# FINAL Report on Carcinogens Background Document for



# o -Nitrotoluene

June 20, 2008

U.S. Department of Health and Human Services Public Health Services National Toxicology Program Research Triangle Park, NC 27709This Page Intentionally Left BlankRoC Background Document for o -Nitrotoluene

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### FOREWORD
The Report on Carcinogens (RoC) is prepared in response to Section 301 of the Public Health Service Act as amended. The RoC contains a list of identified substances (i) that either are known to be human carcinogens or are reasonably be anticipated to be human carcinogens and (ii) to which a significant number of persons residing in the United States are exposed. The Secretary, Department of Health and Human Services (HHS), has delegated responsibility for preparation of the RoC to the National Toxicology Program (NTP), which prepares the report with a ssistance from other Federal health and regulatory agencies and nongovernmental institutions. 1
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22 23 24 25 26 27 28 Nominations for (1) listing a new substance, (2) reclassifying the listing status for a substance already listed, or (3) removing a substance already listed in the RoC are reviewed in a multi-step, scientific review proces s with multiple opportunities for public comment. The scientific peer-review gr oups evaluate and make independent recommendations for each nomination according to specific RoC listing criteria. This background document was prepared to assist in the review of ortho -nitrotoluene. The scientific information used to prepare Sections 3 through 5 of this document must come from publicly available, peer-reviewed s ources. Information in Sections 1 and 2, including chemical and physical properties , analytical methods, production, use, and occurrence may come from published and/or unpublished sources. For each study cited in the background document from the peer-rev iewed literature, information on funding sources (if available) and the authors’ affiliations are provided in the reference section. The draft background document was peer reviewed in a public forum by an ad hoc expert panel of scientists from the public and pr ivate sectors with relevant expertise and knowledge selected by the NTP in accordance with the Federal Advisory Committee Act and HHS guidelines and regulations. This doc ument has been finalized based on the peerreview recommendations of the expert pane l and public comments received on the draft document. Any interpretive conclusions, commen ts, or statistical cal culations made by the authors or peer reviewers of this docum ent that are not contained in the original citation are identified in brackets [ ].RoC Background Document for o -Nitrotoluene

ii 6/20/08 A detailed description of the RoC nomination re view process and a list of all substances under consideration for listing in or delisti ng from the RoC can be obtained by accessing the 12 th RoC at 1
2 http://ntp.niehs.nih.gov/go/9732 . The most recent RoC, the 11th Edition (2004), is available at 3 http://ntp.niehs.nih.gov/go/19914

. 4RoC Background Document for o -Nitrotoluene

CONTRIBUTORS

Project Managers, Authors, and Principal Reviewers National Toxicology Program (NTP) and National Institute of Environmental Health Sciences (NIEHS) Ruth Lunn, Dr.P.H. Director, Report on Carcinogens Group C.W. Jameson, Ph.D. Report on Carcinogens Group (former Director) Leo T. Burka, Ph.D. Laboratory of Pharmacology and Chemistry June Dunnick, Ph.D. Toxicology Operations Branch Robert Sills, D.V.M., Ph.D. Labor atory of Experimental Pathology Constella Group, LLC (Support provided through NIEHS Contract Number NO1- ES-35505) Sanford Garner, Ph.D. Principal Investigator Stanley Atwood, M.S., D.A.B.T. Greg Carter, M.E.M. Susan Goldhaber, M.S. Consultants Douglas Rickert, Ph.D. Independent Consultant Administrative Support Shawn Jeter, B.S. NTP/Report on Carcinogens Group Anna Lee Sabella Kelly Services Ella Darden, B.S. Constella Group, LLC Tracy Saunders, B.S. Constella Group, LLC Editorial Support Susan Dakin, Ph.D. Independent Consultant in Technical & Scientific Writing & Editing

