte necrosis (see Table C. 1) in male rats exposed to tgDNT via diet (CUT, 1982a) for 26 weeks. Table C.2 summarizes the BMD modeling results. All the models fit except the Logistic and Probit models. Among the fitting models, the LogLogistic model has the lowest AIC. Thus, the 75 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 BMDLio of 2.16 mg/kg-day from this model is selected for this end point (see Figure C. 1 and the BMD text output for details). Table C.l. Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 26 Weeks" Low-dose Mid-dose High-dose Control (3.47 mg/kg-d) (13.6 mg/kg-d) (34.6 mg/kg-d) Sample size 10 10 10 10 Incidence 0 0 7 9 "CUT (1982a). Table C.l. BMD Modeling Results on Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 26 Weeks Model Name AIC />-value BMD BMDL10 Scaled Residual of Interest Gamma 25.6203 0.2905 4.54802 1.36 -0.794 Logistic 30.1827 0.0174 5.63874 3.47 -1.058 LogLogistic 24.262 0.5178 5.17793 2.16 -0.61 LogProbit 24.2957 0.5089 5.07807 2.32 -0.569 Multistage 27.0948 0.1987 2.96349 1.14 -1.159 Probit 30.5661 0.029 5.67662 3.59 -1.058 Weibull 26.3218 0.2588 3.60128 1.23 -1.024 76 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Log-Logistic Model with 0.95 Confidence Level Log-Logistic 1 0.8 0.6 0.4 0.2 0 BMDL BMD 0 5 10 15 20 25 30 35 dose 16:25 07/24 2012 Figure C.l. LogLogistic Model for Increased Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 26 Weeks Text Output for LogLogistic Model for Increased Hepatocyte Necrosis in Male Rats After Dietary Exposure to tgDNT for 26 weeks (CUT, 1982a) Logistic Model BMDS Model Run The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))] Dependent variable = Effect Independent variable = Dose Slope parameter is restricted as slope >= 1 Total number of observations = 4 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 77 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 User has chosen the log transformed model Default Initial Parameter Values background = 0 intercept = -5.72644 slope = 2.32112 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -background have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) intercept slope intercept 1 -0.98 slope -0.98 1 Interval Variable Limit background intercept slope Parameter Estimates Estimate -6.69767 2.73682 Std. Err. Indicates that this value is not calculated. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. Full model -9.35947 4 Fitted model Reduced model P-value -10.131 -26.9205 1.54303 35.122 0.4623 <.0001 AIC: 24 .262 Dose Goodness of Fit Est._Prob. Expected Observed Size Scaled Residual 0.0000 3.4700 13.6400 34.6100 0.0000 0.0358 0.6115 0.9527 0.000 0.358 6.115 9.527 0.000 0.000 7.000 9.000 10 10 10 10 0. 000 -0.610 0.574 -0.784 Chi^2 =1.32 d.f. = 2 P-value = 0.5178 Benchmark Dose Computation 78 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Specified effect Risk Type Confidence level BMD BMDL 0.1 Extra risk 0. 95 5.17793 2.16115 79 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 INCREASED INCIDENCE OF HEPATOCYTE NECROSIS IN MALES TREATED WITH tgDNT FOR 104 WEEKS (( 11 1 . 1982a) The procedure outlined above was applied to the data for increased hepatocyte necrosis (see Table C.3) in male rats exposed to tgDNT via diet (CUT. 1982a) for 104 weeks. Table C.4 summarizes the BMD modeling results. As assessed by the % goodness-of-fit^-values, all the models failed to model this data set (see Table C.4). To further attempt to calculate a BMDL based on this data set, BMD models were run with the control and low-dose data points after dropping the mid-dose data point. Table C.5 presents the BMD modeling results. All the models available from the BMDS except the Weibull model successfully fit the data set. The estimated BMDLios from these models differed by more than 3-fold, so the lowest BMDL of 0.363 mg/kg-day (from the Multistage 2, 3) is selected for this endpoint (see Figure C.2 and the BMD text output for Multistage 2 model). Table C.3. Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks3 Low-dose Mid-dose Control (3.51mg/kg-d) (14 mg/kg-d) Sample size 61 70 23 Incidence 0 38 11 aCIIT 0 982a). Table C.4. BMD Results for Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Model Name AIC /j-valuc BMD10 BMDL10 Scaled Residual of Interest Gamma 154.943 0 0.856072 0.677433 0 Logistic 181.412 0 2.98469 2.21859 4.388 LogLogistic 141.63 0.0015 0.479011 0.335441 0 LogProbit 156.474 0 1.27286 1.02557 0 Multistage 2 154.943 0 0.856072 0.677433 0 Multistage 3 154.943 0 0.856072 0.677433 0 Probit 180.738 0 2.85092 2.17342 4.457 Weibull 154.943 0 0.85607 0.677433 0 80 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Table C.5. BMD Results After