United States Environmental Protection 1=1 m m Agency EPA/690/R-13/009F Final 4-10-2013 Provisional Peer-Reviewed Toxicity Values for 2,6-Dinitrotoluene (CASRN 606-20-2) 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 J. Phillip Kaiser, PhD National Center for Environmental Assessment, Cincinnati, OH DRAFT DOCUMENT PREPARED BY ICF International 9300 Lee Highway Fairfax, VA 22031 PRIMARY INTERNAL REVIEWERS Audrey Galizia, DrPH National Center for Environmental Assessment, Washington, DC Zheng (Jenny) Li, PhD, DABT National Center for Environmental Assessment, Washington, DC This document was externally peer reviewed under contract to Eastern Research Group, Inc. 110 Hartwell Avenue Lexington, MA 02421-3136 Questions regarding the contents of this document may be directed to the U.S. EPA Office of Research and Development's National Center for Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300). l 2,6-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) 9 HUMAN STUDIES 13 Oral Exposures 13 Inhalation Exposures 13 ANIMAL STUDIES 13 Oral Exposures 13 Short-term Studies 13 Subchronic Studies 14 Chronic Studies 19 Developmental Studies 20 Reproductive Studies 20 Carcinogenicity Studies 20 Inhalation Exposures 22 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 23 Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity 39 LD50 Toxicity Studies 39 Metabolism/Toxicokinetic Studies 39 Mode-of-Acti on/Mechani sti c Studi es 41 Neurotoxicity 42 DERIVATION 01 PROVISIONAL VALUES 43 DERIVATION OF ORAL REFERENCE DOSES 44 Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 44 Derivation of Chronic Provisional RfD (Chronic p-RfD) 44 DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 44 Derivation of Subchronic Provisional RfC (Subchronic p-RfC) 44 Derivation of Chronic Provisional RfC (Chronic p-RfC) 44 CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 44 MODE-OF-ACTION DISCI SSION 46 DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 46 Derivation of Provisional Oral Slope Factor (p-OSF) 46 Derivation of Provisional Inhalation Unit Risk (p-IUR) 49 APPENDIX A. PROVISIONAL SCREENING VALUES 50 APPENDIX B. DATA TABLES 59 APPENDIX C. BMD OUTPUTS FOR THE SUBCHRONIC AND CHRONIC p-RfDs 72 APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE ORAL SLOPE FACTOR 73 APPENDIX E. REFERENCES 77 li 2,6-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 2,6-Dinitrotoluene ------- FINAL 4-10-2013 PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR 2,6-DINITROTOLUENE (CASRN 606-20-2) 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 2,6-Dinitrotoluene ------- FINAL 4-10-2013 INTRODUCTION 2,6-Dinitrotoluene (also called 2,6-DNT) has several names, including toluene, 2,6-dinitro-; 2-methyl-l,3-dinitrobenzene or benzene, 2-methyl-l,3-dinitro; and 1-methyl- 2,6-dinitrobenzene (U.S. EPA, 1990; IARC, 1996; NLM, 2011). The isomer 2,6-dinitrotoluene often occurs in a mixture with the isomer 2,4-dinitrotoluene (2,4-DNT), made by combining toluene with mixed nitric and sulfuric acids. The isomeric composition of dinitrotoluene (DNT) may vary, but technical grade DNT (CASRN 25321-14-6) refers to a mixture of approximately 76% 2,4-DNT and 19% 2,6-DNT. The remaining 5% is a combination of the remaining four dinitrotoluene isomers: 2,3-DNT, 2,5-DNT, 3,4-DNT, and 3,5-DNT. In the literature, this mixture is also called dinitrotoluene (isomers mixture), DNT or DNT 80/20. In this document, the name technical grade DNT (tgDNT) is used as a representative of this mixture composition (76%) 2,4-DNT and 19% 2,6-DNT). DNT is used to make flexible polyurethane foams used in the bedding and furniture industries, and as a chemical intermediate in the production of toluene diamines and diisocyanates. DNT is also used to produce dyes, explosives, and propellants (IARC, 1996; AT SDR, 1998). The empirical formula for 2,6-DNT is C7H6N2O4, and its structure is shown in Figure 1. A table of the physicochemical properties of 2,6-DNT is provided in Table 1. o Figure 1. 2,6-Dinitrotoluene Structure 2 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 1. Physicochemical Properties for 2,6-Dinitrotoluene (CASRN 606-20-2)a Property (unit) Value Boiling point (°C) 285 Melting point (°C) 66 Density (g/cm3) 1.2833 at lire Vapor pressure (mm Hg at 25°C) 5.67 x icr4 pH (unitless) ND Solubility in water (mg/L at 20°C) 180 Relative vapor density (air =1) 6.28 Molecular weight (g/mol) 182.14 "Values from NLM (2011) and IARC (1996). 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 tgDNT (approximated as 76% 2,4-DNT and 19%) 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. A previous PPRTV for 2,6-DNT was posted in 2004 (U.S. EPA, 2004; see Table 2). The purpose of this PPRTV is to review and update the toxicity of 2,6-DNT (approximately 99% 2,6-DNT). 3 2,6-Dinitrotoluene ------- FINAL 4-10-2013 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 (various compositions of DNTs) 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 (various compositions of DNTs) 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 10~5 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 4 2,6-Dinitrotoluene ------- FINAL 4-10-2013 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 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 Health effect assessment 2.3 x lCT1 per mg/kg-dd and 2.1 x 1CT1 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 4.5 x 10 1 per mg/kg-d (screening p-OSF) NV NV NV Based on a BMDLi0Hed of 0.224 from a 104-wk study in rats with increased incidence of liver hepatocellular carcinomas, liver neoplastic nodules, mammary fibroadenomas and subcutaneous fibromas in males U.S. EPA (2013) 4-3-2013 5 2,6-Dinitrotoluene ------- FINAL 4-10-2013 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 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 1() 3 mg/kg-d8 4 x 10 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-DNT based 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 Drinking water NV 2^10 3 mg/kg-d (1-d Health advisory) 1 x 1CT1 mg/L (Drinking water equivalent level) 1 x 10° mg/L Cl- and 10-d Health advisory for a 10-kg child) lxio 3 mg/kg-d (1-d Health advisory) 4 x 1CT2 mg/L (Drinking water equivalent level) 4 x 1CT1 and 4 x 10~2 mg/L (1- and 10-d Health advisory for a 10-kg child) NV NA U.S. EPA (2011a) 2-6-2013 6 2,6-Dinitrotoluene ------- FINAL 4-10-2013 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 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 (2007) 9-13-2012 OSH A/PEL 1.5 mg/m3 NV NV NV TWA for 8-hr workday OSHA (2006) 9-13-2012 IRIS/Oral RfD NV 2 x 10 3 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 (1993) 9-13-2012 IRIS/Inhalation RfC NV NV NV NV None U.S. EPA (1990) 9-13-2012 HEAST/ subchronic Oral RfD NV 2 x 10 3 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 Health effects assessment NV NV NV NV NA U.S. EPA (1987) 2-6-2013 7 2,6-Dinitrotoluene ------- FINAL 4-10-2013 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 PPRTV 5 x l(T3 mg/kg-d (screening subchronic p-RfD)1 9 x 10 4 mg/kg-d (screening chronic p-RfD)1 NV 1 x 1CT2 mg/kg-d (subchronic p-RfD)k 1 x 10 3 mg/kg-d (chronic p-RfD)k NV 'Based on a BMDL10Hed of 0.52 mg/kg-d for hepatic necrosis in a 26-week oral study in male rats; JBased on a BMDL10Hed of 0.087 mg/kg-d for hepatic necrosis in a 104-week oral study in male rats; kBased on a NOAEL of 4 mg/kg-d for numerous health effects in a 13-wk oral study in male and female dogs tgDNT is U.S. EPA (2013); 2,6-DNT is U.S. EPA (2004) 4-3-2013 and 2-6-2013 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. 8 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Literature searches were conducted on sources published from 1900 through February 2013 for studies relevant to the derivation of provisional toxicity values for 2,6-DNT CAS Number (606-20-2). The following databases were searched by chemical name, synonyms, or CASRN: ACGIH, ANEUPL, AT SDR, BIOSIS, Cal EPA, CCRIS, CD AT, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HERO, HMTC, HSDB, I ARC, 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 toxicity values or exposure limits: 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 2,6-DNT and includes all potentially relevant repeated short-term-, subchronic-, and chronic-duration studies. The phrase, "statistical significance" used throughout the document, indicates ap-value of <0.05. 9 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 3. Summary of Potentially Relevant Data for 2,6-Dinitrotoluene (CASRN 606-20-2) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL" BMDL/ BMCLa LOAEL' Reference (Comments) Notesb Human 1. Oral (mg/kg-d)a Acute0 ND Short-termd ND Long-term6 ND Chronicf ND 2. Inhalation (mg/m3)a Acute0 ND Short-termd ND Long-term0 ND Chronicf ND Animal 1. Oral (mg/kg-d)a Short-term 6/0, Sprague-Dawley rat, gavage, 14 d 0, 4, 7, 14, 35, 68, 134 mg/kg-d Mild anemia at 14 mg/kg-d, increased relative kidney weight at >7 mg/kg-d, decreased body weight at >35 mg/kg-d, increased ALT activity at >68 mg/kg-d, increased absolute and relative spleen weight at >68 mg/kg-d; decreased absolute and relative testes weight and absolute epididymides weight at 134 mg/kg-d; histopathological changes in various organs at >35 mg/kg-d 7 DU 14 Lent et al. (2012a) PR 10 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 3. Summary of Potentially Relevant Data for 2,6-Dinitrotoluene (CASRN 606-20-2) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL" BMDL/ BMCLa LOAEL' Reference (Comments) Notesb Subchronic 16/16, CD rat, dietary, 4 or 13 wk 0, 7, 35, 145 (M); 0, 7, 37, 155 (F) (Adjusted) Lesions in the liver and spleen at >35 mg/kg-d, testicular atrophy and aspermatogenesis in males at 145 mg/kg-d 7 DU 35 Lee et al. (1976a) NPR Subchronic 16/16, Albino Swiss mouse, dietary, up to 13 wk 0, 11,51,289 (M); 0, 11,55, 299 (F) (Adjusted) Mortality at >51 mg/kg-d, decreased relative liver weight at >11 mg/kg-d in males, decreased relative kidney weight at 51 mg/kg-d in males NDr DU 11 Lee et al. (1976b) NPR Subchronic 4/4, Beagle dog, oral (gelatin capsules), d/wk not reported, 4 or 13 wk 0, 4,20,100 (Adjusted) Mortality, neurotoxicity, hematological effects, histopathology of liver, kidney, and testes, anemia, Heinz bodies, and methemoglobinemia at >20 mg/kg-d, splenic extramedullar hematopoiesis at >4 mg/kg-d NDr DU 4 Lee et al. (1976c) PS, NPR Chronic 28/0, F344/CrlBR rat, dietary, 26 or 52 wk 0, 7, 14 (Adjusted) Decreased body weight, increased relative liver weight, increased ALT activity; all at >7 mg/kg-d NDr 0.69 for increased relative liver weight at 52 wk 7 Leonard et al. (1987) PR Developmental ND Reproductive ND Carcinogenicity 30/0, F344 rat, diet (high in pectin, pectin-free, or 5% pectin), up to 12 mo ADD: 0, 0.6-0.7, 3.0-3.5 HED: 0, 0.13-0.15, 0.63-0.74 Increase in hepatocellular carcinomas and neoplastic nodules in rats fed diet high in pectin content at 3.0-3.5 mg/kg-d NA NA NA Goldsworthy et al. (1986); Tumors were only observed in rats fed 2,6-DNT in diet high in pectin content PR 11 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 3. Summary of Potentially Relevant Data for 2,6-Dinitrotoluene (CASRN 606-20-2) Category Number of Male/Female, Strain, Species, Study Type, Study Duration Dosimetry" Critical Effects NOAEL3 BMDL/ BMCLa LOAEL3 Reference (Comments) Notesb Carcinogenicity 28/0, F344/CrlBR rat, dietary, 52 wk ADD: 0, 7,14 HED: 0,1.9, 3.6 Increase in neoplastic nodules and hepatocellular carcinomas at 7 and 14 mg/kg-d NA 0.25 for increased hepatocellular carcinomas NA Leonard et al. (1987) PS, PR Carcinogenicity 26/26, A/J mouse, gavage, 2 d/wk, 12 wk ADD: 0, 343.9, 857.1, 1714 HED: 0, 79, 198, 396 No increase in lung tumor incidence NA NA NA Stoner et al. (1984) PR 2. Inhalation (mg/m3)a Subchronic ND Chronic ND Developmental ND Reproductive ND Carcinogenicity ND ""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects. 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. Values from animal developmental studies are not adjusted to a continuous exposure. bNotes: IRIS = Utilized by IRIS, date of last update; PS = Principal study; PR = Peer reviewed; NPR = Not peer reviewed. 0 Acute = Exposure for 24 hr or less (U.S. EPA, 2002). dSubchronic = 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). fChronic = Repeated exposure for >10% lifespan (U.S. EPA, 2002). DU = Data unsuitable, NA = Not applicable, NV = Not available, ND = No data, NDr = Not determined, NI = Not identified, NP = Not provided, NR = Not reported, NR/Dr = Not reported but determined from data, NS = Not selected, FEL = Frank effect level. 12 2,6-Dinitrotoluene ------- FINAL 4-10-2013 HUMAN STUDIES Oral Exposures No studies investigating the effects of oral exposure to 2,6-DNT in humans have been identified. Inhalation Exposures No data on the effects of pure 2,6-DNT in humans following inhalation exposure are identified. ANIMAL STUDIES Oral Exposures The effects of oral exposure of animals to 2,6-DNT have been evaluated in one short-term (Lent et al. 2012a), one subchronic (Lee et al., 1976), one chronic (Leonard et al., 1987), and three carcinogenicity studies (Goldsworthy et al., 1986; Leonard et al., 1987; Stoner et al., 1984). Short-term Studies Lentetal., 2012a In a peer-reviewed study, Lent et al. (2012a) investigated the effects of short-term oral administration of various DNT isomers including 2,6-DNT in male rats. Groups of Sprague-Dawley rats (6/males/dose level) were gavaged with 2,6-DNT (>99% pure) at doses of 0, 4, 7, 14, 35, 68, 134 mg/kg-day for 14 days. All animals were observed twice a day for clinical signs of toxicity and morbidity. Body weight and food consumption were measured on Days 0, 1, 3, 7, and 14. Blood samples were collected for hematology and clinical chemistry tests at study termination prior to necropsy. Weights of the liver, spleen, kidneys, heart, brain, testes, and epididymides were recorded and relative organ weights were calculated. Various tissues underwent histopathological examination. The study authors observed no clinical signs of toxicity. At study termination, absolute body weight exhibited a biologically relevant decrease (>10%) at 35, 68, and 134 mg/kg-day. This decrease in body weight was statistically significant at 134 mg/kg-day. Food consumption was also statistically significantly decreased at 134 mg/kg-day from Days 0 to 7. The study authors noted the following statistically significant changes in blood parameters: decreased hemoglobin and hematocrit in all dose groups, increased total and percent neutrophils at 68 and 134 mg/kg-day, increased monocytes and percent lymphocytes at 134 mg/kg-day, decreased albumin and total protein in all dose groups, decreased chlorine at 134 mg/kg-day, and increased ALT and AST at 68 and 134 mg/kg-day. Mild anemia was reported at 14 mg/kg-day. Relative kidney weight was statistically significantly increased at 14, 68, and 134 mg/kg-day. Relative spleen weight was statistically significantly increased at 68 and 134 mg/kg-day, and absolute spleen weight was statistically significantly increased at 134 mg/kg-day. Absolute and relative testes and absolute epididymides weights were statistically significantly decreased at 134 mg/kg-day. With respect to histopathological examination, the following changes were observed: tubular degeneration, multinucleated giant cell formation, and interstitial atrophy in the testes at 68 and 134 mg/kg-day and hepatocellular hyperplasia, oval cell hyperplasia, and hepatocellular hypertrophy were observed in the liver at >35 mg/kg-day. Mitotic activity, cell necrosis, and karyocytomegaly were also noted in the liver at >68 mg/kg-day. Proximal tubule degeneration and renal tubular basophilia were observed in the kidney at 134 mg/kg-day, as well as lymphoid hyperplasia at >68 mg/kg-day. Splenic extramedullary hematopoiesis was reported 13 2,6-Dinitrotoluene ------- FINAL 4-10-2013 at 68 and 134 mg/kg-day along with lymphoid depletion of the spleen at 134 mg/kg-day. The study authors did not report statistical analyses for any of the histopathological changes. Based on mild anemia and increased relative kidney weight, a LOAEL of 14 mg/kg-day is determined with a corresponding NOAEL of 7 mg/kg-day. Subchronic Studies Lee etal., 1976 Lee et al. (1976) conducted a series of tests investigating the subchronic oral toxicity of 2,6-DNT in rats, mice, and dogs. For the sake of clarity, in this document, the study is divided into three separate summaries (Lee et al., 1976) based on the species tested. These studies are not considered to be peer reviewed. Lee etal., 1976a Groups of CD rats (16/sex/dose level) were fed diets containing 2,6-DNT (>99% pure) at 0, 0.01, 0.05, or 0.25% for 4 or 13 weeks. These doses were calculated by the study authors to be equivalent to 0, 7, 35, or 145 mg/kg-day for males and 0, 7, 37, or 155 mg/kg-day for females. All animals were observed for clinical signs of toxicity and behavioral changes. Body weight was recorded weekly while food consumption was determined throughout the study. At 4, 8, 13, and/or 17 weeks, blood samples were collected for hematology and clinical chemistry tests. Of the 16 rats/sex/group, 4 were sacrificed at 4 weeks, and 4 were sacrificed at 13 weeks. Additionally, the treatment of 4 rats/sex/group was discontinued at the end of 4 and 13 weeks. These rats were kept for observation for 4 weeks and necropsied for examination at 8 and 17 weeks, respectively, to study the reversibility of any adverse effects and were examined for gross lesions. Weights of the liver, spleen, kidneys, heart, and brain were recorded. Relative organ weights were calculated. Various tissues were removed and stained for microscopic examination of lesions. Lee et al. (1976a) reported that treatment with 2,6-DNT did not cause overt neuromuscular signs, but rats in the high-dose group (145 mg/kg-day [males], 155 mg/kg-day [females]) were less active and had rough coats and signs of malnutrition. There were no deaths during the study. Tables B.l and B.2 provide treatment-related effects on body weights and absolute and relative organ weights. Body weights were markedly and consistently reduced at the mid- and high-doses of 35 and 145 mg/kg-day in males and 37 and 155 mg/kg-day in females throughout the exposure period. At 13 weeks, absolute body weights were 17 and 25% lower than controls in mid-dose males and females, respectively, and 53 and 38% lower than controls in high-dose males and females, respectively. Absolute body weights were also reduced in males and females at the low dose of 7 mg/kg-day for much of the study, but the difference from controls was less than 10% (9.8% in males and 5.5% in females). Lee et al. (1976a) found that after 13 weeks, there were several statistically significant organ weight changes. Statistically significant increases in relative liver weight in males were observed in the liver at 7 mg/kg-day and at 145 mg/kg-day (but not at 35 mg/kg-day) and in females at 155 mg/kg-day. Absolute liver weight was statistically significantly increased in males at 35 and 145 mg/kg-day. Relative organ weights (spleen, kidneys, heart, and brain) were statistically significantly increased in males at 145 mg/kg-day. There were also