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iv 6/20/08 PEER REVIEW The draft background document on ortho -Nitrotoluene was peer reviewed by the Report on Carcinogens expert panel for Captafol and ortho -Nitrotoluene. The panel met in a public forum at the Sheraton Chapel Hill Hotel, Chapel Hill, NC on October 15–16, 2007. Members of the expert panel are as follows: Lauren Zeise, Ph.D. (Chair) California Environmental Protection Agency Office of Environmental Health Hazard Assessment Michael Elwell, D.V.M., Ph.D. Covance Laboratories, Inc. Department of Pathology Penelope A. Fenner-Crisp, Ph.D., D.A.B.T. Independent Consultant (Retired from the International Life Science Institute and the U.S. Environmental Protection Agency) Gregory L. Kedderis, Ph.D. Independent Consultant Steven Markowitz, M.D. Queens College, City University of New York Robert C. Millikan, D.V.M., Ph.D. University of North Carolina, School of Public Health, Department of Epidemiology Shane S. Que Hee, Ph.D. University of California, Los Angeles School of Public Health, Department of Environmental Health Sciences Thomas J. Slaga, Ph.D. University of Texas Health Science Center Department of Pharmacology Alexander W. Teass, Ph.D. (Retired from the National Institute of Occupational Safety & HealthRoC Background Document for o -Nitrotoluene
Criteria for Listing Agents, Substances or Mixtures in the Report on Carcinogens U.S. Department of Health and Human Services National Toxicology Program The criteria for listing an agent, substance, mixture, or exposure circumstance in the RoC are as follows:

Known To Be Human Carcinogen: There is sufficient evidence of carcinogenicity from studies in humans * , which indicates a causal relationship between expo sure to the agent, substance, or mixture, and human cancer. Reasonably Anticipated To Be Human Carcinogen: There is limited evidence of carcinogenicity from studies in humans * , which indicates that causal interpretation is credible, but that alternative explanations, such as chance, bias, or confounding factors, could not adequately be excluded,

or there is sufficient evidence of carcinogenicity from studies in experimental animals, which indicates there is an increased incide nce of malignant and/or a combination of malignant and benign tumors (1) in multiple sp ecies or at multiple tissue sites, or (2) by multiple routes of exposure, or (3) to an unusual degree with regard to incidence, site, or type of tumor, or age at onset,

or there is less than sufficient evidence of ca rcinogenicity in humans or laboratory animals; however, the agent, substance, or mixture belongs to a well-defined, structurally related class of substances whose members are listed in a previous Report on Carcinogens as either known to be a human carcinogen or reasonably anticipated to be a human carcinogen, or there is convincing relevant information that the agent acts through mechanisms i ndicating it would likely cause cancer in humans. Conclusions regarding carcinogenicity in hum ans or experimental animals are based on scientific judgment, with consideration gi ven to all relevant information. Relevant information includes, but is not limited to , dose response, route of exposure, chemical structure, metabolism, pharmacokinetics, sensiti ve sub-populations, genetic effects, or other data relating to mechanism of action or factors that may be unique to a given substance. For example, there may be substances for which there is evidence of carcinogenicity in laboratory animals, but there are compelling data indicati ng that the agent acts through mechanisms which do not operate in humans and would theref ore not reasonably be anticipated to cause cancer in humans.

* This evidence can include traditional cancer epidemiology studies, data from clinical studies, and/or data derived from the study of tissues or cells from humans exposed to the substance in question that can be useful for evaluating whether a relevant ca ncer mechanism is operating in people.

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## Executive Summary
Introduction 1

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22 23 24 25 26 27 o -Nitrotoluene is a nitro arom atic compound consisting of a benzene ring, with a methyl and a nitro group attached ortho to each other. It is one of three isomers of nitrotoluene, with m -nitrotoluene and p -nitrotoluene being the other two. o -Nitrotoluene is an important chemical intermediate used in the sy nthesis of dyes that are used in the textile, paper, and other industries and also in the synthesis of ag ricultural, rubber, and other chemicals.