Dropping Mid-dose Point for Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Model Name AIC /j-valuc BMD10 BMDL10 Scaled Residual of Interest Logistic 100.526 NA 3.07144 1.576 0 Multistage 2 100.526 NA 0.774156 0.363194 0 Multistage 3 102.526 NA 1.02805 0.363194 0 Probit 100.526 NA 2.66223 1.36408 0 0 0.5 1 1.5 2 2.5 3 3.5 dose 10:38 07/23 2012 Figure C.2. Multistage 2 BMD Model for Increased Hepatocyte Necrosis in Male Rats After Dietary Exposure to tgDNT for 104 Weeks (CUT, 1982a) Multistage Model with 0.95 Confidence Level I I Multistage BMDL BMD Text Output for Multistage 2 BMD Model for Increased Hepatocyte Necrosis in Male Rats After Dietary Exposure to tgDNT at 104 weeks (CUT, 1982a) Multistage Model. (Version: 3.2; Date: 05/26/2010) BMDS Model Run 81 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Observation # < parameter # for Multistage model. The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2) ] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose Total number of observations = 2 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.542857 Beta(l) = 0.223008 Beta(2) = 0.0635351 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Background have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Beta(1) Beta (2) Beta (1) NA NA Beta (2) NA NA NA - This parameter's variance has been estimated as zero or less. THE MODEL HAS PROBABLY NOT CONVERGED!!! Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0 * * * Beta(1) 0.111504 * * * Beta(2) 0.0317676 * * * * - Indicates that this value is not calculated. At least some variance estimates are negative. 82 Technical Grade Dinitrotoluene ------- THIS USUALLY MEANS THE MODEL HAS NOT CONVERGED! Try again from another starting point. Error in computing chi-sguare; returning 2 FINAL 4-4-2013 Model Full model Fitted model Reduced model AIC: Analysis of Deviance Table Log(likelihood) # Param's Deviance Test d.f. -48.2628 -48.2628 -78.8908 100.526 0 61.256 P-value NA <.0001 Dose Est. Prob. Goodness of Fit Expected Observed Size Scaled Residual 0.0000 3.5100 Chi^2 = 0.00 0.0000 0.5429 0.000 38.000 d.f. = 0 0.000 38.000 P-value = NA 61 70 0. 000 0. 000 Benchmark Dose Computation Specified effect Risk Type Confidence level BMD BMDL BMDU 0.1 Extra risk 0. 95 0.774156 0.363194 1.48533 Taken together, (0.363194, 1.48533) is a 90 interval for the BMD two-sided confidence 83 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE SCREENING p-OSF MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA The model-fitting procedure for dichotomous cancer incidence data is as follows. The Multistage-Cancer model in the EPA's BMDS (version 2.2.2) is fit to the incidence data using the extra risk option. The Multistage-Cancer model is run for all polynomial degrees up to n - 1 (where n is the number of dose groups including control). An adequate model fit is judged by three criteria: (1) goodness-of-fit/>-value (p > 0.1), (2) visual inspection of the dose-response curve, and (3) scaled residual at the data point (except the control) closest to the predefined benchmark response (BMR). Among all the models providing adequate fit to the data, the BMDL from the best fitting Multistage-Cancer model as judged by the goodness-of-fit />value, is selected as the POD. In accordance with U.S. EPA (2012) guidance, BMDs and BMDLs associated with an extra risk of 10% are calculated. HEPATOCELLULAR CARCINOMAS IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a) Table A.6 shows the dose-response data on hepatocellular carcinomas, liver neoplastic nodules, hepatocellular carcinomas and/or neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas in male F344 rats administered tgDNT via the diet for 104 weeks (CUT. 1982a). Modeling was performed according to the procedure outlined above using BMDS for each individual tumor type based on the ADDs, and Table D.l summarizes the results. For incidence of hepatocellular carcinomas in male rats, the 2-degree Multistage-Cancer model provided an adequate fit (goodness-of-fitp-wdXut >0.1; see Table D.l and Figure D.l). The estimated BMDio value is 3.04 mg/kg-day with a BMDLio of 2.15 mg/kg-day. Table D.l. BMD Results for Hepatocyte Carcinomas in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Degree of Scaled Residual of Model Name polynomial AIC />-value BMD BMDL10 Interest Multistage-Cancer 1 95.5511 0.0006 1.3531 0.998 0.35 Multistage-Cancer 2 81.7442 0.6332 3.03847 2.15 -0.366 84 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation 1 0.8 0.6 0.4 0.2 0 BMDL BMD 0 2 4 6 8 10 12 14 dose 09:50 07/18 2012 Figure D.l. Multistage-Cancer BMD Model for Increased Hepatocellular Carcinomas in Male Rats at 104 Weeks (CUT. 1982a) Text Output