statistically significant increases in absolute spleen, kidney, and heart weights in male rats at 145 mg/kg-day. Relative spleen weights were statistically significantly increased in females at 155 mg/kg-day, 14 2,6-Dinitrotoluene ------- FINAL 4-10-2013 and relative brain weights were statistically significantly increased at 37 and 155 mg/kg-day. Study authors also reported a statistically significant decrease in absolute heart weight in females at 155 mg/kg-day. Lee et al. (1976a) observed no significant and/or consistent treatment-related hematological effects in the low-dose rats (7 mg/kg-day). A statistically significant decrease in reticulocyte count from baseline (baseline refers to data obtained from rats during pretreatment) was observed in control males and males at 7 and 35 mg/kg-day at 4 and 13 weeks (see Table B.3). A statistically significant increase in leukocyte count was observed in males at 35 mg/kg-day at Week 13. In addition, a statistically significant increase in erythrocyte count was observed in males at this dose group (35 mg/kg-day) at Weeks 4 and 13. This increase in erythrocyte count was considered as mild by the study authors and was not seen in male controls or males fed the low or high level of 2,6-DNT. Males at 145 mg/kg-day showed a decrease in erythrocyte count with a compensatory reticulocytosis and an increase in leukocyte count at 4 weeks; these parameters partially recovered at 8 (data not shown) or 13 weeks. Females fed 7 and 37 mg/kg-day had a statistically significant decrease in reticulocyte count at 4 and 13 weeks when compared to baseline. Females fed 37 mg/kg-day had a statistically significant increase in leukocytes at 13 weeks (see Table B.3). Females at 155 mg/kg-day showed an increase in leukocyte and reticulocyte count at 4 weeks; these parameters partially recovered at 13 weeks. A statistically significant increase in methemoglobin was seen in males at 145 mg/kg-day and in females at 155 mg/kg-day after 8 (data not shown) or 13 weeks; however, after recovery for an additional 4 weeks, this increase was no longer observed (data not shown). In summary, the only significant hematological changes from control were increased leukocytes (males), increased reticulocytes (males and females), and decreased erythrocytes (males) following 4 weeks of exposure to the high dose of 2,6-DNT (145 mg/kg-day [males] and 155 mg/kg-day [females]). Lee et al. (1976a) reported extramedullary hematopoiesis in the spleen and liver; also, bile duct hyperplasia in the liver was observed in males at 35 mg/kg-day and in females at 37 mg/kg-day following 13 weeks of exposure (see Tables B.4 and B.5). Hemosiderosis was also observed in the spleen in males at 145 mg/kg-day and in females at 155 mg/kg-day at 13 weeks. Focal atrophy of the testes was observed in control and low dose males at 13 weeks. Retardation of spermatogenesis was observed at 13 weeks in mid-dose males, and atrophy of the testes and aspermatogenesis were observed in males at 145 mg/kg-day. In general, the effects on the testes, spleen, and liver were more frequent and more severe in the high-dose rats than in the mid-dose rats. Only partial recovery of the tissue lesions was seen after the 4-week recovery periods (data not shown). Lee et al. (1976a) observed several effects in animals exposed to 2,6-DNT. Decreases in body weights (greater than 10%) were biologically significant in both males and females; however, study authors suggested that the decreases in body weight could be due to decreased food consumption. Therefore, decreased body weight could be due to the effect of 2,6-DNT on food palatability and not necessarily a systemic toxicological effect of the chemical. Additionally, hematological effects are noted but are only significantly different from control at the high-dose in animals exposed for 4 weeks, with no significant effects, as compared to control, at 12 weeks. Histopathology revealed lesions in the liver (i.e., hematopoiesis and bile duct hyperplasia) that were statistically significantly increased in males exposed to 35 mg/kg-day for 13 weeks and in females exposed to 37 mg/kg-day; incidence of bile duct hyperplasia was 15 2,6-Dinitrotoluene ------- FINAL 4-10-2013 statistically significant at 145 mg/kg-day in males and 155 mg/kg-day in females. Incidence of splenic hemosiderosis was statistically significantly increased as compared to controls in males exposed to 7 mg/kg-day for 13 weeks. However, at the next dose tested of 35 mg/kg-day, splenic hemosiderosis was not seen in any of the rats examined. While effects were also noted in the testes, atrophy observed in the control and low-dose group makes these effects difficult to interpret. Thus, based on the increased incidences of splenic and liver hematopoiesis in male rats, a LOAEL of 35 mg/kg-day is identified from this study with a corresponding NOAEL of 7 mg/kg-day. Lee etal., 1976b Lee et al. (1976b) fed groups of 16 male and 16 female albino Swiss mice a diet containing 0, 0.01, 0.05, or 0.25% 2,6-DNT (>99% pure) for up to 13 weeks. According to the authors, the corresponding intakes of test material were 0, 11, 51, or 289 mg/kg-day for males and 0, 11, 55, or 299 mg/kg-day for females. The basic design and procedure for this study were the same as that described for rats (Lee et al., 1976a); however, blood clinical chemistry tests in mice were not performed. Lee et al. (1976b) reported no compound-related effects in mice at 11 mg/kg-day. However, several deaths occurred during the study, including 3 males in the control group (Week 12), 2 males in the low-dose group of 11 mg/kg-day (Weeks 1 and 3), 8 males and 1 female in the mid-dose group of 51 mg/kg-day (males) and 55 mg/kg-day (females), and 8 males and 6 females in the high-dose group of 289 mg/kg-day (males) and 299 mg/kg-day (females). Furthermore, all males in the high-dose group died before Week 9, while two of eight females in the high-dose group survived the full 13 weeks of feeding. The authors stated that in the mid- and high-dose groups, most of the deaths could be contributed to 2,6-DNT administration. The exact cause of death was not discussed, but most mice that died had low body weight, frequently with significant weight loss a week or two before death. In the mid- and high-dose groups, food consumption was lower than in controls. Blood analyses in the mid- and high-dose groups revealed a number of statistically significant changes relative to the controls at the respective time intervals; however, the authors stated that these changes were mild, inconsistent, and not related to 2,6-DNT exposure. The study authors also observed decreased absolute and relative liver weight (>10%) at 11 mg/kg-day in male mice treated for 13 weeks. Absolute kidney weight in male mice was increased (>10%) at 11 mg/kg-day and then decreased at 51 mg/kg-day. The biological significance of the observed decrease in absolute liver and kidney weights is unknown due to the accompanied increases in body weight that were biologically significant at 11 and 51 mg/kg-day in mice treated for 13 weeks. Relative kidney weight was biologically and statistically significantly decreased at 51 mg/kg-day in male mice treated for 13 weeks. Lee et al. (1976b) noted marked aspermatogenesis in all males at 289 mg/kg-day, and depressed spermatogenesis was seen in one male from the mid-dose group (51 mg/kg-day) treated for 4 weeks and in two males from the low-dose group (11 mg/kg-day) treated for 4 or 13 weeks. Bile duct hyperplasia occurred in the only mouse that survived treatment with the high dose of 2,6-DNT for 13 weeks and in two mice fed the mid-dose for 13 weeks. Bile duct hyperplasia was also observed in two high-dose mice treated for 4 weeks and allowed to recover for 4 weeks, suggesting that this lesion developed slowly. The investigators indicated that extramedullary hematopoiesis in the liver and spleen was seen more often in mice treated with 2,6-DNT than in the controls and that generally, the incidence and severity were dose related. 16 2,6-Dinitrotoluene ------- FINAL 4-10-2013 No testicular lesions were observed in mice treated for 4 weeks and allowed to recover for 4 weeks. Because no high-dose males survived longer then Week 9, it is not known whether testicular lesions would have occurred in this dose group following 13 weeks of treatment. There was partial recovery of the bile duct hyperplasia after the 4-week recovery period, but extramedullar hematopoiesis continued to be observed in the liver and/or spleen. A LOAEL of 11 mg/kg-day is identified based on decreased relative liver weight. Because 11 mg/kg-day is the lowest dose tested, a NOAEL could not be determined. Lee etal., 1976c Lee et al. (1976c) is selected as the principal study for deriving the screening subchronic and chronic p-RfDs. Lee et al. (1976c) dosed groups of beagle dogs (4/sex/dose level) with 2,6-DNT (>99% pure) in gelatin capsules at doses of 0, 4, 20, or 100 mg/kg-day for 4 or 13 weeks. The dogs were evaluated in the same manner as in the rat study (Lee et al., 1976a) with the following exceptions: at the end of 4 and 13 weeks, one male and one female dog from each group were euthanized for necropsy; liver, spleen, kidneys, brain, adrenals, thyroid and gonads were examined and the weights recorded; the treatment for one male and one female dog from each group was discontinued at the end of 4 and 13 weeks and observed until necropsy at the end of 8 and 17 weeks, respectively; food consumption was recorded daily. Lee et al. (1976c) observed the deaths of all dogs receiving 100 mg/kg-day of 2,6-DNT were between Weeks 2 and 8 (one dog died during the second week, and the last dog on this treatment died in the eighth week). The signs exhibited by these dogs consisted of listlessness, incoordination, lack of balance, pale gums, dark urine, and weakness (particularly of the hind limbs); tremors were seen occasionally. Terminal signs seen in some dogs included yellow gums and darkened sclera. Because of the severity of symptoms observed in the dogs exposed to 100 mg/kg-day of 2,6-DNT, they were placed on the reversibility study after 4 weeks and continued for 19 weeks (23 weeks total) before they were sacrificed. Similar symptoms were observed in dogs at 20 mg/kg-day but were less severe. Signs of toxicity in the mid-dose group (20 mg/kg-day) were not seen until Week 4. Two female dogs at 20 mg/kg-day died during Week 9. A Fisher's exact test comparing death in the control and mid-dose groups yielded a />value of 0.233, indicating a nonstatistically significant difference. However, group sizes were too small for the statistical test to have much power to detect an effect and the deaths may have been compound-related, as gross necropsy showed emaciation and jaundice. No significant treatment-related effects occurred in dogs at 4 mg/kg-day (other than mild splenic extramedullar hematopoiesis in some dogs). However, animals at both 20 and 100 mg/kg-day showed clear signs of toxicity (neurological, hematological, and liver histopathology), and the incidence and severity of the effects were dose related. Extramedullar hematopoiesis in the spleen was observed at 4 mg/kg-day and appeared to be reversible depending upon the length of exposure and postexposure recovery period even at the higher doses. Lee et al. (1976c) found no significant alterations in body weight in animals receiving 2,6-DNT at 4 mg/kg-day, but dogs at 20 mg/kg-day began to lose weight during Weeks 4 and 5, which correlated with the adverse effects previously noted. Dogs at 100 mg/kg-day lost weight from the first week of treatment. Food consumption correlated with weight changes. During the reversibility studies, the affected dogs quickly returned to normal food consumption rates. 17 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Due to the mortality observed in one dog in the high-dose group during Week 2, the study authors collected blood samples from all surviving dogs in all dose groups at the end of Week 2, in addition to the scheduled analyses (Lee et al., 1976c). Control dogs and dogs at 4 mg/kg-day showed mild fluctuations in hematology and clinical chemistry parameters, which were not considered to be biologically significant by the study authors. However, significant effects were observed at 2 weeks in dogs exposed to 20 mg/kg-day, including anemia characterized by decreases in hematocrit and hemoglobin with a compensatory reticulocytosis. Small amounts of methemoglobin were seen at Week 8, and Heinz bodies were seen at Week 13. Serum alanine aminotransferase (ALT) activity was increased at Weeks 8 and 13. One of the females that died in Week 9 was severely anemic, with large amounts of Heinz bodies and methemoglobin, and elevated serum ALT, aspartate aminotransferase (AST), and alkaline phosphatase (AP). Blood analysis done on Week 2 in dogs at 100 mg/kg-day showed severe effects, including a 66% reduction in RBC and signs of immature erythrocytes. Also evident was leukocytosis, with an increased percentage of neutrophils and decreased percentage of lymphocytes, and increased serum AP and ALT activities. Laboratory data from dogs in the mid-dose group (20 mg/kg-day) treated for 4 or 13 weeks showed recovery after 4 weeks, but high-dose dogs (100 mg/kg-day) treated for 4 weeks did not recover until Week 19. No significant alterations in organ weights were seen in dogs at 4 mg/kg-day compared to the control group (Lee et al., 1976c). Treatment-related histological alterations in dogs at both 20 and 100 mg/kg-day after 4 weeks of treatment included extramedullary hematopoiesis in the liver and spleen, bile duct hyperplasia, degeneration and/or subacute inflammation in the liver, and degeneration and/or depression of spermatogenesis in the testes. The incidence and severity of these lesions were generally dose related. Lymphoid depletion in the spleen and lymph node, and involution of the thymus were also seen in high-dose animals. A female dog from the low-dose group (4 mg/kg-day) had several graafian follicles but no corpora lutea. This female also had mild extramedullary hematopoiesis in the spleen. Because this effect was observed in dogs given this dose for 13 weeks and in dogs given higher doses, these alterations were considered to be compound related. Treatment up to 13 weeks with the mid- or high-dose of 2,6-DNT caused similar lesions in the liver and spleen. It also caused kidney effects consisting of dilated tubules, foci of inflammation, degeneration, yellow pigment, and/or casts in the tubules. The high-dose caused lesions in the testes, lymph nodes, and thymus. The effects observed in dogs treated with 20 mg/kg-day of 2,6-DNT at 13 weeks were usually more numerous and more severe than those seen at 4 weeks. Mild extramedullary hematopoiesis and lymphoid depletion in the spleen of some dogs at 4 mg/kg-day were also considered compound related by the study authors. Splenic extramedullary hematopoiesis was observed in all dogs treated at 4, 20, and 100 mg/kg-day for 13 weeks. In dogs treated for 4 weeks and allowed to recover, extramedullary hematopoiesis and adverse testicular effects were milder. Two dogs receiving 2,6-DNT at 100 mg/kg-day and allowed to recover for 19 weeks showed complete recovery. Dogs treated for 13 weeks did not show full recovery, as one dog in the mid-dose group still had various lesions in the liver, kidney, and testes, and a low-dose female dog still had minimal bile duct hyperplasia. In dogs treated for 13 weeks and allowed to recover for 4 weeks, splenic extramedullary hematopoiesis was not observed in any dose group. To determine whether treatment with 2,6-DNT causes an allergic reaction, the study authors measured its effects on serum IgE levels. The results revealed no apparent change in serum IgE concentration. 18 2,6-Dinitrotoluene ------- FINAL 4-10-2013 A LOAEL of 4 mg/kg-day is identified based on splenic extramedullary hematopoiesis. However, the biological significance of this endpoint is indefinite. Because 4 mg/kg-day is the lowest dose tested, a NOAEL cannot be identified. Chronic Studies Leonardet al., 1987 Leonard conducted a 1-year chronic toxicity and carcinogenicity study on pure 2,6-DNT and technical grade DNT. The unpublished study was designed to compare the hepatic carcinogenic potential of technical grade DNT, 2,6-DNT, and 2,4-DNT. The tumor data reported in this study are discussed separately under the carcinogenicity studies subheading below. In a peer-reviewed study, Leonard et al. (1987) fed groups of 28 male Fischer (F344)/CrlBR rats a diet containing 2,6-DNT at doses of 0, 7, or 14 mg/kg-day for 52 weeks. The authors stated that purified 2,6-DNT was used, but the actual purity was not specified. Concentrations of 2,6-DNT were adjusted in each diet batch based on food consumption and average body weight to maintain the 2,6-DNT doses at the target levels. Rats were housed as four per cage, and average dietary consumption for each cage was determined weekly. Body weights were measured every 2 weeks throughout the study. The study authors sacrificed 4 animals in each group after 6 and 26 weeks of feeding and measured hepatic microsomal epoxide hydrolase (EH) and cytosolic DT-diaphorase (DTD) activities (these are considered to be phenotypic markers of neoplastic lesions). At the end of the treatment period, all surviving animals were sacrificed and necropsied, selected organs were weighed (liver and lungs), histopathological examination was performed on liver and lung tissue, and hepatic EH and DTD activities were measured. Serum enzyme activities (ALT and glutamyl transferase [GGT]) were also determined. Statistical evaluations were done using the F-test and Dunnett's test (p < 0.05). Hematology and clinical chemistry were not evaluated in rats sacrificed at 1 year. Leonard et al. (1987) reported no treatment-related deaths. Statistically significant changes were seen in body weight, organ weight, and serum chemistry in rats that received 7 or 14 mg/kg-day 2,6-DNT at 26 or 52 weeks, including decreased body weight, increased absolute and relative liver weights and increased serum GGT activity (see Table B. 11). These effects, however, were more pronounced at 52 weeks. For example, terminal body weights of the 7 and 14 mg/kg-day rats were decreased relative to the controls by 5 and 18% and 20 and 32% at Weeks 26 and 52, respectively. It is unclear from the study report if the observed decreased body weight was due to a systemic effect of 2,6-DNT or an effect of the chemical on food palatability and consumption. At 52 weeks, statistically significantly increased serum ALT activity was seen in both the 7- and 14-mg/kg-day dose groups, and statistically significantly increased serum gamma-glutamyl transpeptidase activity was seen at 14 mg/kg-day (see Table B. 11). The study authors did not report the effects of 2,6-DNT exposure for 6 weeks on body weight, liver weight, or serum enzyme activities. Microscopic evaluation of the liver sections from animals that had received 52 weeks of dietary treatment revealed hepatocyte degeneration and vacuolation in the majority of these treated animals at all doses. These effects, however, did not appear