o -Nitrotoluene was nominated for possible lis ting in the Report on Carcinogens based on the results of a 2002 National Toxicology Pr ogram (NTP) two-year feeding bioassay study demonstrating clear evidence of carcinogenicity in rats and mice. Human Exposure

o -Nitrotoluene is a chemical intermediate used in the synthesis of azo dyes. It is also used (either directly or as an intermediate) in the production of other dyes, agricultural chemicals, rubber chemicals, pesticides, petrochemicals, pharmaceuticals, and explosives. o -Nitrotoluene is produced principally by the nitration of toluene with a mixture of nitric acid and either sulf uric, aromatic sulfonic, or phosphoric acid. o Nitrotoluene is a high production volume (H PV) chemical, and its U.S. production was 10 to 50 million pounds per year for every f our-year reporting period from 1986 to 2002. Exposure to o- nitrotoluene in the United States is primarily a result of occupational exposure during the production a nd use of this chemical. Little information is available on environmental occurrence of o -nitrotoluene or on human exposure. The compound has been detected in the ambient air in Idaho and at U.S. chemical manufacturing plants where it is used, and in surface water and gr oundwater in the United States, France, the Netherlands, and Germany. o- Nitrotoluene, which can be formed as a breakdown product of di- or trinitrotoluenes, has been detected in the efflue nt or wastewater of plants producing these chemicals. The uses of di- and trinitrotoluenes in clude the production of commercial and military explosives, and o -nitrotoluene has been found in theRoC Background Document for o -Nitrotoluene

viii 6/20/08 groundwater, private well water, and surface water and soil at or near munitions production facilities and/or military training grounds. o- Nitrotoluene also was detected in mainstream smoke of a U.S. nonfilter cigarette in one study. 1
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22 23 24 25 26 27 28 29 Human Cancer Studies No studies on the relationship between human cancer and specific exposure to o nitrotoluene were identified. o -Nitrotoluene may be used to manufacture magenta and thus magenta manufacturing workers may be exposed to o -nitrotoluene. IARC reviewed magenta manufacturing in 1987 and 1993 and concluded that there is sufficient evidence in humans that the manufacture of magenta enta ils exposures that ar e carcinogenic. Their assessment was based on two cohort studies and a case-control study, all of which reported an excess risk of bladder can cer; however, only one study specifically mentioned that the workers were exposed to o -nitrotoluene as a part of the manufacturing process. [These studies are limited for the evaluation of the carcinogenicity of o nitrotoluene in humans because the workers we re also exposed to other chemicals, such

as o -toluidine, 2,5-diaminotoluene, and 4,4'-met hylenebis(2-methylaniline), and exposure to o -nitrotoluene was not specifically assessed.] Studies in Experimental Animals The carcinogenicity of o -nitrotoluene was evaluated in feeding studies in rats and mice. Malignant mesothelioma was first observed in male rats administered o -nitrotoluene for 13 weeks. Tumors at multiple tissues sites were observed in a stop-exposure study in male rats and two-year chronic studies in both sexes of rats and mice. The NTP concluded that there was clear evidence of carcinogenic activity of o- nitrotoluene in male and female rats, based on increased incide nces of subcutaneous skin neoplasia and mammary-gland fibroadenoma in both sexes, and malignant mesothelioma and liver tumors in males. Increased incidences of lung tumors in males and hepatocellular adenoma in female rats also were consid ered to be exposure related. The NTP also concluded that there was clear evidence of carcinogenic activity of o -nitrotoluene in male and female mice, based on increased incide nces of hemangiosarcoma in both sexes, carcinoma of the large intestine (cecum) in males, and hepatocellular tumors in females.RoC Background Document for o -Nitrotoluene