for Multistage-Cancer BMD Model for Increased Hepatocellular Carcinomas in Male Rats (CUT. 1982a) Multistage Cancer Model BMDS_Model_Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2) ] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose Total number of observations = 3 85 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.000900222 Beta(l) = 0 Beta(2) = 0.012451 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Beta(l) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Background Beta (2) Background 1 -0.32 Beta (2) -0.32 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.0147051 * * * Beta(1) 0 * * * Beta(2) 0.0114121 * * * * - Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -38.7541 -38.8721 -77.3384 # Param's 3 2 1 Deviance Test d.f. 0.236087 77.1687 P-value 0. 627 <.0001 AIC: 81.7442 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.0147 0.897 1.000 61 0.110 3.5100 0.1439 10.076 9.000 70 -0.366 14.0000 0.8948 20.580 21.000 23 0.286 86 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Chi^2 =0.23 d.f. = 1 P-value = 0.6332 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 3.03847 BMDL = 2.14507 BMDU = 3.67911 Taken together, (2.14507, 3.67911) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0466186 87 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 LIVER NEOPLASTIC NODULES IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a) Modeling was performed according to the procedure outlined above using BMDS based on the ADDs, and Table D.2 summarizes the results. For incidence of liver neoplastic nodules in male rats, the 2-degree Multistage-Cancer model provided an adequate fit (goodness-of-fit p-wdXut >0.1; see Table D.2 and Figure D.2). The estimated BMDio value is 4.86 mg/kg-day with a BMDLio of 2.69 mg/kg-day. Table D.2. BMD Results for Liver Neoplastic Nodules in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Degree of Scaled Residual of Model Name polynomial AIC /j-valuc BMD BMDL10 Interest Multistage-Cancer 1 150.829 0.0262 2.43085 1.58 -1.606 Multistage-Cancer 2 146.021 0.5454 4.85647 2.69 -0.45 Multistage Cancer Model with 0.95 Confidence Level 0.9 Multistage Cancer Linear extrapolation 0.8 0.7 0.6 0.5 0.4 0.3 0.2 1 BMDL BMD 0 0 2 4 6 8 10 12 14 dose 11:30 07/25 2012 Figure D.2. Multistage-Cancer BMD Model for Increased Neoplastic Nodules in Male Rats at 104 Weeks (("11 1 . 1982a) 88 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Text Output for Multistage-Cancer BMD Model for Increased Neoplastic Nodules in Male Rats (('11 1 . 1982a) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/USEPA/BMDS220/Data/104 week/msc_104 week male neoplastic nodules_Opt.(d) Gnuplot Plotting File: C:/USEPA/BMDS220/Data/104 week/msc_104 week male neoplastic nodules_Opt.pit Wed Jul 25 11:30:27 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2) ] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.128217 Beta(l) = 0 Beta(2) = 0.00468028 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Beta(l) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Background Beta(2) Background 1 -0.3 6 Beta (2) -0.36 1 Parameter Estimates 89 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.131201 * * * Beta(1) 0 * * * Beta(2) 0.00446721 * * * * - Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -70.8268 -71.0106 -82.5378 # Param's Deviance Test d.f. P-value 3 2 0.367614 1 0.5443 1 23.422 2 <.0001 AIC: 146.021 Dose Goodness of Fit Est._Prob. Expected Observed Size Scaled Residual 0.0000 3.5100 14.0000 0.1312 0.1777 0.6380 8.003 12.441 14.675 9.000 11.000 15.000 61 70 23 0.378 -0.450 0.141 Chi^2 =0.37 d.f. = 1 P-value = 0.5454 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 4.85647 BMDL = 2.68723 BMDU = 6.53922 Taken together, (2.68723, 6.53922) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.037213 90 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 COMBINED HEPATOCELLULAR CARCINOMAS AND LIVER NEOPLASTIC NODULES IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (( 11 1 . 1982a) Modeling was performed according to the procedure outlined above using BMDS based on the ADDs, and Table D.3 summarizes the results. For incidence of combined hepatocellular carcinomas and liver neoplastic nodules in male rats, the 2-degree Multistage-Cancer model provided an adequate fit (goodness-of-fitp-value >0.1; see Table D.3 and Figure D.3). The estimated BMDio value is 2.42 mg/kg-day with a BMDLio of 1.68 mg/kg-day. Table D.3. BMD Results for Combined Hepatocellular Carcinomas and Liver Neoplastic Nodules in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Model Name Degree of polynomial AIC />-value BMD BMDL Scaled Residual of Interest Multistage-Cancer 1 156.06 0.0008 0.991314 0.721042 0.828 Multistage-Cancer 2 142.255 0.2389 2.41883 1.67831 -0.762 91 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation 1 0.8 0.6 0.4 0.2 BMDL BMD 0 0 2 4 6 8 10 12 14 dose 11:41 08/01 2012 Figure D.3. Multistage-Cancer BMD Model for Increased Combined Hepatocellular Carcinomas and Neoplastic Nodule in Male Rats at 104 Weeks (('11 1 . 