to be dose-related and were also seen in controls. Over 90% of the treated animals had acidophilic and basophilic hepatocyte foci. Neither type of foci was apparent in the controls. The study authors also noted bile duct hyperplasia in most animals fed 2,6-DNT. No specific mention was made of nonneoplastic lesions in the lungs. A LOAEL of 7 mg/kg-day based on a biological (>10% change) and statistically significant increase in relative liver weight observed at 12 months is identified from this study. Because 7 mg/kg-day is the 19 2,6-Dinitrotoluene ------- FINAL 4-10-2013 lowest dose tested, a NOAEL cannot be identified from this study. It is unclear if the noncancer liver effects observed in this study were due to the presence of hepatocellular tumors caused by 2,6-DNT treatment. Developmental Studies No studies were identified. Reproductive Studies The studies by Lee et al. (1976) report limited reproductive toxicological endpoints (e.g., aspermatogenesis). These studies are summarized in the Subchronic Studies section. Carcinogenicity Studies Goldsworthy et al., 1986 Goldsworthy et al. (1986) conducted a study to evaluate the effect of diets varying in pectin content on the induction of foci and hepatic tumor by 2,6-DNT. Six groups of 30 male F344 (CDF/CrlBR) rats were placed on one of three diets containing sufficient quantities of 2,6-DNT (purity of 99.9%) to produce daily doses of 0.6-0.7 or 3-3.5 mg/kg-day. The diets used were NIH-07, an open formula cereal-based diet high in pectin content; AIN-76A, a purified pectin-free diet; or AP, which is A1N-76A supplemented with 5% pectin. These three diets served as the control diets for the addition of 2,6-DNT (see Table B.12). The study authors incorporated 2,6-DNT into the diets by premixing the 2,6-DNT into 100 grams of the test diet followed by blending the mixture. Body weight and food consumption were recorded monthly. The study authors screened the rats for the absence of viral titers throughout the study. Ten animals from each group were sacrificed at 3, 6, and 12 months, and the livers were evaluated histopathologically. Quantitative stereology was used to assess the number of hepatic foci per liver. Three markers commonly used to detect hepatic preneoplasia—GGT, ATP, G6P—were used to score and quantitate foci. The study authors performed statistical analysis on the number -3 of foci per cm liver using a Newman-Keuls multiple comparison test (p < 0.05). Goldsworthy et al. (1986) reported no deaths in the control or treatment groups. Body weight was increased in all rats fed all three diets (all animals in the 6 groups) for 3, 6, and 12 months. All groups receiving the high-dose of 2,6-DNT (3.0-3.5 mg/kg-day) gained approximately 10% less weight than their respective controls at 12 months (data on body and liver weight were presented in a graphical format by the study authors and not in tables). Liver weights were not significantly altered throughout the study compared to the control groups, except for the high-dose NIH-DNT (3-3.5 mg/kg-day) treated group, which showed a marked increase in liver weights at 12 months. In the other dose groups, the liver weights remained constant, and the liver/body-weight ratio was decreased in rats throughout the treatment periods. The study authors reported that no changes in monthly food consumption were observed in treated rats during the treatment period (data were not shown in the study). No further information was provided regarding nonneoplastic effects. Goldsworthy et al. (1986) examined the effect of the control and DNT-diets on the fraction of animals with foci (see Table B. 13). The fraction of animals with hepatocyte foci (i.e., GGT, ATP, G6P) was increased in a dose- and time-dependent manner in animals administered 2,6-DNT in the test diet with NIH > AP > AIN. Animals fed AIN and AP diets, with or without 2,6-DNT, had few or no GGT foci throughout the study. At 12 months, the number of ATP and G6P foci was approximately equal in all DNT-treated groups, and the number of GGT foci in the 20 2,6-Dinitrotoluene ------- FINAL 4-10-2013 high dose NIH-DNT group (3-3.5 mg/kg-day) was impossible to quantitate accurately, which was explained by the study authors to be due to the presence of neoplastic nodules and hepatocellular carcinomas. Hepatocellular carcinomas and neoplastic nodules were observed only in rats fed the NIH "3 diet containing 2,6-DNT (statistical analysis was only done on the number of foci per cm in the liver and not on the hepatocellular carcinomas and neoplastic nodules). At 12 months, the treatment group on the NIH diet that received the high dose of 2,6-DNT (3-3.5 mg/kg-day) exhibited a 100% incidence of hepatic foci, including 6/10 rats with hepatocellular carcinomas and 6/10 with neoplastic nodules; the low dose of 2,6-DNT (0.6-0.7 mg/kg-day) exhibited 3/10 rats with neoplastic nodules, each rat with a single nodule; and the control diet had no tumors or neoplastic nodules present in 10 rats. No tumors or neoplastic nodules were observed in rats receiving the control AIN diet with or without added pectin or added 2,6-DNT in these diets. Based on these results, the study authors concluded that 2,6-DNT is a potent hepatocarcinogen in male F344 rats, and its carcinogenic potency differs depending on whether rats are fed an NIH or AIN (with or without pectin) diet. They stated that the carcinogenicity of 2,6-DNT would not have been observed in the study if tested only in the pectin purified diets and scored by the GGT marker alone. Leonardet al., 1987 The carcinogenic study by Leonard et al. (1987) is selected as the principal study for deriving the oral slope factor (p-OSF). In a peer-reviewed study, Leonard et al. (1987) fed groups of 28 male Fischer (F344)/CrlBR rats a diet containing 2,6-DNT at doses of 0, 7, or 14 mg/kg-day for 52 weeks. Other details concerning the study methodology are presented in the Chronic Studies section. Leonard et al. (1987) noted that administration of 2,6-DNT at doses of 7 and 14 mg/kg-day to male rats in the diet for 52 weeks resulted in an increased incidence of neoplastic lesions in the liver (see Table B. 14). Neoplastic nodules were found in 18/20 rats at the low-dose (7 mg/kg-day) and 15/19 rats at the high-dose (14 mg/kg-day). Hepatocellular carcinomas, described as trabecular, occurred in 17/20 rats at 7 mg/kg-day and 19/19 rats at 14 mg/kg-day, and one tumor described as an adenocarcinoma was found in a rat at 7 mg/kg-day. Cholangiocarcinomas occurred in 2/20 rats at 7 mg/kg-day. Liver tumors metastasized to the lung in 3/20 rats at 7 mg/kg-day and 11/19 at 14 mg/kg-day. These results indicated that overall neoplastic nodule incidence paralleled tumor incidence, with the exception of the high-dose 2,6-DNT-treated group. According to the study authors, this difference is due to the extensive tumor involvement in the livers from the high-dose group. Hepatic microsomal EH and cytosolic DTD activity are induced following treatment with a number of hepatocarcinogens and are considered to be phenotypic markers of neoplastic nodules (Leonard et al., 1987). The study authors noted a dose-related increase in EH activity in rats treated with 7 mg/kg-day and 14 mg/kg-day for 26 weeks to 380% and 520% of controls, respectively. This increase was sustained at similar levels from 6 weeks to 1 year (data in the study on hepatic microsomal EH and cytosolic DTD activity were presented in graphical format and not in a table). At 1 year, EH activity in the high-dose group was lower than that observed at 6 weeks and 26 weeks (220% of controls). The study authors attributed this change to the 21 2,6-Dinitrotoluene ------- FINAL 4-10-2013 extensive tumor burden in the livers from these animals. They also stated that this observation is consistent with the lower nodule incidence observed in these animals, and this provides indirect evidence to support the suggestion that EH elevations occur in the nodules but not the tumors. In contrast to EH activities, DTD activity was increased in the high-dose 2,6-DNT-treated rats at 6 weeks. This increase was enhanced to a somewhat greater extent in both 2,6-DNT-treated groups at 26 weeks. At 12 months, DTD activities were maximally enhanced in the low- and high-dose 2,6-DNT-treated animals to 420 and 650% of controls, respectively. The study authors stated that the increase in DTD activity appeared to be linked to the presence of nodules and tumors, particularly at 12 months. In summary, administration of 2,6-DNT at oral doses of 7 and 14 mg/kg-day produced hepatocellular carcinomas in 85% and 100% of male rats, respectively. The majority of tumors had a trabecular pattern, and pulmonary metastases. Similar to the 2,6-DNT results, rats fed a diet containing 35 mg/kg-day technical grade DNT (equivalent to 7 mg/kg-day 2,6-DNT) yielded a positive hepatocarcinogenic response. The 2,6-DNT isomer induced hepatocellular carcinomas in twice as many animals as did the technical grade DNT, and 2,4-DNT was not hepatocarcinogenic when fed to rats at twice the high dose of 2,6-DNT (2 mg/kg-day) over the same time period. The study authors concluded that 2,6-DNT is a complete carcinogen, capable of both initiation and promotion, and the hepatocarcinogenicity of technical grade DNT is mainly due to 2,6-DNT. Stoner et al., 1984 In a 12-week study, Stoner et al. (1984) administered 0, 1200, 3000, or 6000 mg/kg 2,6-DNT (98%) purity) to A/J mice (26 mice/sex/dose group) for 2 days/week for 12 weeks by gavage. The corresponding duration-adjusted doses are 0, 342.9, 857.1, or 1714 mg/kg-day. The animals were sacrificed after 30 weeks, and the lungs were examined. Lung tumors, which appeared as white nodules on the surface, were counted and randomly sampled for histopathological evaluation and confirmation of adenoma. In addition, liver, kidneys, spleen, intestines, thymus, stomach, and endocrine glands were examined grossly. If gross lesions were observed, the organs were examined histologically for the presence of neoplasms. The lung tumor response (percentage of mice that developed lung tumors and the number of lung tumors per mouse) in experimental and control groups was compared by Student's t-test. No increase in lung tumor incidence or in the number of lung tumors per mouse, as compared to controls, was observed. Ellis et al., 1979 Ellis et al. (1979) performed a 2 year carcinogenicity study in which Sprague Dawley rats (38/sex/dose), CD-I Swiss mice (58/sex/dose), and beagle dogs (6/sex/dose) were treated with 2,4-DNT (approximately 98%> 2,4-DNT and 2%> 2,6-DNT) in the diet. The rat portion of the study was used as the principal study by IRIS to derive the p-OSF for the 2,4-/2,6-DNT mixture (various compositions of DNTs; U.S. EPA, 1990). Because the focus of this PPRTV is to review the toxicity of only 2,6-DNT, the Ellis et al. (1979) study is not considered as a potential principal study because of the low amount (2%>) of 2,6-DNT that was in the test compound. Inhalation Exposures No studies were identified. 22 2,6-Dinitrotoluene ------- FINAL 4-10-2013 OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) Studies on the genotoxicity, carcinogenicity by routes other than oral or inhalation, short-term toxicity, toxicokinetics, and mode of action/mechanism of 2,6-DNT are available. These are summarized in Tables 4A and 4B. Table 4A. Summary of 2,6-Dinitrotoluene Genotoxicity Studies 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/plate + (TA98, TA100, TA1535, TA1538) + (TA98, TA100, TA1535, TA1537, TA1538) Couch et al. (1981) Reverse mutation S. typhimurium strains TA98, TA1537, TA1538 NR - (TA98, 1537) + (TA1538) - (TA98, TA1537, TA1538) Ellis etal. (1978, as cited in ATSDR, 1998) Reverse mutation S. typhimurium strain TA100 50-1000 (ig/plate + ND Simmon et al. (1977) Reverse mutation S. typhimurium strains TA98, TA100 5-2000 (ig/plate - - Sayama et al. (1989, 1992) Reverse mutation S. typhimurium strains TA98, TA100, YG1021, YG1024, YG1026, YG1029, YG1041, YG1042 0-5 \M - (TA98, TA100, YG1021) + (YG1024, YG1026, YG1029, YG1041, YG1042) ND Highest degree of mutagenicity in YG1041 andYG1042 Sayama et al. (1998) Reverse mutation S. typhimurium strains TA98, TA100 NR + (TA98, TA100) + (TA98) - (TA100) Tokiwa et al. (1981) 23 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4A. Summary of 2,6-Dinitrotoluene Genotoxicity Studies Resultsb Endpoint Test System Dose Concentration" Without Activation With Activation Comments References Reverse mutation S. typhimurium strains TA98, TA100, TA1535, TA1537, TA1538, TA100NR3 10-5000 (ig + (TA100) - (TA98, TA1535, TA1537, TA1538, TA100NR3) + (TA100) - (TA98, TA1535, TA1537, TA1538, TA100NR3) Spanggord et al. (1982) Reverse mutation S. typhimurium strains TA98, TA100 0.1-10 (imol/plate - (TA98, TA100) + (TA98) - (TA100) In TA98, it was negative with rat liver activation and positive with hamster liver activation and in TA100 it was negative with both rat and hamster liver activation Dellarco and Prival (1989) Reverse mutation S. typhimurium strains TA98, TA1538 1-1000 nmol/plate + (weakly positive at highest doses) + (weakly positive at highest doses) Whong and Edwards (1984) Reverse mutation S. typhimurium strains TA98, TA98NR, TA98/1,8-DNP6, YG1021, YG1024 NR + (TA98, YG1021, YG1024, T A9 8/1,8 -DNP6) - (TA98NR) ND Einistoe et al. (1991) Reverse mutation S. typhimurium strains NR NR NT + Pearson et al. (1979, as cited by ATSDR, 1998) Reverse mutation S. typhimurium strains TA98, YG1021, YG1024, YG1041 10-100 (ig/plate + (TA98, (YG1021 YG1024, YG1041) ND Hagiwara et al. (1993) 24 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4A. Summary of 2,6-Dinitrotoluene Genotoxicity Studies Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation Reverse mutation S. typhimurium strains TA98, TA100 50-500 (ig/plate - (TA98) + (TA100) - (TA98) + (TA100) George et al. (2001) Reverse mutation S. typhimurium strain TA100 1-150 (ig/mL - ND Padda et al. (2003) Forward mutation TM 677 500 (ig/mL + + 2,6-DNT and technical-grade DNT Couch et al. (1981) SOS repair induction ND Genotoxicity studies in nonmammalian eukaryotic organisms Mutation ND Recombination induction ND Chromosomal aberration ND Chromosomal malsegregation ND Mitotic arrest ND Genotoxicity studies in mammalian cells—in vitro Mutation Chinese hamster ovary/HGPRT 2.5 mM - - Abernathy and Couch (1982) Mutation Chinese hamster ovary NR - ND Lee et al. (1976) Mutation P388 mouse lymphoma cells 1.6-1000 (ig/mL - - 2,6-DNT and technical grade DNT Styles and Cross (1983) Morphological transformation Syrian hamster embryo cells up to 100 (ig/mL — ND 2,6-DNT and technical grade DNT Holen et al. (1990) 25 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4A. Summary of 2,6-Dinitrotoluene Genotoxicity Studies Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation Chromosomal aberrations Human peripheral lymphocytes 0.002-0.10 mmol/L + ND Huang et al. (1995) Sister chromatid exchange (SCE) ND DNA damage Rat germ cells 0.000032-0.02 mmol/L + ND DNA strand breaks showed a dose-response relationship Yang et al. (2005) DNA adducts ND DNA repair Primary rat hepatocytes 1 x 10~4, 1 X l(T3 M - ND Bermudez et al. (1979) DNA repair Primary rat hepatocytes 0.1, 1.0 mM - - Butterworth et al. (1989) DNA repair Human hepatocytes 0.01-1.0 mM - - Butterworth et al. (1989) Unscheduled DNA synthesis Rat spermatocytes 10-1000 (iM ND Working and Butterworth (1984) DNA and protein synthesis Chinese hamster ovary cells 10-1000 (ig/mL + (weak) ND Garrett and Lewtas (1983) Genotoxicity studies in mammals—in vivo Chromosomal aberrations CD rats (sex not reported) 35-37 mg/kg-d + ND Lee et al. (1976) Sister chromatid exchange (SCE) ND DNA damage Male Sprague-Dawley rats 35, 68, and 134 mg/kg-d (gavage) + (liver) ND Lent et al. (2012b) 26 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4A. Summary of 2,6-Dinitrotoluene Genotoxicity Studies Endpoint Test System Dose Concentration" Resultsb Comments References Without Activation With Activation DNA adducts Male F344 rats 1.2 mmol/kg i.p. + ND La and Froines (1993) Mouse biochemical or visible specific locus test ND Dominant lethal ND Unscheduled DNA synthesis Male F344 rats 5-100 mg/kg (gavage) + ND Mirsalis and Butterworth (1982) Unscheduled DNA synthesis Male F344 rats 20 mg/kg (gavage) ND Working and Butterworth (1984) Micronucleus test Male F344 rats 125, 250 mg/kg (gavage) + (liver) - (peripheral blood) ND Takasawa et al. (2010) Micronucleus test Male Sprague-Dawley rats 35, 68, and 134 mg/kg-d (gavage) - (peripheral blood) ND Lent et al. (2012b) Genotoxicity studies in subcellular systems DNA binding ND aLowest effective dose for positive results, highest dose tested for negative results. b+ = Positive, ± = Equivocal or weakly positive, - = Negative, T = Cytotoxicity, NA = Not applicable, ND = No data, NDr = Not determined, NR = Not reported, NR/Dr = Not reported, but determined from data. 27 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Carcinogenicity other than oral/inhalation 26/26 A/J mice were administered 2,4-DNT (92-95% pure, with the major impurity being 2,6-dinitrotoluene), 2,6-DNT (98% pure), and 2:1 mixture of 2,4-DNT and 2,6-DNT by i.p. injection at doses of MTD (maximal tolerated dose), 0.5 MTD, and 0.2 MTD three times per week for 8 wk. The total doses were 0, 600, 1500, and 3000 (maximum tolerated dose, MTD) mg/kg bw for 2,4-and 2,6-DNT. For the 2:1 mixture of 2,4-DNT and 2,6-DNT, the total doses were 0, 960, 2400, and 4800 (MTD) mg/kg bw. Animals were killed after 30 wk, and the lungs, liver, kidneys, spleen, intestines, thymus, stomach, salivary, and endocrine glands were examined grossly. If gross lesions were observed, they were examined histologically for the presence of neoplasms. No increase in lung tumor incidence or in the number of lung tumors per mouse was observed compared to controls in all three compounds. No lesions were observed in any other organ site. 2,6-DNT, 2,4-DNT, and the 2:1 mixture of 2,4-DNT and 2,6-DNT did not induce lung tumors in A/J mice when given by i.p. injection. According to the study authors, the inability of these dinitrotoluenes to induce lung tumors in A/J mice is expected because it is known that hepatocarcinogens are either inactive or only weakly active for lung tumor induction in strain A mice. Stoner et al. (1984) 28 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Carcinogenicity other than oral/inhalation Study authors performed a number of initiation-promotion liver foci assays in CDF (F344)/CrlBR rats using 4 initiation-promotion protocols. The study aimed to evaluate the relative initiating potential of each DNT isomer (2,4 and 2,6) and compare it with the initiating potential of technical grade DNT. A group of 8-10 male (F344)/CrlBR rats were administered by gavage a single dose of 75 mg/kg bw in corn oil of either technical grade DNT or 2,6-DNT (purity >99.4%), or 2,4-DNT (purity >99.4%) at 12 hr post partial hepatectomy. The numbers of gamma glutamyltranspeptidase-positive (GGT) foci were quantified. Technical grade DNT was a weak initiator when administered as a single oral dose (75 mg/kg) at 12 hr post hepatectomy. Significant dose-related increases in the number of GGT+ foci were observed compared with control rats administered 2,6-DNT. No initiating activity was demonstrated with 2,4-DNT. 2,6-DNT and technical grade DNT were weak tumor initiators with comparable initiating activity. Thus, the initiating activity of 2,6-DNT likely accounts for the initiating activity in technical grade DNT. 2,4-DNT was not a tumor initiator. Leonard et al. (1983) 29 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Carcinogenicity other than oral/inhalation A rat hepatic initiation-promotion protocol, 8-10 male CDF (F344)/CrlBR rats were initiated with a single dose of 150 mg/kg bw diethylnitrosamine by i.p. injection and permitted to recover for 2 wk. After the recovery period, the animals were placed on diets containing: • 27 mg/kg-d of 2,4-DNT (994% pure) or • 2.8, 7 or 14 mg/kg-d 2,6-DNT (994% pure) or • 14 or 35 mg/kg-d technical grade DNT Rats receiving technical-grade DNT were killed after 3 or 6 wk of feeding and those receiving the purified isomers after 6 or 12 wk of feeding. Sections from three liver lobes of each animal were stained for gamma glutamyl transferase (GGT), and the number of GGT+ foci per cm3 was calculated. Initiation-promotion assay: 1) Technical grade DNT: at 3 wk, dose-dependent increase in number of GGT+ foci and foci volume; at 6 wk, time-dependent increase in the number of foci and foci volume relative to 3 wk treatment. 