6/20/08 ix Absorption, Distribution, Metabolism, and Excretion 1

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27 28 29 Metabolites of o -nitrotoluene, which include o- nitrobenzoic acid and o- nitrobenzyl alcohol, have been detected in the urine of factory workers, indicating that absorption occurs in humans from sk in contact and inhalation. o -Nitrotoluene is absorbed after oral administration to rats an d mice. The half-life of o -nitrotoluene in plasma of rats is fairly short, approximately 1.5 hours, and the primary route of excretion is in urine, occurring mainly in the first 24 hours after exposure. Excretion of o -nitrotoluene metabolites also occurs through feces and expired air. A study in rats showed that more than 85% of the total oral dose of o- nitrotoluene was recovered in the urine within 24 hours, with lesser amounts in the feces (4.6%) and expired air ( 0.1%). Metabolites excreted into the feces are subject to reabsorption and further me tabolism, most likely leading to urinary excretion. o -Nitrotoluene or its metabolites are di stributed to the liver, as indicated by recovery of radiolabel from o -nitrotoluene in the bile afte r oral administration and by its binding to hepatic DNA. No data documenting the di stribution of o -nitrotoluene to other tissues was found. However, it has been reported that o -nitrotoluene or its metabolites may also form hemoglobin adducts in humans and rats implying distribution to other tissues. The major urinary metabolites found in rats and mice are o -nitrobenzoic acid and o nitrobenzyl glucuronid e; however, other metabolites, such as S -( o -nitrobenzyl)- N

acetylcysteine and o -aminobenzyl alcohol, are found only in rats. Female rats excreted less than half as much of the dose as o -aminobenzyl alcohol or as S -( o -nitrobenzyl)- N

acetylcysteine as did male rats. The major biliary metabolite of o- nitrotoluene was o nitrobenzyl glucuronide, which accounted for 22.1% of the dose in males and 8.3% of the dose in females. The next most abundant metabolite was S -( o -nitrobenzyl) glutathione, which accounted for 4.9% of the dose in males and 0.4% in females. Toxicity

o -Nitrotoluene caused more treatment-related toxi city in male rats than in female rats, and most toxic effects occurred at or greater than 2,500 pp m in a 13-week study. o Nitrotoluene exposure resulted in toxic effect s in the kidney (hyalin e droplet nephropathyRoC Background Document for o -Nitrotoluene

x 6/20/08 and increased renal α 2u -globulin levels) and liver (hep atocyte vacuolization, oval-cell hyperplasia, PGST+ foci, and increased seru m bile acids, sorbitol dehydrogenase, and alanine aminotransferase. Hematopoiesis and pigmentation in the spleen were noted in male and female rats exposed to o- nitrotoluene for two years, but no liver lesions were noted in female rats at any concentration (up to 10,000 ppm tested). No kidney or splenic lesions occurred in females at conc entrations less than 2,500 ppm. 1
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o -Nitrotoluene did not cause mutations in prokaryotic systems. In mammalian in vitro systems, it induced (1) sister chromatid exch ange in Chinese hamster ovary (CHO) cells; (2) chromosomal aberrations in Chinese ha mster lung cells (CHL) and human peripheral lymphocytes (but not in CHO cells); (3) micronuclei in CH L cell line (but not in CHOK1 cells), and (4) DNA damage in L5178Y mouse lymphoma cells. It did not induce DNA repair in rat or human hepatocytes. In rats exposed to o -nitrotoluene in vivo , DNA adducts and increased DNA repair were detected in males but not females. o -Nitrotoluene induced a slight increase in normochro matic micronuclei in high-dose male mice (equivocal response) but did not induce micr onuclei in the bone marrow of male rats (polychromatic), male mice (polychromatic ), or female mice (normochromatic). The genotoxicity of o -nitrotoluene, as measured by the in vivo–in vitro DNA repair assay in rats, depends on metabolism (both mammalia n and bacterial) and is sex specific. DNA repair was induced only in male rats with an intact intestinal mi croflora. Incubation of o nitrotoluene in vitro with hepatocytes isolated from male rats failed to induce DNA repair. Biliary excretion is an im portant step in the activation of o- nitrotoluene. Interruption of bile flow into the intestine by cannulation of the bile duct decreased the covalent binding of o -nitrotoluene–related material at 12 hours post-administration to 7% (in males) or 22% (in females) of that seen in sham-operated animals. Moreover, deleterious effects of o -nitrotoluene generally are more severe in male rats than in females and include changes in hepatic, renal, or splenic histopathology, and tumor incidence. The toxicity of o -nitrotoluene, particularly in male rats, likely involves its metabolism by oxidation of the methyl group to an alc ohol, conjugation of o -nitrobenzyl alcohol with glucuronic acid and ex cretion in bile, deconjugation of o -nitrobenzylRoC Background Document for o -Nitrotoluene