1982a) Text Output for Multistage-Cancer BMD Model for Increased Combined Hepatocellular Carcinomas and Neoplastic Nodule in Male Rats (CUT, 1982a) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/USEPA/BMDS220/Data/msc_livercombined_Msc2-BMR10.(d) Gnuplot Plotting File: C:/USEPA/BMDS220/Data/msc_livercombined_Msc2- BMR10.pit Wed Aug 01 11:41:18 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2) ] The parameter betas are restricted to be positive 92 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Dependent variable = Response Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0 Beta(l) = 0 Beta(2) = 5.25101e+017 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Beta(l) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Background Beta (2) Background 1 -0.3 6 Beta (2) -0.36 1 Parameter Estimates Interval Variable Limit Background Beta(1) Beta(2) Estimate 0.143194 0 0.0180081 Std. Err. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -68.1416 -69.1276 -98.4788 # Param's 3 2 1 Deviance Test d.f. P-value 1.97201 60.6745 0.1602 <.0001 AIC: 142 .255 Goodness of Fit Scaled 93 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Dose Est. Prob. Expected Observed Size Residual 14.0000 3.5100 0.0000 0.9749 0.3137 0.1432 22.422 21.957 8 .735 23.000 19.000 10.000 23 70 61 0.770 -0.762 0. 462 Chi^2 = 1.39 d.f. = 1 P-value = 0.2389 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 2.418 83 BMDL = 1.67831 BMDU = 3.09631 Taken together, (1.67831, 3.09631) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0595837 94 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 MAMMARY FIBROADENOMAS IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a) Modeling was performed according to the procedure outlined above using BMDS based on the ADDs, and Table D.4 summarizes the results. For incidence of mammary fibroadenomas in male rats, both 1- and 2-degree Multistage-Cancer models provided an identical fit (goodness-of-fit /rvalue >0.1; see Table D.4 and Figure D.4). The estimated BMDio value is 7.37 mg/kg-day with aBMDLio of 3.73 mg/kg-day. Table D.4. BMD Results for Mammary Fibroadenomas in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Degree of Scaled Residual Model Name polynomial AIC /j-valuc BMD BMDL10 of Interest Multistage-Cancer 1 97.5334 0.9081 7.37334 3.73 0.085 Multistage-Cancer 2 97.5334 0.9081 7.37334 3.73 0.085 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation 0.4 0.3 0.2 0.1 0 BMDL BMD 0 2 4 6 8 10 12 14 dose 10:28 07/25 2012 Figure D.4. Multistage-Cancer BMD Model for Increased Mammary Fibroadenomas in Male Rats at 104 Weeks (CUT. 1982a) 95 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Text Output for Multistage-Cancer BMD Model for Increased Mammary Fibroadenomas in Male Rats (("11 1 . 1982a) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C :/USEPA/BMDS220/Data/msc_Maittmary_Mscl-BMR10 . (d) Gnuplot Plotting File: C:/USEPA/BMDS220/Data/msc_Mammary_Mscl-BMR10.pit Wed Aug 01 15:18:00 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Response Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 2 Total number of specified parameters = 0 Degree of polynomial = 1 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.0518599 Beta(1) = 0.0137724 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(l) Background 1 -0.62 Beta (1) -0.62 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.0505334 * * * Beta(1) 0.0142894 * * * * - Indicates that this value is not calculated. 96 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -46.7601 -46.7667 -49.1781 Param's 3 2 1 Deviance Test d.f. 0.0133121 4.83601 P-value 0.9081 0.0891 AIC: 97.5334 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.0505 3.083 3.000 61 -0.048 3.5100 0.0970 6.789 7.000 70 0. 