2) 2,6-DNT: time- and dose-dependent increase in the number of GGT+ foci and foci volume at 6 wk 3) 2,4-DNT: time-dependent increase in the number of GGT+ foci at 6 wk. Technical grade DNT, 2,4-DNT, and 2,6-DNT had hepatocyte foci-promoting activity. 2,6-DNT is a complete hepatocarcinogen while 2,4-DNT was a pure promoter. Leonard et al. (1986) LD50 studies Rat (gavage) Mouse (gavage) LD50 (rat) = 795 mg/kg tol80 mg/kg LD50 (mouse) = 621 mg/kg to 807 mg/kg LD50 (rat) = 795 to 180 mg/kg-d LD50 (mouse) = 621 to 807 mg/kg Lee et al. (1975, as cited in ATSDR, 1998); Ellis et al. (1978, as cited in ATSDR, 1998); Vernot et al. (1977, as cited in ATSDR, 1998) 30 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Metabolism/ toxicokinetic Male and female F344 rats were administered by gavage (14C)-ring-labeled 2,6-DNT (99% radio chemically pure) at a dose of 10 mg/kg-bw. Urine and fecal samples were collected and analyzed for metabolites. The major route of excretion of 14C after a single dose was via the urine (males, 53.6 ± 2.6%; females, 54.0 ± 4.8%). Fecal excretion accounted for 17.9% (males) and 19.8% (females) of the dose. The urinary metabolites of 2,6-DNT identified were • 2,6-dinitrobenzyl (21.7%); • 2,6-dinitrobenzoic acid (21.1%); and • 2-amino-6-nitrobenzoic acid (14.0%). Results were compared with to those found after administration of 10 mg/kg bw 2,4-DNT to male and female F344 rats. The only major difference in the disposition of the two isomers was that no A'-ace ty la mi no ni trobe nzo ic acid was found after administration of 2,6-DNT in vitro. This may reflect steric hindrance to iV-acetylation of an amino group adjacent to a methyl group. Urine is the major route of 2,6-DNT excretion. Disposition of 2,4-DNT and 2,6-DNT is not the same Long and Rickert (1982, as cited in IARC, 1996) Metabolism/ toxicokinetic Six male F344 rats received an i.p. injection of either sulfotransferase inhibitor: DCNP (2,6-dichloro-4-nitrophenol) or PCP (pentachlorophenol) (40 |imol/ kg). After 45 min, three of these animals were administered orally [3-3H]-2,6-DNT at a dose of 28 mg/kg and were euthanized 12 hr later. Urine was collected for analysis of metabolites. Prior administration of PCP had no significant effect on the excretion of the benzyl glucuronide or benzoic acid metabolites of 2,6-DNT. (The effects of DCNP on 2,6-DNT excretion were not tested.) Prior administration of PCP had no significant effect on the excretion of urinary metabolites of 2,6-DNT. Kedderis et al. (1984) 31 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Metabolism/ toxicokinetic The study authors examined metabolites formed by anaerobic incubation of 2,6-DNT or 2,4-DNT with intestinal microflora of male Wistar rats in vitro. The metabolites formed with the incubation of 2,6-DNT were • 2-nitroso-6-nitrotoluene (reached peak at 2 hr of the anaerobic incubation); • 2-hydroxyl amino-6-nitrotoluene (reached peak at 5 hr); • 2-amino-6-nitrotoluene (reached peak at 6 hr); and • 2,6-diaminotoluene (reached peak at 12 hr). Two nitroazoxy compounds: 2,2'-dimethyl- 5,5'-dinitroazoxybenzene and 4,4'-dimethyl- 3,3'-dinitroazoxybenzene, in addition to known metabolites (nitrosonitrotoluenes, hydroxylaminonitrotoluenes, aminonitrotoluenes, and diaminotoluene), were detected in the incubation of 2,4-DNT with intestinal microflora. 2,6-diaminotoluene is the terminal intestinal metabolite of 2,6-DNT. Sayama et al. (1993, as cited in ATSDR, 1998) Metabolism/ toxicokinetic Groups of six male F344 rats were pretreated with Aroclor 1254 at a dose of 25 mg/kg for 1 week and then administered 75 mg/kg 2,6-DNT in DMSO by gavage for 5 wk. Urine was collected for analysis. Interim sacrifices were carried out at 2 and 4 wk of treatment, and the liver, small intestine, large intestine, and cecum of each rat was excised at autopsy for analysis. Gastrointestinal enzyme activities were measured, and DNA adducts from liver were also determined. A significant increase in the excretion of mutagenic urinary DNT metabolites was observed after the first week of Aroclor 1254 treatment, peaked at Wk 2 and then declined by nearly 25% at Wk 4. However, at the end of the treatment, a 4-fold increase in the formation of hepatic DNA adducts was observed. This increase in DNA adducts and decrease in urinary mutagens was due to the significant elevation in hepatic metabolism and to the increase in B-glucuronidase activity in the small intestine and cecum at 4 wk. Pretreatment of F344 rats with Aroclor 1254 significantly altered select intestinal enzyme activity, stimulated hepatic enzyme activity, accelerated the biotransformation and bioactivation of 2,6-DNT, and potentiated the formation of 2,6-DNT -derived DNA adducts in the liver. The authors noted that hepatic metabolism alone likely did not account for the potentiated bioactivation of 2,6-DNT. Chadwick et al. (1993) 32 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Metabolism/ toxicokinetic 2,6-DNT metabolism by human liver and male F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubations conditions was examined. Under aerobic conditions, • The major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); and • Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132 pmol/min per mg protein, respectively. Under anaerobic conditions, • 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite; and • Rates of 2Am6NT formation by human and rat liver microsomes were 292 and 285 pmol/min per mg protein, respectively. Anaerobic reduction of 2,6-DNT and 2Am6NT by rat and human liver microsomes is mediated by cytochrome P-450. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. The major metabolites isolated from microsomal fractions of human and rat liver preparations incubated with 2,6-DNT were 2,6-dinitrobenzyl alcohol and 2 -amino -6 -nitro toluene. Chapman et al. (1992, 1993) 33 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Metabolism/ toxicokinetic Male Wistar rats (n = 6) were administered orally a single dose of 2,6-DNT or 2,4-DNT (75 mg/kg). Urine samples were collected after 24 hr and were analyzed for conjugated and unconjugated metabolites using high performance liquid chromatography. The major urinary metabolite identified after oral administration of 2,6-DNT was 2,6-dinitrobenzyl glucuronide, which accounted for about 17.4% of the administered dose. Other metabolites identified were • 2,6-dinitrobenzyl alcohol (0.53%); • 2-amino-6-nitrotoluene (0.44%); and • 2,6-dinitrobenzoic acid (0.17%). Urinary excretion of oxidized and Y-acctylatcd derivatives for 2,4-DNT was observed but not after administration of 2,6-DNT, demonstrating metabolic differences in male Wistar rats for the two isomers. 2,6-dinitrobenzyl glucuronide is the major urinary metabolite of 2,6-DNT. Metabolic differences for 2,4-DNT and 2,6-DNT exist in male Wistar rats. Metabolism of 2,6-DNT differs between two strains of rat (Wistar and F344 [Rickertetal., 1983]). Mori et al. (1996) 34 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Metabolism/ toxicokinetic Ninety-nine Chinese workers exposed to dinitrotoluenes and 61 nonmatched, nonexposed controls working in the same factory manufacturing TNT were examined and subjected to questionnaires inquiring about exposure history and other health and lifestyle factors. Blood samples were collected, and the levels of hydrolyzable Hb adducts were determined. Female Wistar rats (n = 3) were administered a single dose of 0.5 mmol/kg of 2,6-DNT or 2,4-DNT by gavage. The rats were killed after 24 hr, and hydrolyzable hemoglobin (Hb) adducts were determined for each of the compounds investigated. The Hb adduct profile in rats was compared to those in Chinese workers. Hydrolysis of Hb from rats dosed with 2,4-DNT yielded 4-amino-2-nitrotoluene (4A2NT), 24TDA, and 4-acetylamino- 2-aminotoluene (4AA2AT). Hydrolysis of Hb from rats dosed with 2,6-DNT yielded three amines, 2-amino-6-nitrotoluene (2A6NT), 26TDA and 2-acetylamino- 6-aminotoluene (2AA6AT) with 2A6NT being the predominant adduct. Hb adduct levels in rats dosed with 2,4-DNT were higher than adduct levels in rats dosed with 2,6-DNT. A similar Hb adduct pattern was found in Chinese workers exposed to dinitrotoluenes, although 2-acetylamino-6-aminotoluene (2AA6 AT) was not found in the workers. 2A6NT was the predominant adduct in 2,6-DNT exposed workers, and 4A2NT was the predominant adduct in 2,4-DNT exposed workers. Quantification of DNT-Hb adducts provided an effective biomarker of toxicity (inertia, somnolence, nausea, and dizziness) and could be used to estimate the risk associated with a particular exposure to DNT. Jones et al. (2005) Distribution and absorption/ toxicokinetic Male F344 rats (CDF (F344)/CrlBR) (36/group) were administered gavage doses of 10 or 35 mg/kg of radiolabeled 2,6-DNT. Three rats from each group were killed at 1, 2, 4, 8, 12, 24, 48, 96, 192, and 384 hr after the dose. Livers and intestine were removed and analyzed for metabolites. Increase in hepatic concentrations of radioactivity in male rats in 2 stages, with the first peak occurring 1-2 hr and a second peak occurring 8-12 hr after the dose. The second peak was followed by a gradual decline up to 16 d and was thought to be the result of enterohepatic cycling. The rapid disappearance of radioactivity from the first quarter of the small intestine of rats following the oral administration of uniformly [14C]-ring-labeled 2,4- or 2,6-DNT indicates rapid and fairly complete absorption. Rickert et al. (1983) 35 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Distribution and excretion/ toxicokinetic Male A/J mice were given 1-, 10-, or 100-mg/kg doses of the radiolabeled [3H] 2,6-DNT (2.5 (iCi/mouse) by oral or i.p. exposure. The amount of 3H in blood, liver, kidney, lung, and intestine was measured. Distribution of radioactivity in the blood, liver, kidneys, lungs, and intestines was the same 8 hr after dosing, with very low levels of radioactivity in brain, heart, and spleen. Orally administered 2,6-DNT was eliminated primarily via urine (approximately 50% of the administered dose after 8 hr). 2,6-DNT was rapidly and extensively metabolized following both routes of administration. The liver and intestines appear to be the primary organ sites for metabolism. Schut et al. (1983, as cited in U.S. EPA, 2011a, ORNL, 1995) Mode of action/ mechanistic Male F344 rats (CDF (F344)/CrlBR) (36/group) were administered by gavage doses of 10 or 35 mg/kg of radiolabeled 2,4- or 2,6-DNT. Three rats from each group were killed at 1, 2, 4, 8, 12, 24, 48, 96, 192, and 384 hr after the dose. Livers and intestine were removed and analyzed for metabolites. Hepatic covalent binding to RNA, DNA, and protein were measured. Also, intestinal disposition of [14C] dinitrotoluenes was determined. DNT-related material is covalently bound to hepatic DNA, RNA, and protein after administration of either isomer. Covalent binding to each macromolecular species was proportional to dose. Terminal half-lives of radioactivity indicated that macromolecular damage produced by 2,6-DNT was no more persistent than that produced by 2,4-DNT. However, 2,6-DNT was 10 times more potent than 2,4-DNT in producing unscheduled DNA synthesis after 12 hr. Results indicate that DNT-related material is covalently bound to hepatic DNA, RNA, and protein after administration of either isomer, but that the degree of binding after 2,6-DNT is greater than after 2,4-DNT. Rickert et al. (1983) Mode of action/ mechanistic Six male F344 rats received an i.p. injection of either sulfotransferase inhibitor: DCNP (2,6-dichloro-4-nitrophenol) or PCP (pentachlorophenol). After 45 min, three of these animals were administered orally [3-3H]-2,6-DNT at a dose of 28 mg/kg and killed 12 hr later. Livers were excised and minced, and covalently bound radiolabel to hepatic macromolecular and hepatic DNA was determined. No signs of toxicity were observed in any of the animals receiving 2,6-DNT or sulfotransferase inhibitors at the doses administered. Prior administration of the sulfotransferase inhibitors DCNP or PCP resulted in a significant decrease in the hepatic macromolecular covalent binding of 2,6-DNT by 65 to 70%. Prior administration of the sulfotransferase inhibitors DCNP or PCP decreased the binding of 2,6-DNT to DNA by 95%. These results suggest that a sulfotransferase-dependent pathway is responsible for the majority of the covalent binding of 2,6-DNT to hepatic DNA. Kedderis et al. (1984) 36 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Mode of action/ mechanistic Male F344 rats and Male A/J mice were administered 150 mg/kg of [3-3H]-2,6-DNT and 2,4-DNT by i.p. The animals were killed by cervical dislocation after 12 or 24 hr (two animals/time point), and their liver, lungs, and small and large intestines were removed. Covalently bound radiolabel to DNA from these tissues was determined. Treatment in F344 rat resulted in • a covalent binding of 2,6-DNT and 2,4-DNT to DNA of the liver; and • lower binding to DNA of the lungs and the intestine. Treatment of A/J mice resulted in • lower binding in the liver; • no detectable binding of 2,6-DNT in extrahepatic tissues; and • low amounts of binding of 2,4-DNT to lung and intestinal DNA. Binding of 2,6-DNT to liver DNA requires its prior reductive metabolism, probably by intestinal microorganisms, and that the higher binding of 2,6-DNT in the F344 rat than in the A/J mouse may, in part, be responsible for the high susceptibility of the F344 rat to 2,6-DNT carcinogenesis. Dixit et al. (1986) Mode of action/ mechanistic F344 rats were given 219 mg/kg of 2,6-DNT or 2,6-diaminotoluene by single i.p. injection. In another experiment, 2,6-DNT was also given to the animals by gavage. In both experiments, DNA adduct formation in the liver was determined. Four adducts were detected following administration of 2,6-DNT. No adducts were observed following administration of 2,6-diaminotoluene. 2,6-DNT produced extensive hemorrhagic necrosis in the liver, whereas no evidence of hepatocellular necrosis was detected following administration of 2,6-diaminotoluene. No quantitative or qualitative differences in adduct formation were found when treatment occurred by gavage or i.p. injection. The differences between the two compounds in both DNA binding and cytotoxicity were consistent with the differences in their carcinogenicity: 2,6-DNT is a potent hepatocarcinogen while 2,6-diaminotoluene is not carcinogenic. La and Froines (1993); La and Froines (1992, as cited in ATSDR, 1998) 37 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 4B. Other Studies Test Materials and Methods Results Conclusions References Mode of action/ mechanistic Male B6C3Fi mice (n = 5) were dosed orally with 50 mg/kg 2,6-DNT daily for 3 consecutive days. CD-I mice (n = 4) were given a single oral dose of 75 mg/kg 2,6-DNT. F344 rats (n = 6) were treated orally with 75 mg/kg 2,6-DNT three times at biweekly intervals. DNA adduct formation in the liver was determined. Two distinct hepatic DNA adducts were detected in B6C3Fi which differed from the four adducts observed in hepatic DNA from 2,6-DNT treated F344 rats. This difference in the number of adducts in B6C3Fi mice in comparison with F344 rats was explained to be due to • the differences in dosing regimen; and • 80% of the dose administered to B6C3Fi mice is excreted in feces. Different number of adducts observed in mice compared to rats George et al. (1996) Immunotoxicity ND Neurotoxicity ND ND = No data 38 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity The in vitro mutagenicity or genotoxicity of 2,6-DNT has been evaluated in studies with bacterial and mammalian cell systems. It has shown mixed results in the Salmonella typhimurium Ames assay using several strains with and without metabolic activation, see Table 4A. It was negative in most studies in mammalian cell systems, including studies for mutations in Chinese hamster ovary cells (Abernathy and Couch, 1982; Lee et al., 1976), only weakly positive in a study by Garrett and Lewtas (1983) and negative in mouse lymphoma cells (Styles and Cross, 1983) and Syrian hamster embryo cells (Holen et al., 1990). It was also negative for DNA repair in rat and human hepatocytes (Bermudez et al., 1979; Butterworth et al., 1989) and for unscheduled DNA synthesis in rat spermatocytes (Working and Butterworth, 1984). Positive results were reported for chromosomal aberrations in human peripheral lymphocytes (Huang et al., 1995) and DNA strand breaks in rat germ cells (Yang et al., 2005). 2,6-DNT has also been tested in vivo in mutagenicity and genotoxicity studies, with mixed results. In rats, 2,6-DNT induced positive results for chromosomal aberrations (Lee et al., 1976), DNA adducts (La and Froines, 1993), DNA damage (Lent et al., 2012b), and micronuclei (Takasawa et al., 2010) in the liver, ,but was negative for unscheduled DNA synthesis (Working and Butterworth, 1984) and micronuclei in peripheral blood (Takasawa et al., 2010, Lent et al., 2012b). In terms of tests evaluating the carcinogenicity of 2,6-DNT, Stoner et al. (1984) did not report an increase in lung tumor induction in mice exposed intraperitoneally (i.p.) to 2,6-DNT, 2,4-DNT, or a mixture of 2,4-DNT and 2,6-DNT. In hepatic tumor initiation-promotion protocols, both 2,6-DNT and technical grade DNT were reported to have tumor promoting and tumor initiating activity (Leonard et al., 1983). In contrast, 2,4-DNT was a hepatic tumor promoter but not a tumor initiator in the same in vivo hepatic initiation-promotion protocol. Leonard et al. (1986) reported that 2,6-DNT, technical grade DNT, and 2,4-DNT have hepatocyte foci promoting activity by the i.p. route. 