6/20/08 xi glucuronide and reduction of the nitro group by intestinal ba cteria, and final activation of

o -aminobenzyl alcohol by the formation of o -aminobenzyl sulfate. 1
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27 28 o -Nitrotoluene caused significantly increased inci dences of tumors in tissues other than the liver in both rats and mice, including ma mmary gland, skin, lung, large intes tine, and hemangiosarcomas in various tissues. This s uggests that there are other mechanisms of activation for o -nitrotoluene.

Mutations in the p53 and β -catenin genes and production of th ese proteins in mice were detected in o -nitrotoluene–induced hemangiosarcomas and colon tumors from mice but not in spontaneous hemangiosarcomas; K- ras gene mutations and cyclin D1 protein production also were detected in the colon tumors. Mutations in p53 , β -catenin , and Kras genes may be a result of the genotoxic effects of o -nitrotoluene. The pattern of mutations is consistent with targeting of gua nine for adduct formation since mutations in the p53 gene in hemangiosarcomas mainly involved G·C → A·T transitions, and almost all the mutations in the K- ras gene in cecal carcinomas were G·C → T·A transversions. Human colorectal cancers also have a high frequency of mutations in the K- ras and p53 genes, and the β -catenin and cyclin D1 genes are upregulated. As a result of these genetic effects, both human and mouse colon tumors have alterations in pathways that are considered important for the progression of cells from a normal state to cancer; these pathways include the β -catenin / Wnt signaling pathway, ras / MAP kinase pathway, and cell-cycle checkpoint genes (e.g., the cyclin D1 and p53 genes). Studies in rats have also provided evidence th at cellular and molecular events involved in the induction of mesotheliomas ar e similar in both experimental animals (rats exposed to

o- nitrotoluene) and humans. Microarray analys is of peritoneal mesotheliomas from F344 rats treated with o- nitrotoluene identified the following carcinogenic pathways: insulinlike growth factor 1 (IGF-1), p38 MAPK, Wnt/ β -catenin, and integrin signaling pathways. In studies of early gene expr ession in mouse liver, a dose-de pendent loss of expression of the fragile histidine triad gene ( Fhit ) and the WW domain-containing oxidoreductaseRoC Background Document for o -Nitrotoluene

xii 6/20/08 ( Wwox ) gene was seen from o -nitrotoluene exposure. The au thors noted that these genes are human tumor suppressor genes and are of ten lost together in many human cancers. Further, o -nitrotoluene treatment also resulted in strong up-regu lation of the cell-cycle genes, cyclin G1 (Ccng1) and p21 ( Cdkn1a ), down-regulation of the epidermal growth factor ( Egfr ) gene, down-regulation of the transcri ption factor early growth response 1 ( Egr1 ) gene, and down-regulation of inhibin β -A ( Inhba ), a member of the TGF- β superfamily, and Jun-B oncogene ( Junb ), a negative regulator of proliferation genes. 1
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## Abbreviations

ACGIH: American Conference of G overnmental Industrial Hygienists APC: adenomatous polyposis coli b.w.: body weight CHO: Chinese hamster ovary CIIT Chemical Industry Institute of Toxicology CRASF: Charles River altered Schaedler flora DOT: Department of Transportation EPA: Environmental Protection Agency
g: gram GI: gastrointestinal HPV high production volume IARC: International Agency for Research on Cancer kg: kilogram
K oc : soil organic adsorption coefficient L: liter

m 3 : cubic meter mg: milligram mL: milliliter mol wt: molecular weight NADPH: nicotinamide adenine dinucleotide phosphate NCEs: normochromatic erythrocytes NCI National Cancer Institute NIEHS: National Institute of Environmental Health Sciences NIOSH: National Institute for Occupational Safety and Health