085 14.0000 0.2227 5.122 5.000 23 -0.061 Chi^2 = 0.01 d.f. = 1 P-value = 0.9081 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 7.37334 BMDL = 3.72 678 BMDU = 33.6009 Taken together, (3.72678, 33.6009) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0268328 97 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 SUBCUTANEOUS FIBROMAS IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a) Modeling was performed according to the procedure outlined above using BMDS based on theADDs, and Table D.5 summarizes the results. For incidence of subcutaneous fibromas in male rats, the 1-degree Multistage-Cancer model provided an adequate fit (goodness-of-fit /rvalue >0.1; see Table D.5 and Figure D.5). The estimated BMDio value is 2.01 mg/kg-day with a BMDLio of 1.38 mg/kg-day. Table D.5. BMD Results for Subcutaneous Fibromas in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks Degree of Scaled Residual of Model Name polynomial AIC />-value BMD BMDL10 Interest Multistage-Cancer 1 140.118 0.4138 2.01413 1.38 -0.582 Multistage-Cancer 2 141.438 NA 2.79287 1.45 0 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 BMDL BMD 0 2 4 6 8 10 12 14 dose 11:36 07/25 2012 Figure D.5. Multistage-Cancer BMD Model for Increased Subcutaneous Fibromas in Male Rats at 104 Weeks (CUT. 1982a) 98 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Text Output for Multistage-Cancer BMD Model for Increased Subcutaneous Fibromas in Male Rats (('II I . 1982a) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/USEPA/BMDS220/Data/104 week/msc_Subcutaneous fibromas_Opt.(d) Gnuplot Plotting File: C:/USEPA/BMDS220/Data/104 week/msc_Subcutaneous fibromas_Opt.pit Wed Jul 25 10:38:29 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 2 Total number of specified parameters = 0 Degree of polynomial = 1 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.0491346 Beta(1) = 0.0625806 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(l) Background 1 -0.5 6 Beta (1) -0.56 1 Parameter Estimates 95.0% Wald Confidence Std. Err. Lower Conf. Limit Upper Conf. k k k -k Interval Variable Estimate Limit Background 0.0738705 Beta(1) 0.0523107 99 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 * - Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -67.7191 -68.0589 -80. 0153 # Param's 3 2 1 Deviance Test d.f. 0.679587 24.5923 P-value 0.4097 <.0001 AIC: 140.118 Dose Goodness of Fit Est._Prob. Expected Observed Size Scaled Residual 0.0000 3.5100 14.0000 0.0739 0.2292 0.5547 4.506 16.045 12 .759 5.000 14.000 14.000 61 70 23 0.242 -0.582 0.521 Chi^2 =0.67 d.f. = 1 P-value = 0.4138 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 2.01413 BMDL = 1.37981 BMDU = 3.2 9703 Taken together, (1.37981, 3.29703) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.0724738 100 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 MSCOMBO-MULTIPLE TUMOR BMD MODEL FOR ALL TUMOR TYPES IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a) MSCombo-multiple tumor BMD modeling was used to combine tumor incidence data for combined hepatocellular carcinomas and/or neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas in male rats. For each tumor type, the best-fitting multistage model (i.e., the degree of Poly setting) was maintained in the MSCombo model run. The calculated combined tumor BMDLio based on the MS Combo model is 0.852 mg/kg-day (see MS Combo text output for details). This BMDLio is used as the POD to derive the p-OSF. Text Output for MS COMBO Multiple Tumor Model for Combined Tumors in Male Rats MS_COMBO. (Version: 1.5 Beta; Date: 01/25/2011) Input Data File: C:\USEPA\BMDS220\Data\New.(d) Gnuplot Plotting File: C:\USEPA\BMDS22 0\Data\New.plt Wed Aug 01 15:35:40 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2) ] The parameter betas are restricted to be positive Dependent variable = Response Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0 Beta(l) = 0 Beta(2) = 5.25101e+017 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Beta(l) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Background Beta (2) 101 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Background Beta(2) 1 -0.36 -0.36 1 Parameter Estimates Interval Variable Limit Background Beta(1) Beta(2) Estimate 0.143194 0 0.0180081 Std. Err. 95.0% Wald Confidence Lower Conf. Limit Upper Conf. Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -68.1416 -69.1276 -98.4788 # Param's Deviance Test d.f. P-value 3 2 1.97201 1 0.1602 1 60.6745 2 <.0001 AIC: 142 .255 Log-likelihood Constant 63.914608924352244 Dose Goodness of Fit Est._Prob. Expected Observed Size Scaled Residual 14.0000 3.5100 0.0000 Chi^2 = 1.39 0.9749 0.3137 0.1432 d.f. = 1 22.422 23.000 23 21.957 19.000 70 8.735 10.000 61 P-value = 0.2389 0.770 -0.762 0. 462 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 2.418 83 BMDL = 1.67831 BMDU = 3.09631 Taken together, (1.67831, 3.09631) is a 90 % two-sided confidence interval for the BMD 102 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 MS_COMBO. (Version: 1.5 Beta; Date: 01/25/2011) Input Data File: C:\USEPA\BMDS220\Data\New.