2,6-DNT was approximately 10 times more potent than 2,4-DNT. LD50 Toxicity Studies Characteristic signs of 2,6-DNT toxicity in animals include central nervous system depression, respiratory depression, and ataxia (U.S. EPA, 1986, as cited in U.S. EPA, 2004). The following LD50 values were identified for 2,6-DNT (Lee et al., 1975; Ellis et al., 1978; Vernot et al., 1977, all as cited in ATSDR, 1998): 535 and 795 mg/kg for male and female CD rats, respectively; 180 mg/kg for male Sprague-Dawley rats; 621 and 807 mg/kg for male and female CD mice, respectively; and 1000 mg/kg for CF-mice. Metabolism/Toxicokinetic Studies Information on the toxicokinetics of 2,6-DNT is available in several reviews (ATSDR, 1998; U.S. EPA, 2004, 1987; OECD, 2004; Rickert et al., 1984, as cited in ATSDR, 1998) and is described in Table 4B. Results of the available studies indicate that DNT, including the isomer 2,6-DNT and technical grade DNT, is absorbed through the gastrointestinal tract, respiratory tract, and skin in most species. Metabolism of 2,6-DNT is believed to occur in the liver and the intestine. Urine appears to be the major route of 2,6-DNT excretion. The main urinary metabolites of 2,6-DNT are the corresponding dinitrobenzyl alcohol glucuronide, dinitrobenzoic acid, and aminonitrobenzoic acid (Rickert and Long, 1982, as cited in Mori et al., 1996). Figure 2 illustrates the proposed pathway for the metabolism of 2,6-DNT in rats from gastric absorption 39 2,6-Dinitrotoluene ------- FINAL 4-10-2013 to urinary excretion (Rickert et al., 1984, as cited in ATSDR, 1998; Sayama et al., 1993, as cited in ATSDR, 1998; Chapman et al., 1993; La and Froines, 1993). After 2,6-DNT is absorbed from the gastrointestinal tract, it is metabolized by the following steps: (1) Oxidation of the aliphatic methyl group of 2,6-DNT by hepatic cytochrome P-450 to form dinitrobenzyl alcohol, which is then conjugated with glucuronic acid, partially excreted in the bile and subsequently transferred to the intestine; (2) In the intestine, hydrolyzation of the glucuronide and reduction of one nitro group occur by intestinal microflora to form aminonitrobenzyl alcohol; (3) A portion of this metabolite is reabsorbed from the intestine and circulated back to the liver by enterohepatic circulation; and (4) In the liver, the amine group is A'-hydroxylated by cytochrome P450 to form an unstable sulfate conjugate (Kedderis et al., 1984). The sulfate conjugate can decompose and form carbonium or nitrenium ions, which then can bind to hepatic macromolecules, leading to mutations and subsequently to liver tumors. 40 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Liver ch2 ch2oh o2n no2 o2N no2 P-459 2,6-Dinitrotoluene 2,6-Dinitrobenzylalcohol UDP-Glucuronosyl- transf erase CH20-glucuronide 0,N -NO, ^ bonding Hepatic < [Activated macromolecules metabolite] 2,6-Dinitrobenzylalcohol glucuronide ch2oh H2N' x-NHOH 2-hydroxylamino-6-nitrobenzyl alcohol Intestine ch2oh ch2oh 02N -N02 02N\ /NH2 ISitroreductase 2,6-Dinitrobenzylalcohol 2-amino-6-nitrobenzyl alcohol beta-glucuronidase Nitroreductase CH20-glucuronide CH2OH 02N -NO, H2N\ /-NH2 2,6-Dinitrobenzylalcohol 2,6-Diaminobenzyl alcohol glucuronide co2h co2H 0,N "i 02N\ ^ NOz Nitroreductase 2-amino-6-nitrobenzoic 2,6-Dinitrobenzoic acid acid Figure 2. Metabolism Pathway of 2,6-DNT Mode-of-Action/Mechanistic Studies Ellis et al. (1979, as cited in ATSDR, 1998; U.S. EPA, 2004) described the mechanism by which DNT (in general) induced hematotoxicity in animals. DNT compounds or their metabolites can oxidize the ferrous ion in hemoglobin and produce methemoglobin. Hydroxylamine is probably the oxidizing species, because it is an intermediate in the reduction of nitrogen compounds to amines. Methemoglobin can form aggregates of hemoglobin degradation products called Heinz bodies, which are sensitive indicators of hemoglobin destruction. High levels of methemoglobin lead to the development of anemia, which is compensated by reticulocytosis. When reticulocytosis cannot compensate adequately, then frank anemia develops. This hematotoxic syndrome is a common effect of exposure to aromatic amines and most organic and inorganic nitrates. 41 2,6-Dinitrotoluene ------- FINAL 4-10-2013 The principal mechanism that is thought to be responsible for the genotoxicity of 2,6-DNT involves the bioactivation of 2,6-DNT to reactive metabolites, which are capable of covalent binding to hepatic macromolecules. As illustrated in Figure 2, conjugation, biliary excretion, microbial metabolism in the gut, and intestinal reabsorption are prerequisites to hepatic binding of DNT. Swenberg et al. (1983) demonstrated covalent binding of 2,6-DNT to rat hepatocyte RNA following oral dosing with 2,6-DNT, with hepatocytes of female rats showing slightly less binding than male rats. Rickert et al. (1983) reported similar hepatic binding of 2,6-DNT to protein, RNA, and DNA of rats. Hepatic binding may be greater for 2,6-DNT than for 2,4-DNT (Rickert et al., 1983). Diet (i.e., as it affects microbial activity and number) also may influence the degree to which binding of DNT metabolites occurs. Hepatic 32 DNA adducts have been detected by P-postlabeling technique in 2,6-DNT-treated male B6C3Fi and CD-I mice and F344 rats (George et al., 1996). Further information on these studies is provided in Table 4B. Neurotoxicity In animal studies, 2,6-DNT has been shown to affect the nervous system of mice and dogs (Lee et al., 1976b,c). Clinical signs in dogs have included incoordination and stiffness of the hind legs leading to complete paralysis, cerebellar vacuolation, hypertrophy, and focal gliosis, and cerebellar and brain stem hemorrhage. In mice, depression and hyperexcitability were observed, while some rats administered 2,6-DNT showed neuromuscular symptoms. Further details on this study are presented in Table 3 and in the Subchronic Studies section. There are no data on the biochemical events involved in the toxicity of the nervous system. 42 2,6-Dinitrotoluene ------- FINAL 4-10-2013 DERIVATION OF PROVISIONAL VALUES Tables 5 and 6 present a summary of noncancer reference and cancer values, respectively. IRIS data are indicated in the table, if available. Table 5. Summary of Reference Values for 2,6 Dinitrotoluene (CASRN 606-20-2) Toxicity Type (Units) Species/Sex Critical Effect p-Reference Value POD Method POD UFC Principal Study Screening Subchronic p-RfD (mg/kg-d) Dog/M and F Increased incidence of splenic extramedullary hematopoiesis 3 x 1(T3 LOAELhed 3 1,000 Lee et al. (1976c) Screening Chronic p-RfD (mg/kg-d) Dog/M and F Increased incidence of splenic extramedullary hematopoiesis 3 x 1(T4 LOAELhed 3 10,000 Lee et al. (1976c) Subchronic p-RfC (mg/m3) NDr Chronic p-RfC (mg/m3) NDr NDr = Not determined. Table 6. Summary of Cancer Values for 2,6 Dinitrotoluene (CASRN 606-20-2) Toxicity type Species/Sex Tumor type Cancer value Principal study p-OSF F344 Rat/M Hepatocellular carcinomas 1.5 x 10° (mg/kg-d)"1 Leonard et al. (1987) p-IUR NDr NDr = Not determined. 43 2,6-Dinitrotoluene ------- FINAL 4-10-2013 DERIVATION OF ORAL REFERENCE DOSES Derivation of Subchronic Provisional RfD (Subchronic p-RfD) There are three subchronic-duration studies (Lee et al. 1976) presented in one report on 2,6-DNT in rats, mice, and dogs (see Table 3). Lee et al. (1976) is considered inadequate for p- RfD derivation because it is a nonpeer-reviewed and unpublished report. However, the Lee et al. (1976c) study is suitable for the derivation of a screening subchronic toxicity value. Appendix A provides details on the screening subchronic p-RfD. Derivation of Chronic Provisional RfD (Chronic p-RfD) One chronic oral study in rats is available (Leonard et al., 1987), but it is not a comprehensive study and only investigated effects in the liver. Leonard et al. (1987) is not considered to derive the chronic p-RfD because it is unclear if the limited noncancer effects in the liver could be attributed to the carcinogenic effects of 2,6-DNT. The subchronic study in dogs by Lee et al. (1976c) (see discussion in the derivation of the subchronic p-RfD section above), is not used to derive the chronic p-RfD because it is a nonpeer-reviewed and unpublished report. However, the Lee et al. (1976c) dog study is used to derive the screening chronic p-RfD. Details are provided in Appendix A. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS No studies were identified that could be used to derive provisional inhalation RfCs for 2,6-DNT. Available epidemiological studies consist primarily of occupational studies in which workers were exposed to the technical grade DNT mixture, which consists primarily of the 2,4-DNT isomer. In these studies, dermal as well as inhalation exposure was investigated, and exposure to 2,6-DNT was not quantified. No animal inhalation studies are available for 2,6-DNT. Derivation of Subchronic Provisional RfC (Subchronic p-RfC) The available data do not support derivation of any inhalation toxicity values. Derivation of Chronic Provisional RfC (Chronic p-RfC) The available data do not support derivation of any inhalation toxicity values. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR Table 7 identifies the cancer weight-of-evidence (WOE) descriptor for 2,6-DNT. 44 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 7. Cancer WOE Descriptor for 2,6-DNT Possible WOE Descriptor Designation Route of Entry (Oral, Inhalation, or Both) Comments "Carcinogenic to Humans " ND ND No human cancer studies on pure 2,6-DNT are available by any route of exposure. "Likely to Be Carcinogenic to Humans " NA NA The cancer weight of evidence does not meet the examples to be considered "Likely to be Carcinogenic to Humans." "Suggestive Evidence of Carcinogenic Potential" Selected Both3 As described below, 2,6-DNT is considered to have "Suggestive Evidence of Carcinogenic Potential" "Inadequate Information to Assess Carcinogenic Potential" NA NA There is evidence to assess the carcinogenic potential of 2,6-DNT. "Not Likely to Be Carcinogenic to Humans " NA NA Evidence of the carcinogenic potential of 2,6-DNT is available in animals. aAlthough data on the carcinogenic effects of 2,6-DNT via the inhalation route are limited to human exposures to a DNT mixture, 2,6-DNT is considered to have suggestive evidence of carcinogenic potential by all routes of exposure based on EPA's 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 descriptor may apply to all routes of exposure that have not been adequately tested at sufficient doses. NA = Not applicable, ND = No data. No human cancer studies of pure 2,6-DNT are available. Results from experimental animal studies showed that 2,6-DNT: (1) increased the incidence of hepatocellular neoplastic nodules and carcinomas in a chronic dietary exposure bioassay with male F344 rats (Leonard et al., 1987); (2) is a tumor initiator and promoter in rat liver using the in vivo hepatic initiation-promotion assay (Leonard et al., 1983, 1986); (3) is mutagenic in bacteria and induces DNA damage and mutations in mammalian cells in culture (Rickert et al., 1984, as cited in AT SDR, 1998; Sayama etal., 1998). As stated in the EPA's cancer guidelines (U.S. EPA, 2005), one of the examples for a chemical to be considered to have suggestive evidence of carcinogenic potential is: "a small, and possibly not statistically significant, increase in tumor incidence observed in a single animal or human study that does not reach the weight of evidence for the descriptor 'Likely to Be Carcinogenic to HumansBased on these guidelines and the carcinogenicity data from available animal studies, the WOE descriptor of suggestive evidence of carcinogenic potential is appropriate for 2,6-DNT. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) indicates that for tumors occurring at a site other than the initial point of contact, the cancer 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 45 2,6-Dinitrotoluene ------- FINAL 4-10-2013 routes. Information available on the carcinogenic effects of 2,6-DNT demonstrates that tumors occur in tissues remote from the site of absorption. 2,6-DNT has been shown to be a hepatocarcinogen in rats in bioassays of various experimental designs by oral exposure. An excess of hepatobiliary cancer was found among munition workers exposed to dinitrotoluenes in which exposures are presumed to be predominantly inhalation with contributions from the dermal route. Information on the carcinogenic effects of 2,6-DNT via the dermal route in humans and animals is limited or absent. Data on the absorption of 2,6-DNT show that the chemical is readily absorbed via all routes of exposure, including oral, inhalation, and dermal. Therefore, based on the observance of liver tumors following oral exposure and absorption by all routes of exposure, it is assumed that an internal dose will be achieved regardless of the route of exposure. Therefore, 2,6-DNT is considered to have suggestive evidence of carcinogenic potential 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" (p. 1-10). 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" (p. 1-10). The potential mode of action for 2,6-DNT is unclear. Table 4A summarizes the studies examining genotoxicity (e.g., clastogenicity, mutagenicity) of 2,6-DNT. 2,6-DNT was shown to be both positive and negative for mutagenicity in S. typhimurium strains. 2,6-DNT was not mutagenic in mammalian cell systems (i.e., Chinese hamster ovary (CHO) cells and p388 mouse lymphoma cells). 2,6-DNT did not cause morphological transformations in Syrian hamster embryo cells but did induce chromosomal aberrations in human peripheral lymphocytes in vitro. 2,6-DNT also caused DNA damage in rats both in vitro (germ cells) and in vivo (liver) but was negative for DNA repair in rat and human hepatocytes. Assays of unscheduled DNA synthesis in rat spermatocytes and CHO cells showed a negative response under in vitro conditions. 2,6-DNT induced both chromosomal aberrations and DNA adducts in rats in vivo. Assays of unscheduled DNA synthesis were both positive and negative in rats. 2,6-DNT caused micronuclei formation in the liver of rats but not in the peripheral blood. Taken together, the available data do not provide a definitive conclusion regarding the mode-of-action for 2,6-DNT-induced carcinogenicity. Therefore, a detailed mode-of-action discussion for 2,6-DNT is precluded and a linear approach is applied as recommended by the U.S. EPA (2005). DERIVATION OF PROVISIONAL CANCER POTENCY VALUES Derivation of Provisional Oral Slope Factor (p-OSF) As noted in Table 7, EPA concluded that there is suggestive evidence of carcinogenic potential for 2,6-DNT. The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) state: "When there is suggestive evidence, the Agency generally would not attempt a dose-response assessment, as the nature of the data generally would not support one; however, when the evidence includes a well-conducted study, quantitative analyses may be useful for some purposes, for example, providing a sense of the magnitude and uncertainty of potential risks, ranking potential hazards, or setting research priorities. In each case, the rationale for the 46 2,6-Dinitrotoluene ------- FINAL 4-10-2013 quantitative analysis is explained, considering the uncertainty in the data and the suggestive nature of the weight of evidence. These analyses generally would not be considered Agency consensus estimates." In this case, although there are no epidemiologic studies that have evaluated the carcinogenicity of 2,6-DNT in humans, its carcinogenicity has been evaluated in studies in both rats and mice. As highlighted in Table 3, these studies indicate that there are differing results regarding the carcinogenic potential of 2,6-DNT. However, the study by Leonard et al. (1987) is a well-conducted study showing evidence of increased incidence of hepatic carcinomas in male rats at multiple treatment levels. The data from this study are adequate to support a quantitative cancer dose-response assessment. Considering these data and the uncertainty associated with the suggestive nature of the tumorigenic response, EPA concluded that quantitative analyses may be useful for providing a sense of the magnitude of potential carcinogenic risk. Based on the weight of evidence, a dose-response assessment of the carcinogenicity of 2,6-DNT is deemed appropriate. The study by Leonard et al. (1987) is selected as the principal study for deriving the p-OSF, with a cancer endpoint of hepatocellular carcinomas in male rats. This study is peer reviewed, examined a sufficient number of animals (28 per dose group), was well conducted, and was of sufficient duration (52 weeks). It was not stated whether the study was performed under GLP standards, but the study appears scientifically sound. Details are provided in the Carcinogenicity Studies section. The study by Goldsworthy et al. (1986) was not selected as the principal study because the reported carcinogenicity of 2,6-DNT was enhanced by the content of pectin in diet and was not solely related to pure 2,6-DNT. In this study, 2,6-DNT was provided in diets with varying pectin content, which is believed to promote or enhance 2,6-DNT-induced carcinogenesis. The hepatocellular carcinomas and neoplastic nodules were observed only in rats fed 2,6-DNT in diets high in pectin content (NIH-2,6-DNT), and, therefore, the reported liver tumor incidences cannot be used to derive a p-OSF for 2,6-DNT. The Stoner et al. (1984) study was not selected as the principal study because it was of insufficient duration (12 weeks) to determine carcinogenic effects. In the study by Leonard et al. (1987), 28 male F344 rats were fed 2,6-DNT (purity unknown) in the diet for 1 year, and the results were compared with an untreated control group of 28 rats. 2,6-DNT induced hepatocellular carcinomas in 100% (19/19) of the high-dose rats (14 mg/kg-day) and 85% (17/20) of the low-dose rats (7 mg/kg-day), compared to no incidence (0/20) in controls. Statistical significance tests conducted by the EPA indicated that incidence of hepatocellular carcinomas was statistically significant at both the high- and low-dose groups compared to controls. The dose-response data for hepatocellular carcinomas in male rats (see Table 8 and B.14) can be used to derive a p-OSF for 2,6-DNT. Statistical analyses performed for these data were done by Fisher's Exact test. Table 8 presents BMD input data for incidence of hepatocellular carcinomas in male rats exposed to 2,6-DNT orally for 1 year. The model result and BMD output text are provided in Appendix D. 47 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table 8. BMD Input for Incidence of Hepatocellular Carcinomas in the Male (F344) Crlbr Rat Exposed to 2,6-DNT Orally for 1 Year" Doscadj (mg/kg-day) Number of Subjects Response (Hepatocellular Carcinoma) 0 20 0 7 20 17* 14 19 19* 'Leonard et al. (1987). *p < 0.001 by Fisher's Exact Test performed by EPA. Table 9 shows the BMD modeling results. Adequate model fit is obtained for the hepatocellular carcinoma incidence data using the multistage-cancer model. The modeling results yield a BMDio of 2.7 mg/kg-day and a BMDLio of 0.25 mg/kg-day. This BMDLio was further converted from an animal dose to an HED and then used as the PODhed to derive the p-OSF for 2,6-DNT. Table 9. Goodness-of-Fit Statistics and BMDio and BMDLio Values for Dichotomous Model for Hepatocellular Carcinoma in the Male (F344)/Crlbr Rat Exposed to 2,6-DNT Orally for 1 Year" Multistage Cancer Goodness-of-fit BMD10 BMDL10 Model />-valucb AIC (mg/kg-day) (mg/kg-day) Hepatocellular 1.0 18.91 2.7 0.25 carcinoma aLeonard et al. (1987). bValues >0.1 meet conventional goodness-of-fit criteria. PODhed = BMDLio (mg/kg-day) x DAF = BMDLio (mg/kg-day) x (BWa1/4 - BWh1/4) = BMDLio (mg/kg-day) x (0.3761/4 - 701/4) = 0.25 mg/kg-day x 0.27 = 0.068 mg/kg-day Note: The BWa of 0.376 kg is the mean body weight from the low-dose male group at Week 104 (see Table B. 11). p-OSF = 0.1 ^ BMDLiohed = 0.1 0.068 mg/kg-day = 1.5 x 10° (mg/kg-day)-1 The p-OSF is 1.5 x 10° (mg/kg-day)_1, as calculated based on BMD modeling from Leonard et al. (1987). 