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ng: nanogram NTP: National Toxicology Program OSHA: Occupational Safety a nd Health Administration PAPS: 3'-phosphoadenosine 5'-phosphosulfate PCEs: polychromatic erythrocytes PGST: placental glutathione S -transferase ppb: parts per billion ppm: parts per million RR relative risk RTECS: Registry of Toxic Eff ects of Chemical Substances SCE: sister chromatid exchange SIR: standardized incidence ratio SMR: standardized mortality ratio TNT: trinitrotoluene UDS: unscheduled DNA synthesis
μ g: microgram

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Table of Contents 1 Introduction................................................................................................................. .............. 1 1.1 Chemical identification.............................................................................................. 1 1.2 Physical-chemical properties ..................................................................................... 2 1.3 Identification of metabolites and analogues .............................................................. 3 2 Human Exposure............................................................................................................... ........ 7 2.1 Use ........................................................................................................................ ..... 7 2.2 Production................................................................................................................. . 7 2.3 Environmental occurrence and fate ........................................................................... 9 2.3.1 Air............................................................................................................. 10 2.3.2 Water......................................................................................................... 10 2.3.3 Soil............................................................................................................ 12 2.4 General population exposure ................................................................................... 13 2.5 Occupational exposure............................................................................................. 14 2.6 Biological indices of exposure................................................................................. 14 2.7 Regulations and guidelines ...................................................................................... 16 2.7.1 Regulations ............................................................................................... 16 2.7.2 Guidelines................................................................................................. 16 2.8 Summary.................................................................................................................. 17 3 Human Cancer Studies......................................................................................................... ... 19 3.1 Studies of magenta manufacturing workers............................................................. 19 3.2 Studies of workers expose d to dinitrotoluenes or o -toluidine ................................. 21 3.2.1 Dinitrotoluenes ......................................................................................... 21 3.2.2 o -Toluidine hydrochloride........................................................................ 22 3.3 Discussion and summary ......................................................................................... 23 4 Studies of Cancer in Experimental Animals........................................................................... 25 4.1 Rats ....................................................................................................................... ... 25 4.1.1 Subchronic exposure................................................................................. 25 4.1.2 Chronic exposure...................................................................................... 29 4.2 Mice ....................................................................................................................... .. 34 4.3 Summary.................................................................................................................. 37 5 Other Relevant Data.......................................................................................................... ...... 39 5.1 Absorption, distribution, meta bolism, and excretion............................................... 39 5.1.1 Human data............................................................................................... 39 5.1.2 Rodent data ............................................................................................... 40 5.1.3 In vitro metabolism of o -, m -, and p -nitrotoluene .................................... 41 5.1.4 In vivo metabolism of o -nitrotoluene in rodents....................................... 43 5.1.5 In vivo metabolism of m - and p -nitrotoluene in rodents........................... 48 5.1.6 Comparison of o -, m -, and p -nitrotoluene metabolism in rodents............ 49

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5.1.7 Biliary excretion of o -, m -, and p -nitrotoluene in rats .............................. 52 5.2 Bioactivation of o -nitrotoluene................................................................................ 53 5.3 Genetic damage and related effects ......................................................................... 57 5.3.1 Prokaryotic systems.................................................................................. 57 5.3.2 Mammalian systems ................................................................................. 58 5.4 Mechanistic studies and considerations................................................................... 64 5.4.1 Potential mechanisms of carcinoge nicity and genotoxicity related to the bioactivation of o -nitrotoluene ....................................................... 64 5.4.2 Gene expression and mutation studies...................................................... 64 5.5 Carcinogenicity and genotoxicity of o -nitrotoluene analogues and metabolites ............................................................................................................... 73 5.5.1 p -Nitrotoluene........................................................................................... 73 5.5.2 Dinitrotoluenes ......................................................................................... 75 5.5.3 o -Toluidine hydrochloride........................................................................ 78 5.6 Toxicity................................................................................................................... . 78 5.7 Summary.................................................................................................................. 80 5.7.1 Absorption, distribution, me tabolism, and excretion................................ 80 5.7.2 Bioactivation of o-nitrotoluene................................................................. 81 5.7.3 Genetic damage and related effects .......................................................... 81 5.7.4 Mechanistic studies and considerations.................................................... 82 5.7.5 Carcinogenicity and genotoxicity of o -nitrotoluene analogues and metabolites................................................................................................ 83 5.7.6 Toxicity..................................................................................................... 84 6 References................................................................................................................... ............ 85 Glossary of Terms......................................................................................................................... 99