(d) Gnuplot Plotting File: C:\USEPA\BMDS22 0\Data\New.plt Wed Aug 01 15:35:40 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Response Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 2 Total number of specified parameters = 0 Degree of polynomial = 1 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.0518599 Beta(1) = 0.0137724 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(l) Background 1 -0.62 Beta (1) -0.62 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.0505334 * * * Beta(1) 0.0142894 * * * * - Indicates that this value is not calculated. Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value 103 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Full model Fitted model Reduced model -46.7601 -46.7667 -49.1781 0.0133121 4.83601 0.9081 0.0891 AIC: 97.5334 Log-likelihood Constant 41.819300823452593 Goodness of Fit Scaled Dose Est. Prob. Expected Observed Size Residual 0.0000 0.0505 3.083 3.000 61 -0.048 3.5100 0.0970 6.789 7.000 70 0. 085 14.0000 0.2227 5.122 5.000 23 -0.061 Chi^2 = 0.01 d.f. = 1 P-value = 0.9081 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 7.37334 BMDL = 3.72 678 BMDU = 33.6009 Taken together, (3.72678, 33.6009) is a 90 % two-sided confidence interval for the BMD MS_COMBO. (Version: 1.5 Beta; Date: 01/25/2011) Input Data File: C:\USEPA\BMDS220\Data\New.(d) Gnuplot Plotting File: C:\USEPA\BMDS22 0\Data\New.plt Wed Aug 01 15:35:40 2012 BMDS Model Run The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*doseAl) ] The parameter betas are restricted to be positive Dependent variable = Response Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 2 Total number of specified parameters = 0 104 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Degree of polynomial Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0.0491346 Beta(1) = 0.0625806 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(l) Background 1 -0.5 6 Beta (1) -0.56 1 Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.0738705 * * * Beta(1) 0.0523107 * * * * - Indicates that this value is not calculated. Analysis of Deviance Table Model Full model Fitted model Reduced model Log(likelihood) -67.7191 -68.0589 -80. 0153 # Param's Deviance Test d.f. P-value 3 2 0.679587 1 0.4097 1 24.5923 2 <.0001 AIC: 140.118 Log-likelihood Constant 62.107410554719884 Dose Goodness of Fit Est._Prob. Expected Observed Size Scaled Residual 0.0000 3.5100 14.0000 Chi^2 =0.67 0.0739 0.2292 0.5547 d.f. = 1 4.506 5.000 61 16.045 14.000 70 12.759 14.000 23 P-value = 0.4138 0.242 -0.582 0.521 Benchmark Dose Computation Specified effect = 0.1 105 Technical Grade Dinitrotoluene ------- Risk Type = Extra risk Confidence level = 0.95 BMD = 2.01413 BMDL = 1.37981 BMDU = 3.2 9703 Taken together, (1.37981, 3.29703) is a 90 % two-sided confidence interval for the BMD **** Start of combined BMD and BMDL Calculations.**** Combined Log-Likelihood -183.95322559798808 Combined Log-likelihood Constant 167.84132030252471 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 1.19552 BMDL = 0.852494 FINAL 4-4-2013 106 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 APPENDIX E. REFERENCES Abernethv. D; Couch. D. (1982). Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese hamster ovary cells. Mutat Res 103: 53-59. http://dx.doi.org/10.1016/Q165- 7992(82)90087-2 Ahrenholz. S. (1980). Health hazard evaluation determination, report no. HE-79-1 13-728, 01 in Chemical Company, Brandenburg, Kentucky [TSCA Submission], (HE-79-113-728). Cincinnati, OH: National Institute of Occupational Safety and Health. Ahrenholz. SH; Mever. CR. (1982). Health hazard evaluation report no HETA 81-295-1 155, Olin (formerly Allied) Chemical Co, Moundsville, West Virginia. Cincinnati, OH: National Institute for Occupational Safety and Health. http://www2a.cdc. gov/hhe/select.asp?PitName=5509&bFlag=0&IP=3049 Ashbv. J; Burlinson. B; Lefevre. PA; Topham. J. (1985). Non-genotoxicity of 2,4,6- trinitrotoluene (TNT) to the mouse bone marrow and the rat liver: implications for its carcinogenicity. Arch Toxicol 58: 14-19. AT SDR (Agency for Toxic Substances and Disease Registry). (1998). Toxicological profile for 2,4-dinitrotoluene and 2,6-dinitrotoluene (Update) [ATSDR Tox Profile], Atlanta, GA: Agency for Toxic Substances & Disease Registry. Bannasch. P. (1976). Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules. Cancer Res 36: 2555-2562. Bannasch. P; Moore. \1A: Klimek. F; Zerban. H. (1982). Biological markers of preneoplastic foci and neoplastic nodules in rodent liver. Toxicol Pathol 10: 19-34. http://dx.doi. org/10.1177/019262338201000204 Bermudez. E; Tillerv. D; Butterworth. BE. (1979). The effect of 2,4-diaminotoluene and isomers of dinitrotoluene on unscheduled DNA synthesis in primary rat hepatocytes. Environ Mol Mutagen 1: 391-398. http://dx.doi.Org/l0.1002/ern.2860010412 Bruning. T; Chronz. C; Thier. R; Havelka. J; Ko. Y; Bolt. HM. (1999). Occurrence of urinary tract tumors in miners highly exposed to dinitrotoluene. J Occup Environ Med 41: 144- 149. Bruning. T; Thier. R; Mann. H; Melzer. H; Brode. P; Dallner. G; Bolt. HM. (2001). Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene. J Occup Environ Med 43:610-615. Cat EPA (California Environmental Protection Agency). (2009). Appendix A: Hot spots unit risk and cancer potency values. Sacramento, CA: Office of Environmental Health Hazard Assessment, http://www.oehha.ca.gov/air/hot spots/2009/AppendixA.pdf Cat EPA (California Environmental Protection Agency). (2012a). All OEHHA acute, 8-hour and chronic reference exposure levels (chRELs) as on February 2012. Sacramento, CA: Office of Environmental Health Hazard Assessment. http://www.oehha.ca.gov/air/allrels.html Cat EPA (California Environmental Protection Agency). (2012b). OEHHA toxicity criteria database. Sacramento, CA: Office of Environmental Health Hazard Assessment. http ://www. oehha.ca. gov/tcdb/ CUT (Chemical Industry Institute of Toxicology). (1982a). 104-week chronic toxicity study in rats: Dinitrotoluene, final report, volume I of II. (86940000342). Research Triangle Park, NC. 107 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 CUT (Chemical Industry Institute of Toxicology). (1982b). Teratological and postnatal evaluation of dinitrotoluene in Fischer 344 rats [TSCA Submission], (FYI-OTS-1282- 0221). Research Triangle Park, NC. CUT (Chemical Industry Institute of Toxicology). (1983). A thirty day toxicology study in Fischer-344 rats given dinitrotoluene, technical grade [TSCA Submission], (878212040). Research Triangle Park, NC. http://www.ntis. gov/search/product.aspx?ABBR=QTS0205947 Couch. D; Allen. P; Abernethv. P. (1981). The mutagenicity of dinitrotoluenes in Salmonella typhimurium. MutatRes 90: 373-383. http://dx.doi.ore/10.1016/0165-1218(81)90060-4 Hamill. PVV; Steinberaer. E; Levine. RJ; Rodriauez-Riaau. LJ; Lemeshow. S; Avrunin. JS. (1982). The epidemiologic assessment of male reproductive hazard from occupational exposure to TDA and DNT. J Occup Environ Med 24: 985-993. Hamilton. CM; Mirsalis. JC. (1987). Factors that affect the sensitivity of the in vivo-in vitro hepatocyte DNA repair assay in the male rat. Mutat Res 189: 341-347. Harth. ¥; Bolt. HM; Brunina. T. (2005). Cancer of the urinary bladder in highly exposed workers in the production of dinitrotoluenes: a case report. Int Arch Occup Environ Health 78: 677-680. http://dx.doi.org/10.1007/s00420-005-0012- IARC (International Agency for Research on Cancer). (1996). 2,4-Dinitrotoluene, 2,6- Dinitrotoluene and 3,5-Dinitrotoluene [IARC Monograph], In Some chemicals used in plastics and elastomers (pp. 309-368). Lyon, France. http://monoaraphs.iarc.fr/ENG/Monoaraphs/vol65/mono65-9.pdf Kliaerroan. AD; Wilmer. JL; Erexson. GL. (1982). The use of rat and mouse lymphocytes to study cytogenetic damage after in vivo exposure to genotoxic agents. In BA Bridges; BE Butterworth; IB Weinstein (Eds.), Banbury Report 13: Indicators of genotoxic exposure (pp. 277-291). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Leonard. I B; Graichen. ME; Popp. J A. (1987). Dinitrotoluene isomer-specific hepatocarcinogenesis in F344 rats. J Natl Cancer Inst 79: 1313-1319. Levine. R. (1983). The reproductive experience of workers exposed to dinitrotoluene and toluene diamine. (NTIS OTS 214308). Research Triangle Park: Department of Epidemiology, Chemcial Industry Institute of Toxicology. Levine. RJ'; Andielkovich. DA; Kersteter. SL; Arp. EW. Jr; Batoah. SA; Blunden. PB; Stanley. JM. (1986). Heart disease in workers exposed to dinitrotoluene. J Occup Environ Med 28: 811-816. Levine. RJ'; Corso. ROD; Blunden. PB. (1985). Fertility of workers exposed to dinitrotoluene and toluenediamine at three chemical plants. In DE Rickert (Ed.), Toxicity of nitroaromatic compounds (pp. 243-254). New York, NY: Hemisphere Publishing. McGee. LC; McCausland. A; Plume. CA; Marlett. NC. (1942). Metabolic disturbances in workers exposed to dinitrotoluene. Am J Dig Dis 9: 329-332. http://dx.doi.ora/10.1007/BF02998020 McGee. LC; Reed. HL; Nereim. TJ; Plume. CA; McCausland. A. (1947). Metabolic disturbances in workers exposed to dinitrotoluene during World War II. Gastroenterology 8: 293-295. Mirsalis, JC; Butterworth, BE. (1982). Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene. Carcinogenesis 3: 241-245. http://dx.doi.ora/10.1093/carcin/3.3.241 Mirsalis, JC; llamm. TE; Sherrilt. JM; Butterworth. BE. (1982). Role of gut flora in the genotoxicity of dinitrotoluene. Nature 295: 322-323. 108 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 Mirsaiis. JC; Tyson. CK; Stein met/. KL; Loh. EK; Hamilton., CM; Bakke. IP; Spalding. JW. (1989). Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds. Environ Mol Mutagen 14: 155-164. http://dx.doi.ore/10.1002/em.2850140305 NIOSH (National Institute for Occupational Safety and Health). (2007). NIOSH pocket guide to chemical hazards. (2005-149). Cincinnati, OH. http://www.cdc.gov/niosh/docs/2005-149/ NLM (National Institutes of Health, National Library of Medicine). (201 1). Hazardous Substances Data Bank (HSDB). Bethesda, MD. http://toxnet.nlm.nih.gov N TP (National Toxicology Program). (201 1). Report on carcinogens: Twelfth edition. Washington, DC: U.S. Department of Health and Human Services. http://ntp.niehs.nih.gov/ntp/roc/twelfth/rocl2.pdf OSHA (Occupational Safety & Health Administration). (2006). Table Z-l limits for air contaminants. Occupational Safety and Health Administration. http://www.osha.gov/pls/oshaweb/owadisp.show document?p table STA\I)ARDS&p id=9992 Price. CJ: Tvl. RW; Marks. TA; Paschke. LL; Ledoux. TA; Reel. JR. (1985). Teratologic evaluation of dinitrotoluene in the Fischer 344 rat. Fundam Appl Toxicol 5: 948-961. http://dx.doi.org/10.1016/0272-0590(85)90176-9 Spares. ER; Lock. LF. (1980). Lack of an indication of mutagenic effects of dinitrotoluenes and diaminotoluenes in mice. Environ Mol Mutagen 2: 111-124. http://dx.doi.org/10.1002/em.28600202Q3 Stavner. L; Dannenberg. A; Thun. M; Reeve. G; Bloom. T; Boeniger. M; Hatperin. W. (1992). Cardiovascular mortality among munitions workers exposed to nitroglycerin and dinitrotoluene. Scand J Work Environ Health 18: 34-43. Stavner. LT; Dannenberg. AL; Bloom. T; Thun. M. (1993). Excess hepatobiliary cancer mortality among munitions workers exposed to dinitrotoluene. J Occup Med 35: 291-296. Styles. J; Cross. M. (1983). Activity of 2,4,6-trinitrotoluene in an in vitro mammalian gene mutation assay. Cancer Lett 20: 103-108. http://dx.doi.org/10.1016/0304-3835(83)90194- 5 U.S. EPA (U.S. Environmental Protection Agency). (1987). Health effects assessment for 2,4- and 2,6-dinitrotoluene [EPA Report], (EPA600888032). Cincinnati, OH. U.S. EPA (U.S. Environmental Protection Agency). (1988). Recommendations for and documentation of biological values for use in risk assessment [EPA Report], (EPA/600/6- 87/008). Cincinnati, OH. http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm7deid 34855 U.S. EPA (U.S. Environmental Protection Agency). (1990). Integrated Risk Information System (IRIS) for 2,4-/2,6-Dinitrotoluene mixture. Washington, DC. http://www.epa.gov/iris/subst/0397.htm U.S. EPA (U.S. Environmental Protection Agency). (1993). 2,4-Dinitrotoluene (CASRN 121-14- 2). Washington, DC: Integrated Risk Information System (IRIS). http ://www. epa. gov/iris/sub st/ tm U.S. EPA (U.S. Environmental Protection Agency). (1994). Chemical assessments and related activities (CARA) [EPA Report], (600R94904; OHEA-I-127). Washington, DC. http://nepis.epa. gov/Exe/ZyPURL.cgi?Dockey=6Q0QlG8L.txt U.S. EPA (U.S. Environmental Protection Agency). (2002). A review of the reference dose and reference concentration processes [EPA Report], (EPA/630/P-02/002F). Washington, DC. http://cfpub epa.gov/ncea/cfm/recordisplay.cfm?deid 51717 109 Technical Grade Dinitrotoluene ------- FINAL 4-4-2013 U.S. EPA (U.S. Environmental Protection Agency). (2003). Health Effects Assessment Summary Tables (HEAST). Available online at http://epa-heast.ornl.gov/ U.S. EPA (U.S. Environmental Protection Agency). (2005). Guidelines for carcinogen risk assessment [EPA Report], (EPA/630/P-03/001F). Washington, DC. http ://www. epa. gov/cancergui delines/ U.S. EPA (U.S. Environmental Protection Agency). (201 la). 201 1 Edition of the drinking water standards and health advisories [EPA Report], (EPA 820-R-11-002). Washington, DC. http://water.epa.gov/action/advisories/drinking/upload/dwstandards2011.pdf U.S. EPA (U.S. Environmental Protection Agency). (201 lb). Recommended use of body weight 3/4 as the default method in derivation of the oral reference dose [EPA Report], (EPA/100/R11/0001). Washington, DC. http://www.epa.gov/raf/publications/interspecies-extrapolation.htm U.S. EPA (U.S. Environmental Protection Agency). (2012). Benchmark dose technical guidance. (EPA/100/R-12/001). Washington, DC. http://www.epa.gov/raf/publications/pdfs/benchmark dose guidance.pdf U.S. EPA (U.S. Environmental Protection Agency). (2013). Provisional peer-reviewed toxicity values for 2,6-dinitrotoluene. Cincinnati, OH: National Center for Environmental Assessment. WHO (World Health Organization). (2012). Online catalog for the Environmental Health Criteria Series. Available online at http://www.who.int/ipcs/publications/ehc/en/ 110 Technical Grade Dinitrotoluene ------- |