48 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Derivation of Provisional Inhalation Unit Risk (p-IUR) No human and animal studies examining the carcinogenicity of 2,6-DNT following inhalation exposure are available located. Therefore, derivation of a p-IUR is precluded. 49 2,6-Dinitrotoluene ------- FINAL 4-10-2013 APPENDIX A. PROVISIONAL SCREENING VALUES For the reasons noted in the main PPRTV document, it is inappropriate to derive a provisional subchronic or chronic p-RfD for 2,6-DNT. However, information is available for this chemical which, although insufficient to support derivation of a provisional toxicity value, under current guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk Technical Support Center summarizes available information in an appendix and develops a "screening value." Appendices receive the same level of internal and external scientific peer review as the PPRTV documents to ensure their appropriateness within the limitations detailed in the document. Users of screening toxicity values in an appendix to a PPRTV assessment should understand that there is considerably more uncertainty associated with the derivation of an appendix screening toxicity value than for a value presented in the body of the assessment. Questions or concerns about the appropriate use of screening values should be directed to the Superfund Health Risk Technical Support Center. DERIVATION OF SCREENING PROVISIONAL ORAL REFERENCES DOSES Derivation of Screening Subchronic Provisional RfD (Subchronic p-RfD) The 13-week toxicity study in dogs (Lee et al., 1976c) is selected as the principal study for the derivation of the screening subchronic p-RfD. The critical effect is increased incidence of splenic extramedullary hematopoiesis in male and female dogs. No human studies are available on oral exposure to 2,6-DNT. One subchronic animal oral study is available (Lee et al., 1976), which, for the sake of clarity in this document, is divided into three separate study summaries based on the species tested: Lee et al. (1976a) in rats, (1976b) in mice, and (1976c) in dogs. Lee et al. (1976) is an unpublished and nonpeer-reviewed study. It is unclear if the study was performed according to Good Laboratory Practice (GLP) guidelines. The dog study (Lee et al. 1976c) is considered to have a small sample size as only one dog/sex/dose level were treated for 13 weeks in the control and low-dose group. At the mid-dose level, only two females and one male were treated for 13 weeks and two dogs/sex/dose level at the high dose. However, this study examined an adequate number of endpoints and has been previously used by both EPA and ATSDR to develop reference values (see Table 2) and is considered to be adequate for the derivation of screening oral reference values. Lee et al. (1976c) (dogs) is selected as the principal study because treatment-related effects observed in the dogs were more sensitive than effects observed in the rats (Lee et al. 1976a) and mice (Lee et al. 1976b). Study details are provided in the "Review of Potentially Relevant Data" section. The most sensitive treatment-related effect observed in the Lee et al. (1976c) study was increased incidence of splenic extramedullary hematopoiesis in male and female beagle dogs. Splenic extramedullary hematopoiesis was observed in every dog treated at 4 (2/2), 20 (3/3), and 100 (4/4) mg/kg-day for 13 weeks compared to zero incidence in the controls (see Table B.8). These data could not be modeled by Benchmark Dose Software (BMDS version 2.1.2) due to the lack of a dose-response. Because these data were not amenable to BMD modeling, a NOAEL/LOAEL approach was employed to identify a potential point of departure (POD). For increased incidence of splenic extramedullary hematopoiesis in male and female beagle dogs, there was an increase at the low-dose group, identifying a LOAEL of 4 mg/kg-day. A Fisher's exact test comparing splenic extramedullary hematopoiesis in the control and treated groups indicated a nonstatistically significant difference. However, group sizes were too small for the statistical test to have much power to detect an effect. 50 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Dogs are considered to be the most sensitive species to the toxicological effects of 2,6-DNT compared to rats and mice. For rats, the most sensitive potential POD is increased incidences of liver and splenic extramedullary hematopoiesis in male rats with a LOAEL of 35 mg/kg-day and a corresponding NOAEL of 7 mg/kg-day. In mice, decreased relative liver weight that was biologically significant (>10%) is the most sensitive effect with a LOAEL of 11 mg/kg-day. Effects in dogs, rats, and mice were modeled by BMDS (version 2.1.2) for consideration of a potential POD when data were amenable to BMD modeling. Details of the modeling methods are provided in Appendix C. Potential PODs for dogs, rats, and mice are listed in Table A.l. Of the toxicological effects observed in dogs, rats, and mice in the subchronic study by Lee et al. (1976), the most sensitive is increased incidence of splenic extramedullary hematopoiesis in male and female beagle dogs with a LOAEL of 4 mg/kg-day. The selection of increased incidence of splenic extramedullary hematopoiesis is supported by the observation that the severities of this effect increased with dose in dogs (see Table B.8). At 4 mg/kg-day in male and female dogs, the severity of splenic extramedullary hematopoiesis ranged from minimal to mild; at 20 mg/kg-day, the severity ranged from minimal to moderate; at 100 mg/kg-day, the severity ranged from marked to markedly severe. Furthermore, splenic extramedullary hematopoiesis was not present in male and female dogs that were treated with 2,6-DNT for 13 weeks and allowed to recover for 4 weeks, suggesting that this effect is treatment-related. Splenic extramedullary hematopoiesis was reported in rats gavaged with 2,6-DNT for 14 days at doses of 68 (1/6) and 134 (6/6) mg/kg-day, the incidence of this lesion in controls was not reported by the study authors (Lent et al., 2012a). Splenic extramedullary hematopoiesis was also observed in male and female dogs treated at 4 (1/2), 20 (2/2), and 100 (2/2) mg/kg-day for 4 weeks compared to zero incidence in controls. Splenic extramedullary hematopoiesis was also statistically significantly increased in both male and female rats treated for 13 weeks (see Tables B.4 and B.5). Further support for splenic extramedullary hematopoiesis as a critical effect following 2,6-DNT treatment is provided by hematological data in Tables B.3. 2,6-DNT statistically significantly increased reticulocytes (an indicator of hematopoiesis) in male and female dogs at 100 mg/kg-day following 2 and 4 weeks of treatment. Additionally, 2,6-DNT statistically significantly increased the amount of reticulocytes in male and female rats compared to controls at the highest dose tested following 4 weeks of exposure. Therefore, the LOAEL of 4 mg/kg-day based on increased incidence of splenic extramedullary hematopoiesis in male and female dogs is chosen as the POD to derive a screening subchronic p-RfD. It is important to note that the selection of the LOAEL of 4 mg/kg-day for increased incidence of splenic extramedullary hematopoiesis in male and female beagle dogs as the POD would also be protective against the 2,6-DNT-induced mortality that was observed in mice and dogs. In mice, 8 of 16 males at the high-dose (289 mg/kg-day) died before Weeks 9, and 6 of 8 females died at the high-dose (299 mg/kg-day) before the end of the study. In addition, 8 of 16 males and 1 of 16 females died at the mid-dose (51 and 55 mg/kg-day, respectively), and 2 of 16 males died at the low-dose (11 mg/kg-day). The study authors stated that in the mid- and high-dose groups, most of the deaths could be contributed to 2,6-DNT administration. These data suggest an FEL of 51 mg/kg-day for the mouse study (Lee et al. 1976b). In dogs, all animals (2 males and 2 females) in the high dose group (100 mg/kg-day) died between Weeks 2 and 8, and 2 of 3 dogs (both females) in the mid-dose group (20 mg/kg-day) died during Week 9. These data suggest an FEL of 20 mg/kg-day for the dog study (Lee et al. 1976c). 51 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table A.l. Potential Subchronic PODs in Animals Following 13 Weeks of Treatment to 2,6-DNT Effect Sex/Species NOAEL (mg/kg-day) LOAEL (mg/kg-day) BMDL10 (mg/kg-day) Comment Decreased Body Weight Males/Rats 7 35 13 Due to decreased palatability possibly due to chemical exposure Increased Relative Liver Weight Males/Rats Not determined 7 Not run No dose-response, effect most likely due to decreased body weight Increased Relative Spleen Weight Males/Rats 35 145 No fit Effect most likely due to decreased body weight Decreased Relative Kidney Weight Males/Rats Not determined 7 Not run No dose-response, effect reverses at higher doses Increased Relative Heart Weight Males/Rats 35 145 Not run No dose-response, effect most likely due to decreased body weight Increased Relative Brain Weight Males/Rats 35 145 No fit Not a valid toxicological endpoint Splenic Hemosiderosis Males/Rats Not determined 7 Not run No dose-response Splenic Hematopoiesis Males/Rats 7 35 Not run Data not suitable for BMD modeling Liver Hematopoiesis Males/Rats 7 35 Not run Data not suitable for BMD modeling Decreased Body Weight Females/ Rats 37 155 64 Due to decreased palatability possibly due to chemical exposure Increased Relative Liver Weight Females/ Rats Not determined 7 No fit Effect most likely due to decreased body weight Increased Relative Spleen Weight Females/ Rats 37 155 48 Effect most likely due to decreased body weight Increased Relative Kidney Weight Females/ Rats 7 37 Not run No dose-response, effect most likely due to decreased body weight Decreased Relative Heart Weight Females/ Rats 155 Not determined Not Run No dose-response Increased Relative Brain Weight Females/ Rats 7 37 3.1 Not a valid toxicological endpoint Splenic Hemosiderosis Females/ Rats 37 155 Not run Data not suitable for BMD modeling Splenic Hematopoiesis Females/ Rats 7 37 Not run Data not suitable for BMD modeling Liver Hematopoiesis Females/ Rats 37 155 Not run Data not suitable for BMD modeling 52 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table A.l. Potential Subchronic PODs in Animals Following 13 Weeks of Treatment to 2,6-DNT Effect Sex/Species NOAEL (mg/kg-day) LOAEL (mg/kg-day) BMDL10 (mg/kg-day) Comment Decreased Relative Liver Weight Males/Mice Not determined 11 No fit Decreased Relative Kidney Weight Males/Mice 11 51 Not run LOAEL is 10-fold higher than LOAEL for splenic extramedullary hematopoiesis in dogs Mortality Males/Mice 11 51 (FEL) Not run Mortality Females/ Mice 55 299 (FEL) Not run Liver Hematopoiesis Both/Dogs 4 20 Not run Data not suitable for BMD modeling Liver bile duct hyperplasia Both/Dogs 4 20 Not run Data not suitable for BMD modeling Liver degeneration Both/Dogs 4 20 Not run Data not suitable for BMD modeling Splenic Hematopoiesis Both/Dogs Not determined 4 Not run Data not suitable for BMD modeling Mortality Both/Dogs 4 20 (FEL) Not run 53 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Dosimetric adjustment for daily exposure: The following dosimetric adjustments were made for each dose in the principal study for dietary treatment to adjust for daily exposure. Dosimetric adjustment for 4 mg/kg-day is presented below. (DOSEadj) = DOSELeeetai., 1976c x [conversion to daily dose] = 4 mg/kg-day x (days of week dosed ^ 7) = 4 mg/kg-day x (7 -h 7) = 4 mg/kg-day In EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 201 lb), 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 PBPK model for 2,6-DNT is not available for use in extrapolating doses from animals to humans. The selected critical effect of splenic extramedullary hematopoiesis was associated with the parent compound or a stable metabolite. Furthermore, this splenic effect is not a portal-of-entry or developmental effect. Therefore, scaling by BW3 4 is relevant for deriving human equivalent doses (HEDs) for these effects. Following U.S. EPA (201 lb) guidance, the POD for splenic extramedullary hematopoiesis in adult animals is converted to a HED through 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 :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, 201 lb), rate-related processes scale across species in a manner related to both the direct (BW171) and allometric scaling (BW3'4) aspects such that BW3'4 ^ BW1'1 = BW ! converted to a DAF = BWa'74 - BWhI/4. 54 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Using a BWa of 12 kg for dogs and a BWh of 70 kg for humans (U.S. EPA, 1988), the resulting DAF is 0.63. Applying this DAF to the LOAEL identified for the critical effect in mature dogs yields a LOAELhed as follows: LOAELhed = 4 mg/kg-day x DAF = 4 mg/kg-day x 0.63 = 3 mg/kg-day The screening subchronic p-RfD for 2,6-DNT is derived as follows: Screening Subchronic p-RfD = LOAELhed + UFC = 3 mg/kg-day ^ 1,000 = 3 x 10~3 mg/kg-day The UFc of 1,000 is presented in Table A.2. Table A.2 summarizes the UFs for the screening subchronic p-RfD for 2,6-DNT. Confidence in the screening value is by definition, low. Table A.2. UFs for Screening Subchronic p-RfD of 2,6-DNTa UF Value Justification ufa 3 For the POD based on an increased incidence of splenic extramedullary hematopoiesis (Lee et al., 1976c), a UFA of 3 (100 5) has been applied to account for uncertainty in characterizing the toxicodynamic differences between dogs and humans following oral 2,6-DNT 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 Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 201 lb). ufd 10 A UFd of 10 has been applied because there are no acceptable two-generation reproductive toxicity or developmental toxicity studies via the oral route. UFh 10 A 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 2,6-DNT in humans. ufl 3 A UFl of 3 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a LOAEL based on splenic extramedullary hepatopoiesis, for which the biological significance is not entirely clear. UFS 1 A UFS of 1 has been applied because a subchronic-duration study was selected as the principal study. UFC 1,000 aLee et al. (1976c). Derivation of Screening Chronic Provisional RfD (Chronic p-RfD) The 13-week toxicity study in dogs (Lee et al., 1976c) is selected as the principal study for the derivation of the screening chronic p-RfD. The critical effect is increased incidence of splenic extramedullary hematopoiesis in male and female dogs. No human chronic studies are available for 2,6-DNT. There is an available chronic oral study in male rats by 55 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Leonard et al. (1987) that investigated the carcinogenic and noncancer effects of only 2,6-DNT on the liver. The carcinogenic effects are described in the "Derivation of Provisional Oral Slope Factor" section. Regarding the noncancer effects, Leonard et al. (1987) reported increased absolute and relative liver weight, decreased body weight, and increased ALT activity (only at 12 months) in male rats at both 6 and 12 months of exposure to 2,6-DNT. Decreased body weight cannot be considered as a critical effect because it is not clear if this effect is due to direct treatment with 2,6-DNT or to a reduction in food consumption. Decreased food consumption was observed in rats in the subchronic study by Lee et al. (1976a), suggesting that decreased body weight in rats reported in the Leonard et al. (1987) study could be due to reduced food consumption. Because there were statistically and biologically significant changes in body weight, changes in absolute organ weights are not considered for potential POD selection. The data for increased relative liver weight were analyzed by the EPA's BMDS (version 2.1.2) continuous-variable models. For increased relative liver weight at 6 months, a LOAEL of 7 mg/kg-day based on a biologically (>10% change) and statistically significant change is a potential POD. Following 12 months of treatment with 2,6-DNT, BMDS calculated a BMDLio of 0.69 mg/kg-day for increased relative liver weight. For increased ALT activity at 12 months, a LOAEL of 7 mg/kg-day based on a statistically significant change is a potential POD. Complete modeling methods and results are in presented Appendix C. The study authors also reported nonneoplastic lesions (e.g., bile duct hyperplasia, basophilic foci, etc) in the liver but presented no quantitative data for these effects that could be used to derive a chronic p-RfD. Based on the BMD modeling results, the most sensitive effect following chronic exposure to 2,6-DNT appears to be increased relative liver weight (BMDL = 0.69 mg/kg-day). However, the chronic study by Leonard et al. (1987) is not a comprehensive study and only reports carcinogenic and noncancer effects in the liver, as well as evaluation of pulmonary metastases. It is also unclear if the reported noncancer effects in the liver may be due to the hepatocarcinogenic effects of 2,6-DNT because increased relative liver weight and increased ALT activity were observed at the same doses (7 and 14 mg/kg-day) as hepatocellular carcinomas following 12 months of exposure. Whereas increased relative liver weight was also observed at 6 months, the study authors did not report pathology results for this time period so it is possible that hepatocellular carcinomas may have been present. From the subchronic-duration study by Lee et al. (1976), it is clear that the spleen is a target organ for 2,6-DNT toxicity; the most sensitive subchronic effect from that study was increased incidence of splenic extramedullary hematopoiesis in dogs with aNOAEL of 4 mg/kg-day, which is more sensitive than splenic and liver effects observed in rats. Because the chronic-duration study by Leonard et al. (1987) did not investigate splenic effects in any species, the sensitivity of spleen toxicity following chronic 2,6-DNT exposure is unknown. Therefore, to protect against potential splenic effects from chronic 2,6-DNT exposure, the LOAEL of 4 mg/kg-day based on increased incidence of splenic extramedullary hematopoiesis in dogs from the subchronic-duration study by Lee et al. (1976c) is used as the POD to derive a screening chronic p-RfD. For the same reasons listed in the screening subchronic p-RfD discussion above, the study by Lee et al. (1976c) meets standards of study design and performance. Details are provided in the "Review of Potentially Relevant Data" section. 56 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Dosimetric adjustment for daily exposure: The following dosimetric adjustments were made for each dose in the principal study for dietary treatment to adjust for daily exposure. Dosimetric adjustment for 4 mg/kg-day is presented below. (DOSEadj) = DOSELeeetai., 1976c x [conversion to daily dose] = 4 mg/kg-day x (days of week dosed ^ 7) = 4 mg/kg-day x (7 -h 7) = 4 mg/kg-day Following U.S. EPA (201 lb) guidance, the POD for splenic extramedullar hematopoiesis in adult animals is converted to a HED through 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 12 kg for dogs and a BWh of 70 kg for humans (U.S. EPA, 1988), the resulting DAF is 0.63. Applying this DAF to the LOAEL identified for the critical effect in mature dogs yields a LOAELhed as follows: LOAELhed = 4 mg/kg-day x DAF = 4 mg/kg-day x 0.63 = 3 mg/kg-day The screening chronic p-RfD for 2,6-DNT based on a LOAELhed of 3 mg/kg-day for splenic extramedullary hematopoiesis in male and female dogs, is derived as follows: Screening Chronic p-RfD - LOAELhed ~=~ UFc = 3 mg/kg-day ^ 10,000 = 3 x 10~4 mg/kg-day 57 2,6-Dinitrotoluene ------- FINAL 4-10-2013 The UFc of 10,000 is presented in Table A.3. Table A.3 summarizes the UFs for the screening chronic p-RfD for 2,6-DNT. Confidence in the screening value is by definition, low. Table A.3. UFs for Screening Chronic p-RfD of 2,6-DNTa UF Value Justification ufa 3 For the POD based on an increased incidence of splenic extramedullary hematopoiesis (Lee et al., 1976c), a UFA of 3 (10°5) has been applied to account for uncertainty in characterizing the toxicodynamic differences between dogs and humans following oral 2,6-DNT 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 Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 201 lb). ufd 10 A UFd of 10 has been applied because there are no acceptable two-generation reproductive toxicity or developmental toxicity studies via the oral route. UFh 10 A 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 2,6-DNT in humans. ufl 3 A UFl of 3 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a LOAEL based on splenic extramedullary hepatopoiesis, for which the biological significance is not entirely clear. UFS 10 A UFS of 10 has been applied to account for the extrapolation from less than chronic exposure. UFC 10,000 aLee et al. (1976c). 58 2,6-Dinitrotoluene ------- FINAL 4-10-2013 APPENDIX B. DATA TABLES Table B.l. Body Weight and Absolute and Relative Organ Weight in Male CD Rats Fed 2,6-DNT for 4 or 13 Weeks" Parameter Exposure Group, mg/kg-d 0 7 35 145 Male Rat—4 Wks Sample size 4 4 4 4 Body weightb (g) 391 ±8 364 ± 18 306 ± 9* 225 ± 52* Absolute liverb (g) 12.6 ±0.6 12.7 ±0.6 11.7 ± 0.5 9.2 ±0.6* Relative liverb (g organ/100 g body wt) 3.2 ±0.1 3.5 ±0.3 3.8 ±0.1 4.1 ±0.2* Absolute spleenb (g) 0.81 ±0.03 0.72 ±0.03 0.64 ±0.03 0.97 ±0.09 Relative spleenb (g organ/100 g body wt) 0.21 ±0.01 0.20 ± 0.02 0.21 ±0.01 0.43 ±0.03* Absolute kidneys'3 (g) 3.26 ±0.08 3.01 ±0.12 3.04 ±0.21 2.29 ±0.20* Relative kidneys'3 (g organ/100 g body wt) 0.83 ±0.01 0.83 ±0.05 0.97 ±0.05 0.02 ±0.08 Absolute heartb (g) 1.34 ±0.10 1.26 ±0.03 1.06 ±0.02* 0.79 ±0.05* Relative heartb (g organ/100 g body wt) 0.34 ±0.02 0.35 ±0.02 0.35 ±0.00 0.35 ±0.02 Absolute brainb (g) 2.08 ±0.04 2.02 ±0.09 2.05 ±0.04 1.94 ±0.07 Relative brainb (g organ/ 100 g body wt) 0.53 ±0.02 0.56 ±0.03 0.67 ±0.02* 0.86 ±0.02* Male Rat—13Wks Sample size 4 4 4 4 Body weightb (g) 545 ± 21 486 ± 24 451± 13* 256±12* Absolute liverb (g) 15.4 ±0.5 17.0 ± 1.3 10.7 ±0.2* 8.9 ±0.3* Relative liverb (g organ/100 g body wt) 2.8 ±0.0 3.5 ±0.3* 2.4 ±0.1 3.5 ± 0.1* Absolute spleenb (g) 0.95 ±0.06 1.02 ±0.03 0.90 ±0.10 0.65 ± 0.02* Relative spleenb (g organ/100 g body wt) 0.17 ±0.00 0.21 ±0.01 0.20 ±0.02 0.26 ± 0.02* Absolute kidneys'3 (g) 3.09 ±0.10 2.33 ±0.18 3.05 ±0.02 2.11 ±0.07 Relative kidneysb (g organ/100 g body wt) 0.57 ±0.03 0.48 ±0.03 0.68 ±0.02 0.83 ± 0.06* Absolute heartb (g) 1.56 ±0.09 1.60 ±0.15 1.27 ±0.01 0.99 ±0.08* Relative heartb (g organ/100 g body wt) 0.29 ±0.01 0.33 ±0.03 0.29 ±0.00 0.39 ±0.02* 59 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.l. Body Weight and Absolute and Relative Organ Weight in Male CD Rats Fed 2,6-DNT for 4 or 13 Weeks" Parameter Exposure Group, mg/kg-d 0 7 35 145 Absolute brainb (g) 2.09 ±0.10 2.17 ±0.07 2.18 ± 0.07 1.97 ±0.05 Relative brainb (g organ/ 100 g body wt) 0.39 ±0.03 0.45 ± 0.02 0.48 ±0.00 0.78 ±0.05* aLee etal. (1976a). bMeans ± SE. Significantly different from corresponding control values at p< 0.05 (Dunnett's multiple comparison procedure). 60 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.2. Body Weight and Absolute and Relative Organ Weight in Female CD Rats Fed 2,6-DNT for 4 or 13 Weeks" Parameter Exposure Group, mg/kg-d 0 7 37 155 Female Rat—4 Wks Sample size 4 4 4 4 Body weightb (g) 232 ±5 210 ±7 194 ± 8* 157±10* Absolute liverb (g) 6.9 ±0.3 6.5 ±0.3 7.1 ±0.2 6.0 ±0.6* Relative liverb (g organ/100 g body wt) 3.0 ±0.1 3.1 ±0.2 3.7±0.1* 4.2 ±0.2* Absolute spleenb (g) 0.51 ±0.04 0.54 ±0.04 0.70 ±0.08 0.46 ± 0.02 Relative spleenb (g organ/100 g body wt) 0.22 ±0.02 0.26 ± 0.02 0.36 ±0.05* 0.29 ±0.02 Absolute kidneys'3 (g) 1.78 ±0.04 1.62 ±0.07 1.58 ± 0.10 1.44 ±0.10* Relative kidneysb (g organ/100 g body wt) 0.77 ±0.03 0.77 ±0.02 0.81 ±0.04 0.91 ±0.03* Absolute heartb (g) 0.92 ±0.08 0.86 ±0.07 0.75 ±0.04 0.58 ±0.07* Relative heartb (g organ/100 g body wt) 0.40 ± 0.04 0.41 ±0.04 0.39 ±0.03 0.37 ±0.04 Absolute brainb (g) 1.88 ±0.05 1.73 ±0.03 1.82 ±0.07* 1.86 ±0.06 Relative brainb (g organ/ 100 g body wt) 0.81 ±0.01 0.83 ± 0.02 0.94 ±0.04 1.20 ±0.07* Female Rat—13Wks Sample size 4 4 4 4 Body weightb (g) 286 ±11 270 ±8 214 ± 17* 176 ±9* Absolute liverb (g) 7.9 ±0.2 8.3 ±0.6 7.1 ±0.4 7.2 ±0.2 Relative liverb (g organ/100 g body wt) 2.8 ±0.0 3.1 ±0.2 3.4 ±0.3 4.1 ±0.1* Absolute spleenb (g) 0.58 ±0.04 0.69 ±0.09 0.54 ±0.05 0.57 ±0.06 Relative spleenb (g organ/100 g body wt) 0.20 ±0.01 0.25 ± 0.03 0.26 ±0.04 0.32 ±0.02* Absolute kidneysb (g) 1.65 ±0.15 1.93 ±0.07 1.71 ±0.04 1.60 ±0.08 Relative kidneysb (g organ/100 g body wt) 0.66 ±0.01 0.62 ±0.11 0.81 ±0.06 0.91 ±0.06* Absolute heartb (g) 0.94 ±0.03 0.86 ±0.06 0.87 ±0.06 0.66 ±0.05* Relative heartb (g organ/100 g body wt) 0.33 ±0.01 0.32 ±0.02 0.41 ±0.03 0.38 ±0.03 Absolute brainb (g) 1.88 ±0.06 2.08 ±0.07 2.00 ±0.04 1.86 ±0.06 Relative brainb (g organ/ 100 g body wt) 0.66 ±0.03 0.77 ±0.05 0.95 ±0.06* 1.06 ±0.03* aLee etal. (1976a). Vleans ± SE. Significantly different from corresponding control values at p< 0.05 (Dunnett's multiple comparison procedure). 61 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.3. Selected Hematology Parameters in the CD Rats Fed 2,6-DNT for 4 or 13 Weeks" Parameter Male Exposure Group, mg/kg-d 0 7 35 145 Male Rat—4 Wks Leukocytes'3 (xl03/MM3) 17.4 ± 1.7 19.5 ± 1.1 22.2 ± 1.2 31.5 ±2.0*'** Reticulocytes b (%) 1.39 ±0.23* 1.73 ±0.13* 0.85 ±0.09* 9.33 ± 1.27*" Erythrocytes'3 (x 10S/MM3) 7.15 ± 0.17 6.94 ±0.03 7.78 ±0.18* 5.80 ±0.35** Methemoglobinb (%) 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 Male Rat—13 Wks Leukocytes'3 (xl03/MM3) 20.7 ± 1.9 25.2 ±4.9 27.4 ±3.1* 25.0 ±4.0 Reticulocytes13 (%) 1.00 ±0.12* 0.85 ± 0.20* 0.81 ±0.16* 0.82 ±0.26 Erythrocytes'3 (x 106/MM3) 6.81 ±0.20 6.88 ±0.57 7.76 ±0.15* 7.40 ±0.14* Methemoglobinb (%) 0.6 ±0.6 0.0 ±0.0 0.0 ±0.0 3.0 ± 1.2* Parameter Female Exposure Group, mg/kg-d 0 7 37 155 Female Rat—4 Wks Leukocytes'3 (xl03/MM3) 19.3 ± 1.7 17.6 ± 1.6 22.7 ±2.1 24.0 ±3.1* Reticulocytes13 (%) 1.18 ±0.20 1.07 ±0.15* 0.85 ± 0.26* 5.24 ± 1.12** Erythrocytes'3 (xl06/MM3) 6.92 ±0.18 6.35 ±0.31 6.78 ±0.20 6.91 ±0.22 Methemoglobin b (%) 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.6 ±0.6 Female Rat—13 Wks Leukocytes'3 (xl03/MM3) 17.5 ±2.7 20.1 ±2.0 26.4 ±2.7* 20.5 ± 1.7 Reticulocytes13 (%) 1.38 ±0.16 1.24 ±0.40* 1.13 ±0.15* 0.89 ±0.10 Erythrocytes'3 (x 106/MM3) 6.49 ±0.44 6.35 ±0.28 6.44 ±0.24 7.06 ± 0.26 Methemoglobin13 (%) 0.6 ±0.6 0.0 ±0.0 0.0 ±0.0 2.4 ± 1.0* aLee etal. (1976a). Vleans ± SE. Significantly different from baseline (Dunnett's multiple comparison procedure). "Significantly different from the controls at the respective time interval (Dunnett's multiple comparison procedure). 62 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.4. Tissue Lesions in Male Rats Fed 2,6-DNT for 13 Weeks" Exposure group, mg/kg-d Lesion Typeb 0 7 35 145 Sample size 4 4 4 4 Heart Focal myocarditis lc 0 0 0 Trachea Tracheitis ld 0 0 0 Lung Chronic murine pneumonia lc 0 0 0 Submaxillary Salivary gland Sialadenitis 0 ld 0 0 Liver Focal mononuclear infiltration 2° 2° r r Extramedullary hematopoiesis 0 0 4°' * 4°' * Bile duct hyperplasia 0 0 3° 4°' * Focal degeneration 0 0 0 lc Kidney Dilatation of pelvis and/or tubules 0 0 0 2° Testis Focal atrophy ld ld 0 0 Degeneration, retardation of spermatogenesis 0 0 lc 0 Atrophy and aspermatogenesis 0 0 0 4d> * Spleen Hemosiderosis 0 4°' * 0 4^ * Extramedullary hematopoiesis 0 0 4°' * 4°' * Bone marrow M/E ratio - to 1.5 - - 1.3-1.5 "Leeetal. (1976a). bFour rats examined per dose group; data are expressed as number of rats exhibiting each type of lesion, except for bone marrow ratio in which the ratio in all four rats is provided as a range. °Lesions were graded by the author as "present, minimal, or mild." dLesions were graded by the author as "moderate, marked, or severe." p < 0.05 by Fisher's Exact Test performed by EPA. 63 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.5. Tissue Lesions in Female Rats Fed 2,6-DNT for 13 Weeks" Exposure group, mg/kg-d Lesion Typeb 0 7 37 155 Sample size 4 4 4 4 Liver Focal mononuclear 2° 2° 2° lc infiltration Extramedullary 0 0 2° 4°, * hematopoiesis Bile duct hyperplasia 0 0 3° 4°' * Focal degeneration 0 0 0 lc Kidney Microcalculi r 0 0 lc Focal mononuclear r 0 0 lc infiltration Uterus Infiltration of 2° 0 0 0 eosinophils Spleen Hemosiderosis 2° 2°, 2d 0 4d, * Extramedullary 0 0 4°' * 4°' * hematopoiesis Bone marrow M/E ratio - to 1.6 - - - to 1.5 aLee et al. (1976a). bFour rats examined per dose group; data are expressed as number of rats exhibiting each type of lesion, except for bone marrow ratio in which the ratio in all four rats is provided as a range. °Lesions were graded by the author as "present, minimal, or mild." dLesions were graded by the author as "moderate, marked, or severe." 64 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.6. Body Weight and Absolute and Relative Liver Weight in Male Albino Swiss Mice Fed 2,6-DNT for 13 Weeks" Parameter Exposure Group, mg/kg-d 0 11 51 289 Sample size 2 4 3 0 Body weightb (g) 27.5 ±2.5 32.5±1.6 31.3±2.2 NA Absolute liverb (g) 1.75 ±0.46 1.51 ±0.08 1.54 ±0.08 NA Relative liverb (g organ/100 g body wt) 6.3 ± 1.1 4.6 ±0.1 4.9 ±0.3 NA Absolute kidneysb (g) 0.87 ±0.01 1.01 ±0.10 0.54 ±0.06 NA Relative kidneys'3 (g organ/100 g body wt) 3.19 ±0.25 3.11 ±0.22 1.73 ±0.13* NA aLee etal. (1976b). bMeans ± SE. Significantly different from corresponding control values at p< 0.05 (Dunnett's multiple comparison procedure). NA=Not applicable. Table B.7. Mortality in Male Albino Swiss Mice Treated with 2,6-DNT in Diet for 4 or 13 Weeks Exposure Group, mg/kg-day No. of Animals No. of Mice Dying During Specified Weeksa'b 0-4 5-8 9-13 14-17 Total0 Males 0 16 0 0 3 0 3 11 16 2 0 0 0 2 51 16 6 0 1 1 8 289 16 0 6 2 0 8 Females 0 16 0 0 0 0 0 11 16 0 0 0 0 0 55 16 1 0 0 0 1 299 16 1 3 2 0 6 aNumber of dead animals. bNumber of animals includes mice treated for 4 and 13 weeks and necropsied and mice allowed to recover for 4 weeks. Calculated for this review from data reported in the study. Source: Lee et al. (1976b) 65 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.8. Tissue Lesions in Male and Female Dogs Treated with 2,6-DNT for 4 or 13 Weeks" Exposure group, mg/kg-d Lesion Typeb 0 4 20 100 4 weeks Sample Size 2 2 2 2 Lung Focal inflammation 2 0 1 1 Liver Extramedullary hematopoiesis 0 0 1 2 Bile duct hyperplasia 0 0 1 0 Degeneration 0 0 0 1 Subacute inflammation 0 0 1 1 Testis Degeneration and/or retardation of spermatogenesis 0 0 1 1 Ovary Several graafian follicles, but no corpea lutea 1 1 1 Thyroid Increase in parafollicular cells 0 1 2 1 Spleen Extramedullary hematopoiesis Lymphoid depletion 0 1 2 1 Tonsil Inflammation 0 1 0 1 Lymph Node Lymphoid degeneration and depletion 0 0 0 1 Thymus Involution Focal hyalinization of the corpuscles 0 0 0 1 1 Bone marrow M/E ratio 1.3 to 1.4 1.1 to 1.3 1.1 - to 0.9 13 weeks Sample size 2 2 3 4 Liver Extramedullary hematopoiesis 0 0 3 4 Bile duct hyperplasia 0 0 2 2 Focal degeneration 0 0 3 4 Kidney Dilatation of pelvis and/or tubules 0 0 0 2 Testis Atrophy and aspermatogenesis 0 0 0 2 Spleen Extramedullary hematopoiesis 0 2° 3d 4e Bone marrow M/E ratio 1.3 to 1.5 1.3 to 1.5 -to 1.3 0.9 to 1.0 "Leeetal. (1976c). bData are expressed as number of dogs exhibiting each type of lesion, except for bone marrow ratio in which the ratio in dogs is provided as a range. °Lesions were graded by the author as "minimal or mild." dLesions were graded by the author as "minimal, mild, or moderate." "Lesions were graded by the author as "marked or markedly severe." 66 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.9. Mortality in Male and Female Dogs Treated with 2,6-DNT in Diet for 13 Weeks Exposure Group, mg/kg-day No. of Animals No. of Dogs Dying During Specified Weeks" 0-4 5-8 9-13 Totalb Males/Females 0 2 0 0 0 0 4 2 0 0 0 0 20 3 0 0 2 2 100 4 0 4 0 4 aNumber of dead animals. Calculated for this review from data reported in the study. Source: Lee et al. (1976c). Table B.10. Reticulocyte Data in Dogs Treated with 2,6-DNT for up to 13 Weeks" Male/Female Exposure Group, mg/kg-day Parameter 0 4 20 100 2 Wks Reticulocytes b (%) 0.76 ± 0.07 1.10 ± 0.13 1.43 ±0.32 16.99 ±3.33*" 4 Wks Reticulocytes b (%) 0.59 ±0.08 0.98 ±0.10 1.67 ±0.43 6.23 ± 1.60" 8 Wks Reticulocytes13 (%) 1.01 ±0.24 0.88 ±0.08 0.84 ±0.30 No data 13 Wks Reticulocytes b (%) 0.56 ±0.11 0.62 ±0.0 1.91 No data aLee et al. (1976c). bMeans ± SE. Significantly different from baseline (Dunnett's multiple comparison procedure). "Significantly different from the controls at the respective time interval (Dunnett's multiple comparison procedure). 67 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.ll. Body Weight, Liver Weight, and Serum Enzyme Activities in the Male (F344)/CrlBR Rat Exposed to Oral 2,6-DNT for 26 and 52 Weeks" Parameter Exposure Group, mg/kg-d 0 7 14 Male Rat—6 Mo Sample size 4 4 4 Terminal body wtb (g) 395 ±2 376 ±9 316 ±9* Liver wtb (g) 9.62 ±0.25 10.52 ±0.47 11.42 ±0.59* Liver/body wt x 100b 2.43 ± 0.07 2.80 ±0.07* 3.62 ±0.08* Serum activity ALTb (IU/liter) 69 ±5 58 ±3 48 ±5 GGTb (IU/liter) 0.2 ±0.2 1.2 ±0.5 3.7 ±0.6* Male Rat—12 Mo Sample size 20 20 20 Terminal body wtb (g) 434 ±3 356 ±5* 297 ± 7* Liver wtb (g) 10.30 ±0.16 21.09 ±0.74* 38.20 ±2.14* Liver/body wt x 100b 2.38 ±0.04 5.99 ±0.29* 13.19 ±0.97* Serum activity ALTb (IU/liter) 133 ± 14 230 ±75* 1,044 ± 163* GGTb (IU/liter) 3.8 ±0.5 43 ± 18 205 ± 46* "Leonard et al. (1987). bMeans ± SEM. Significantly different from corresponding control values at p< 0.05. 68 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.12. Treatment Protocol Adapted from Goldsworthy et al., 1986 Treatment Group" No of Animals Diet Concentration of 2,6-DNT in the Diet, mg/kg-db NIH 30 NIH-07 0 NIH-HD DNT 30 NIH-07 3-3.5 NIH-LD DNT 30 NIH-07 0.6-0.7 AIN 30 AIN-76A 0 AIN-HD DNT 30 AIN-76A 3-3.5 AIN-LD DNT 30 AIN-76A 0.6-0.7 AP 30 AIN-76A + 5% pectin 0 AP-HD DNT 30 AIN-76A + 5% pectin 3-3.5 AP-LD DNT 30 AIN-76A + 5% pectin 0.6-0.7 aThe diets used were NIH-07, an open formula cereal-based diet high in pectin content; AIN-76A, a purified pectin-free diet; or AP, which is A1N-76A supplemented with 5% pectin. These three diets served as the control diets for the addition of 2.6-DNT at either a high-dose (HD DNT) or low-dose (LD DNT). 'TIED are 0, 0.13-0.15, and 0.63-0.74 mg/kg-day for the control, low-dose, and high-dose, respectively. 69 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.13. Effect of the Control and DNT-Diets on the Fraction of Animals with Hepatic Foci Identified by Three Phenotypic Markers3 Treatment Groupb He jatic Phenotypic Markers GGT ATP G6P At 3 months NIH 2/10 6/10 5/10 NIH-LD DNT 6/10 10/10 9/10 NIH-HD DNT 9/10 10/10 8/10 AIN 0/10 4/10 1/10 AIN-LD DNT 0/10 7/10 5/10 AIN-HD DNT 0/10 7/10 7/10 AP 0/10 4/10 5/10 AP-LD DNT 0/10 8/10 7/10 AP-HD DNT 0/10 9/10 10/10 At 6 months NIH 2/10 5/10 5/10 NIH-LD DNT 9/10 8/10 10/10 NIH-HD DNT 8/10 10/10 10/10 AIN 0/10 4/10 3/10 AIN-LD DNT 0/10 7/10 4/10 AIN-HD DNT 3/10 10/10 10/10 AP 0/10 4/10 3/10 AP-LD DNT 0/10 9/10 6/10 AP-HD DNT 3/10 10/10 10/10 At 12 months NIH 10/10 10/10 9/10 NIH-LD DNT 10/10 10/10 10/10 NIH-HD DNT 10/10° 10/10° 10/10° AIN 0/10 9/10 6/10 AIN-LD DNT 5/10 10/10 8/10 AIN-HD DNT 10/10 10/10 10/10 AP 3/10 9/10 9/10 AP-LD DNT 7/10 10/10 10/10 AP-HD DNT 10/10 10/10 10/10 aGoldsworthy et al. (1986). bThe diets used were NIH-07, an open formula cereal-based diet high in pectin content; AIN-76A, a purified pectin-free diet; or AP, which is A1N-76A supplemented with 5% pectin. These three diets served as the control diets for the addition of 2,6-DNT at either a high-dose (HD DNT) or low-dose (LD DNT). °Livers exhibited multi foci, neoplastic nodules (6/10), and hepatocellular carcinomas (6/10). The presence of these lesions did not allow for the accurate quantitation of the foci in these livers. 70 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Table B.14. Incidence of Neoplastic Lesions and Metastases in a 1-Year Oral Study of 2,6-DNT in the Male (F344)/Crlbr Rata Lesion Type Exposure Group, mg/kg-d 0(0) 7 14 Neoplastic nodules 0/20 18/20* 15/19* Hepatocellular carcinoma:Trabecular 0/20 17/20* 19/19* Adenocarcinoma 0/20 1/20 0/19 Cholangiocarcinoma 0/20 2/20 0/19 Pulmonary metastases NA 3/20 11/19 aLeonard et al. (1987). NA = not applicable (no primary tumors present). *p < 0.001 by Fisher's Exact Test performed by EPA. 71 2,6-Dinitrotoluene ------- FINAL 4-10-2013 APPENDIX C. BMD OUTPUTS FOR Till SUBCHRONIC AND CHRONIC p-RfDs MODELING PROCEDURE FOR CONTINUOUS DATA The BMD modeling of continuous data was conducted with EPA's BMDS (version 2.1.2). For these data, all continuous models available within the software were fit using a BMR of 10% relative risk or 1 standard deviation. An adequate fit was judged based on the x goodness-of-fit p-value (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 in 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 screening subchronic and chronic p-RfD. 72 2,6-Dinitrotoluene ------- FINAL 4-10-2013 APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE ORAL SLOPE FACTOR 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 benchmark dose software (BMDS) 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: goodness-of-fit p-walue (p > 0.1), visual inspection of the dose-response curve, and 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 point of departure. In accordance with EPA (2000) guidance, BMDs and BMDLs associated with an extra risk of 10% are calculated. MODEL-FITTING RESULTS FOR HEPATOCELLULAR CARCINOMAS IN MALE F344 RATS (Leonard et al., 1987) Table B. 14 shows the dose-response data on hepatocellular tumors in male F344 rats administered 2,6-DNT via the diet for 12 months (Leonard et al., 1987). Modeling was performed according to the procedure outlined above using BMDS version 2.1.2 with parameter restrictions for rats based on the duration-adjusted animal doses shown in Table 3. Model predictions are shown in Table 9. For incidence of hepatocellular carcinomas in both male rats, the multistage-cancer model provided an adequate fit (goodness-of-fit p-w alue >0.1). The 3-degree polynomial model yielded a BMDio value of 2.7 mg/kg-day with an associated 95% lower confidence limit (BMDLio) of 0.25 mg/kg-day for male rats. The fit of the multistage-cancer models to the hepatocellular carcinoma incidence data for male rats is shown in Table 9 and Figure D. 1. 73 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Multistage Cancer Model with 0.95 Confidence Level Multistage Cancer Linear extrapolation BMD Lower Bound 1 0.8 0.6 0.4 0.2 0 EIMDL BMD 0 2 4 6 8 10 12 14 dose 09:29 01/31 2013 Figure D.l. Multistage Cancer Model for Hepatocellular Carcinomas in Male Rats (Leonard et al., 1987) 74 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Text Output for Multistage Cancer Model for Hepatocellular Carcinomas in Male Rats (Leonard et al., 1987) Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:/Documents and Settings/JKaiser/Desktop/modeling results/msc_2 6dnt_hcar_m_Msc3-BMR10. (d) Gnuplot Plotting File: C:/Documents and Settings/JKaiser/Desktop/modeling results/msc_2 6dnt_hcar_m_Msc3-BMR10.pit Thu Jan 31 09:29:24 2013 BMDS Model Run Observation # < parameter # for Multistage Cancer model. The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -betal*dose/sl-beta2*dose/s2-beta3* doseA3)] The parameter betas are restricted to be positive 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 = 4 Total number of specified parameters = 0 Degree of polynomial = 3 Maximum number of iterations = 250 Relative Function Convergence has been set to: le-008 Parameter Convergence has been set to: le-008 Default Initial Parameter Values Background = 0 Beta(l) = 0 Beta(2) = 0 Beta(3) = 3.92465e+016 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -Background -Beta(l) -Beta(2) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix ) Beta(3) Beta (3) 1 Parameter Estimates 75 2,6-Dinitrotoluene ------- FINAL 4-10-2013 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0 k k k Beta(1) 0 k k k Beta(2) 0 k k k Beta(3) 0.00553091 -k k k * - Indicates that this value is not calculated. Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -8.45418 3 Fitted model -8.45419 1 9.74156e-006 2 1 Reduced model -39.4517 1 61.995 2 <.0001 AIC: 18.9084 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual 0.0000 0.0000 0.000 0.000 20 -0.000 7.0000 0.8500 17.000 17.000 20 0. 000 14.0000 1.0000 19.000 19.000 19 0. 002 Chi^2 = 0.00 d.f. = 2 P-value = 1.0000 Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 2.67071 BMDL = 0.25 04 02 BMDU = 3.13339 Taken together, (0.250402, 3.13339) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 0.399358 76 2,6-Dinitrotoluene ------- FINAL 4-10-2013 APPENDIX E. REFERENCES Abernethy, DJ; Couch, DB. (1982) Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese hamster ovary cells. MutatRes 103:53-59. 700010 ATSDR (Agency for Toxic Substances and Disease Registry). (1998) Toxicological profile for 2,4-dinitrotoluene and 2,6-dinitrotoluene. U.S. Department of Health and Human Services, Public Health Service, Atlanta, GA. Available online at http://www.atsdr.cdc.gov/ToxProfiles/tp 109.pdf. Accessed on November 21, 2012. 595415 Bermudez, E; Tillery, D; Butterworth, BE. (1979) The effect of 2,4-diaminotoluene and isomers of dinitrotoluene on unscheduled DNA synthesis in primary rat hepatocytes. Environ Mot Mutagen 1 (4):391-398. 062838 CalEPA (California Environmental Protection Agency). (2009) Hot spots unit risk and cancer potency values. Appendix A. Office of Environmental Health Hazard Assessment. Available online at http://www.oehha.ca.gov/air/hot spots/pdf/CPFs042909.pdf. Accessed on September 13, 2012. 684164 Cal EPA (California Environmental Protection Agency). (2012a) All OEHHA acute, 8-hour and chronic reference exposure levels (chRELs) as on February 2012. Office of Environmental Health Hazard Assessment, Sacramento, CA. Available online at http://www.oehha.ca.gov/air/allrels.html. Accessed on August 1, 2012. 1259515 Cal EPA (California Environmental Protection Agency). (2012b) OEHHA toxicity criteria database. Office of Environmental Health Hazard Assessment, Sacramento, CA. Available online at http://www.oehha.ca.gov/tcdb. Accessed on August 1, 2012. 783987 Chadwick, RW; George, SE; Kohan, MJ; et al. (1993) Potentiation of 2,6-dinitrotoluene genotoxicity in Fischer-344 rats by pretreatment with Aroclor 1254. Toxicology 80(2-3): 153-171. 699985 Chapman, DE; Michener, SR; Powis, G. (1992) Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions. Xenobiotica 22(8): 1015-1028. 699982 Chapman, DE; Michener, SR; Powis, G. (1993) In vitro metabolism of [(3)H]2,6-dinitrotoluene by human and rat liver. Toxicol in Vitro 7(3 ):213-220. 699986 Couch, DB; Allen, PF; Abernethy, DJ. (1981) Mutagenicity of dinitrotoluenes in salmonella typhimurium. Mutat Res 90(4):373-383. 700009 Dellarco, VL; Prival, MJ. (1989) Mutagencity of nitro compiounds in salmonella typhimurium in the presence of flavin mononucleotide in a preincubation assay. Environ MolMutagen 13(2): 1 16-127. 674985 Dixit, R; Schut, HA; Klaunig, JE; et al. (1986) Metabolism And DNA binding of 2,6-dinitrotoluene in Fischer-344 rats and a mice. Toxicol Appl Pharmacol 82(1):53—61. 699984 77 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Einistoe, P; Watanabe, M; Ishidate, M. Jr; et al. (1991) Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or (9-acety 1 transferase activities. Mutat Res 259:95-102. 700008 Ellis, H.V. Ill, J.H. Hagensen, J.R. Hodgson, J.L. Minor and C.B. Hong. 1979. Mammalian Toxicity of Munitions Compounds. Phase III. Effects of Lifetime Exposure. Part I. 2,4- Dinitrotoluene. Report Order No. AD-A077692. p. 281. 664214 Garrett, NE; Lewtas, J. (1983) Cellular toxicity in Chinese hamster ovary cell cultures. 1. Analysis of cytotoxicity endpoints for twenty-nine priority pollutants. Environ Res 32(2):455-465. 626038 George, SE; Kohan, MJ; Warren, SH. (1996) Hepatic DNA adducts and production of mutagenic urine in 2,6-dinitrotoluene-treated B6C3F1 male mice. Cancer Lett 102(1-2): 107-1 11. 700077 Geroge, SE; Huggins-Clark, G; Brooks, LR. (2001) Use of a Salmonella microsuspension bioassy to detect the mutagenicity of munitions compounds at low concentrations. Mutat Res 490( 1 ):45-56. 630084 Goldsworthy, TL; Hamm, TE, Jr.; Rickert, DE; et al. (1986) The effect of diet on 2,6-dinitrotoluene hepatocarcinogenesis. Carcinogenesis 7( 1 1): 1909-1915. 662284 Hagiwara, Y; Watanabe, Ml, Oda Y; et al. (1993) Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possessing elevated levels of both nitroreductase and acetyltransferase activity. Mutat Res 291(3): 171-180. 699994 Holen, I; Mikalsen, SO; Sanner, T. (1990) Effects of dinitrotoluenes on morphological cell transformation and intercellular communication in Syrian hamster embryo cells. J Toxicol Environ Health 29( 1 ):89-98. 700120 Huang, Q; Wang, L; Han, S. (1995) The genotoxicity of substituted nitrobenzenes and the quantitative structure-activity relationship studies. Chemosphere 30(5):915-923. 629200 IARC (International Agency for Research on Cancer). (1996) 2,4-dinitrotoluene, 2,6-dinitrotoluene, and 3,5-dinitrotoluene. In: Printing processes and printing inks, carbon black and some nitro compounds. IARC monographs on the evaluation of carcinogenic risks to humans, Volume 65. Lyon, France: IARC, p. 309-368. Available online at http://monographs.iarc.fr/ENG/Monographs/vol65/mono65-9.pdf. Accessed on September 13, 2012. 700130 Jones, CR; Liu, YY; Sepai, O; et al. (2005) Hemoglobin adducts in workers exposed to nitrotoluenes. Carcinogenesis 26(1): 133-143. 662333 Kedderis, GL; Dyroff, MC; Ricket, DE. (1984) Hepatic macromolecular covalent binding of the hepatocarcinogen 2,6-dinitrotoluene and its 2,4-isomer in vivo: modulation by the sulfotransferase inhibitors pentachlorophenol and 2,6-dichloro-4-nitrophenol. Carcinogenesis 5(9): 1 199-1204. 700067 78 2,6-Dinitrotoluene ------- FINAL 4-10-2013 La, DK; Froines, JR. (1993) Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene. MutatRes 301(2):79-85. 700007 Lee, CC; Ellis, HV; Kowalski, JJ; Hodgson, JR; Short, RD. (1976a,b,c) Mammalian toxicity of munitions compounds. Phase II: effects of multiple doses. Part III: 2,6-dinitrotoluene. Progress Report No. 4. Midwest Research Institute Project No. 3900-B. 711514 Lent, EM; Crouse, LCB; Quinn, MJ; Wallace, SW. (2012a) Comparison of the repeated dose toxicity of isomers of dinitrotoluene. Int J Toxicol 3 1(2): 143-57. 1466112 Lent, EM; Crouse, LCB; Quinn, MJ; Wallace, SW. (2012b) Assessment of the in vivo genotoxicity of isomers of dinitrotoluenes using the alkaline Comet and peripheral blood micronucleus assay. Mutat Res 742(l-2):54-60. 1005142 Leonard, TB; Lyght, O; Popp, JA. (1983) Dinitrotoluene structure-dependent initiation of hepatocytes in vivo. Carcinogenesis 4(8): 1059-1061. 700073 Leonard, TB; Adams, T; Popp, JA. (1986) Dinitrotoluene isomer-specific enhancement of the expression of diethylnitrosamine-initiated hepatocyte foci. Carcinogenesis 7(11): 1797-1803. 662391 Leonard, TB; Graichen, ME; Popp, JA. (1987) Dinitrotoluene isomer-specific epatocarcinogenesis in F344 rats. J Natl Cancer Inst 79(6): 13 13-13 19. 062891 Mirsalis, JC; Butterworth, BE. (1982) Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene. Carcinogenesis 3(3):241-245. 062892 Mori, MA; Shoji, M; Dohrin, M; et al. (1996) Further studies on the urinary metabolites of 2,4-dinitrotoluene and 2,6-dinitrotoluene in the male Wistar rat. Xenobiotica 26(l):79-88. 699981 NIOSH (National Institute for Occupational Safety and Health). (2007) NIOSH pocket guide to chemical hazards. Department of Health and Human Services, National Institute for Occupational Safety and Health, Cincinnati, OH; DHHS (NIOSH) publication no. 2005-149. Available online at http://www.cdc.eov/niosh/docs/2005-149/pdfs/2005-149.pdf. Accessed September 13, 2012. 192177 NLM (National Library of Medicine). (2011) Hazardous Substances Data Bank (HSDB). Available online at http://toxnet.nlm.nih.gov. Accessed on September 13, 2012. 783990 NTP (National Toxicology Program). (2011) 12th Report on carcinogens. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Available online at http://ntp.niehs.nih.gov/ntp/roc/twelfth/rocl2. Accessed on September 13, 2012. 737606 79 2,6-Dinitrotoluene ------- FINAL 4-10-2013 OECD (Organisation for Economic Cooperation and Development). (2004) Dinitrotoluene (isomers mixture) CAS No 25321-14-6, SIDS (Screening Information Dataset). UNEP (United Nations Environment Programme). Available online at: http://www.inchem.org/documents/sids/sids/2532 L146 pdf. Accessed on November 8, 2011. 700137 ORNL (Oak Ridge National Laboratory). (1995) Toxicity summary for 2,6-dinitrotoluene. Prepared by Rosmarie A. Faust, Health Sciences Research Division, Oak Ridge National Laboratory. Oak Ridge, TN. Available online at http://cira.ornl.gov/documents/26DNT.pdf Accessed on November 8, 201 1. 700131 OSHA (Occupational Safety and Health Administration). (2006) Table Z-l limits for air contaminants: occupational safety and health standards, subpart Z, toxic and hazardous substances. U.S. Department of Labor, Washington, DC; OSHA Standard 1910.1000. Available online at http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=9992. Accessed on September 13, 2012. 670067 Padda, RS; Wang, C; Hughes, JB; et al. (2003) Mutagenicity of nitroaromatic degradation compounds. Environ Toxicol Chem 22(10):2293-2297. 180178 Rickert, DE, Schnell, SR; Long, RM. (1983) Hepatic macromolecular covalent binding and intestinal disposition of [14c]dinitrotoluenes. J Toxicol Environ Health 11 (4—6):555—567. 062896 Sayama, M; Mori, MA; Shirokawa, T; et al. (1989) Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in salmonella typhimurium. MutatRes 226(3): 181-184. 700005 Sayama, M; Inoue, M; Mori, MA; et al. (1992) Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds. Xenobiotica 22(6):633-640. 699980 Sayama, M; Mori, M; Shoji, M; et al. (1998) Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase - and o-acetyltransferase-overproducing Ames test strains. Mutat Res 420( 1 —3):27—32. 631222 Simmon, VF; Kauhanen, K; Tardiff, RG. (1977) Mutagenic activity of chemicals identified indrinking water. In: Scott, D; Bridges, BA; Sobels, FH; eds, Progress in Genetic Toxicology,Vol. 2, Development in Toxicology and Environmental Science, Amsterdam: Elsevier; pp. 249-258. 029451 Spanggord, RJ; Mortelmans, KE; Griffin, AF; et al. (1982) Mutagenicity in salmonella typhimurium and structure-activity relationships of wastewater components emanating from the manufacture of trinitrotoluene. Environ Mutagen 4(2): 163-179. 074995 Stoner, GD; Greisiger, EA; Schut, HAJ; et al. (1984) A comparison of the lung adenoma response in strain A/J mice after intraperitoneal and oral administration of carcinogens. Toxicol App! Pharmacol 72(2):3 13-323. 662574 80 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Styles, JA; Cross, MF. (1983) Activity of 2,4,6-trinitrotoluene in an in vitro mammalian gene mutation assay. Cancer Lett 20:103-108. 700079 Swenberg, JA; Rickert, DE; Baranyi, BL; et al. (1983) Cell specificity in DNA binding and repair of chemical carcinogens. Environ Health Per sped 49:155 -163. 700086 Takasawa, H; Suzuki, J; Ogawa, I; et al. (2010) Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS). MutatRes 698( 1 —2):30—37. 699997 Tokiwa, H; Nakagawa, R; Ohnishi, Y. (1981) Mutagenic assay of aromatic nitro compounds with Salmonella typhimurium. Mutat Res 91 (4—5):321 -325. 700001 U.S. EPA (Environmental Protection Agency). (1986) Guidelines for carcinogen risk assessment. Risk Assessment Forum, Washington, DC; EPA/630/R-00/004. September 1986. Available online at http://epa.gov/raf/publications/pdfs/CA%20GUlPELINES 1986.PDF. 199530 U.S. EPA (Environmental Protection Agency). (1987) Health effects assessment for 2,4- and 2,6-Dinitrotoluene. Prepared by the Office of Research and Development, Environmental Criteria and Assessment Office, U.S. Environmental Protection Agency. Cincinnati, OH. Report No. EPA/600/8-88/032. Available online at nepis.epa.goy/Exe/ZvPURL.cgi?Dockey=2000T88F.txt. Accessed on September 13, 2012. 700134 U.S. EPA (Environmental Protection Agency). (1988) Recommendations for and documentation of biological values for use in risk assessment. Environmental Criteria and Assessment Office, Cincinnati, OH; EPA/600/6-87/008. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=34855#Download. 064560 U.S. EPA (U.S. Environmental Protection Agency). (1990) Integrated risk information system (IRIS) for 2,4-/2,6-dinitrotoluene mixture; no CASRN. Office of Research and Development, National Center for Environmental Assessment, Washington, DC. Available online at http://www.epa.gov/iris/subst/0397.htm. Accessed on September 13, 2012. 700136 U.S. EPA (Environmental Protection Agency). (1993) Integrated risk information system (IRIS) for 2,4-dinitrotoluene (CASRN 121-14-2). Office of Research and Development, National Center for Environmental Assessment, Washington, DC. Available online at http://www.epa.gov/iris/subst/ ten. Accessed on September 13, 2012. 1259516 U.S. EPA (Environmental Protection Agency). (1994) Chemical assessments and related activities (CARA). Office of Health and Environmental Assessment, Washington, DC; EPA/600/R-94/904. Available online at nepis.epa.goy/Exe/ZyPURL.cgi?Dockey=60Q0lG8L.txt. Accessed on September 13, 2012. 596444 81 2,6-Dinitrotoluene ------- FINAL 4-10-2013 U.S. EPA (Environmental Protection Agency). (2000) Benchmark dose technical guidance document [external review draft]. Risk Assessment Forum, Washington, DC; EPA/630/R-00/001. Available online at http://www.epa.gov/nceawwwl/pdfs/bmds/BMD- External 10 13 2000.pdf. 052150 U.S. EPA (Environmental Protection Agency). (2002) A Review of the reference dose and reference concentration process. Final report. Risk Assessment Forum, Washington, DC; EPA/630/P-02/002F. Available online at http://www.epa.gov/raf/publications/pdfs/rfd-final.pdf. 088824 U.S. EPA (Environmental Protection Agency). (2003) Health effects assessment summary tables (HEAST). Prepared by the Office of Research and Development, National Center for Environmental Assessment, Cincinnati OH for the Office of Emergency and Remedial Response, Washington, DC. Available online at http://epa-heast.ornl.gov/. Accessed on Accessed on September 13, 2012. 595422 U.S. EPA (U.S. Environmental Protection Agency). (2004) Provisional peer reviewed toxicity values for 2,6-dinitrotoluene (CASRN 606-20-2) Prepared by the Superfund Health Risk Technical Support Center, National Center for Environmental Assessment, U.S. Environmental Protection Agency. Cincinnati, OH. Available online at http://hhpprtv.ornl.gov/issue papers/Pinitrotoluene26.pdf. Accessed on February 6, 2013. 700135 U.S. EPA (Environmental Protection Agency). (2005) Guidelines for carcinogen risk assessment. Risk Assessment Forum, Washington, DC; EPA/630/P-03/001F. Federal Register 70(66): 17765-17817. Available online at http://www.epa.gov/raf/publications/pdfs/CANCER GUIDELINES FINAL 3-25-05.PDF. 086237 U.S. EPA (Environmental Protection Agency). (201 la) 2011 Edition of the drinking water standards and health advisories. Office of Water, Washington, DC; EPA/820/R-11/002. Available online at http://water.epa.gov/action/advisories/drinking/upload/dwstandards2011.pdf. Accessed on September 13, 2012. 783978 U.S. EPA (Environmental Protection Agency). (201 lb) Recommended use of body weight3 4 as the default method in derivation of the oral reference dose. Office of the Science Advisor, Risk Assessment Forum, Washington, DC; EPA/1000/R-l 1/0001. Available online at http://www.epa.gov/raf/publications/pdfs/recomm.ended-use-of-bw34.pdf. 752972 U.S. EPA (U.S. Environmental Protection Agency). (2013) Provisional peer reviewed toxicity values for technical grade dinitrotoluene (CASRN 25321-14-6) Prepared by the Superfund Health Risk Technical Support Center, National Center for Environmental Assessment, U.S. Environmental Protection Agency. Cincinnati, OH. Accessed on April 3, 2013. 1519123 WHO (World Health Organization). (2012) Online catalogs for the Environmental Health Criteria series. Available online at http://www.who.int/ipcs/publications/ehc/en/. Accessed on Accessed on August 1, 2012. 783977 Whong, WZ; Edwards, GS. (1984) Genotoxic activity of nitroaromatic explosives and related compounds in Salmonella typhimurium. MutatRes 136(3):209-215. 700000 82 2,6-Dinitrotoluene ------- FINAL 4-10-2013 Working, PK; Butterworth, BE. (1984) An assay to detect chemically induced DNA repair in rat spermatocytes. Environ Mutagen 6:273-286. 700105 Yang, L; Xu, J; Zhen, L; et al. (2005) DNA damage of germ cell of rat induced by nitrotoluene chemicals. J Environ Sci 17(1): 84-90. 662653 83 2,6-Dinitrotoluene ------- |