List of Tables Table 1-1. Chemical identification of o -nitrotoluene ..................................................................... 2 Table 1-2. Physical and chemical properties of o -nitrotoluene ...................................................... 3 Table 4-1. Neoplastic lesions identified in F344/N male rats follo wing dietary exposure to o -nitrotoluene for 13 or 26 weeks ......................................................................... 28 Table 4-2a. Neoplastic lesions identified in F344/N rats following dietary exposure to o nitrotoluene for two years.......................................................................................... 32 Table 4-2b. Neoplastic lesions identified in F344/N rats following dietary exposure to o nitrotoluene for two years (continued) ...................................................................... 33 Table 4-3a. Hemangiosarcomas in B6C3F 1 mice following dietary exposure to o nitrotoluene for two years.......................................................................................... 36 Table 4-3b. Neoplastic lesions of the large intestine and liver in B6C3F 1 mice following dietary exposure to o -nitrotoluene for two years....................................................... 36

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Table 4-4. Neoplastic lesions identified in rats and mice following dietary exposure to o nitrotoluene................................................................................................................ 37 Table 5-1. Metabolism of nitrobe nzyl alcohols by rat hepati c glucuronyltransferase and sulfotransferase.......................................................................................................... 43 Table 5-2. Metabolites excreted in urine (% of dose) by ma le and female F344 rats and male B6C3F 1 mice 24 hours after an oral dose of o -nitrotoluene ............................. 44 Table 5-3. Metabolites excreted in urine (% of dose) by rats and mice after an oral dose of 200 mg/kg b.w. of o -, m -, or p -nitrotoluene.......................................................... 50 Table 5-4. Results of genotoxicity testing of o -nitrotoluene in prokaryotic systems ................... 58 Table 5-5. Results of genotoxicity testing of o -nitrotoluene in in vitro mammalian systems ...................................................................................................................... 60 Table 5-6. Results of genotoxicity testing of o -nitrotoluene in in vivo mammalian systems....... 63 Table 5-7. Gene expression in cultured cells or tissues from mice exposed to o nitrotoluene................................................................................................................ 66 Table 5-8. p53, β -catenin, and K- ras mutations in hemangiosarcomas (subcutaneous tissue, skeletal muscle, a nd mesentery) from B6C3F 1 mice orally exposed to o -nitrotoluene ............................................................................................................ 69 Table 5-9. p53, β -catenin, and K- ras mutations in cecal carcinomas from B6C3F 1 mice orally exposed to o -nitrotoluene................................................................................ 70 Table 5-10. Gene mutations and gene expression in o -nitrotoluene-induced tumors in rodents. ...................................................................................................................... 7 2 Table 5-11. Neoplastic lesions identif ied in rats and mice exposed to o -nitrotoluene and p -nitrotoluene ............................................................................................................ 74 Table 5-12. Treatment-related lesions in B6C3F 1 mice following dietary exposure to o nitrotoluene for 13 weeks a ......................................................................................... 80

List of Figures Figure 1-1. Chemical structure of o -nitrotoluene ........................................................................... 2 Figure 1-2. Chemical structures of urinary metabolites of o -nitrotoluene in rats and mice ........... 4 Figure 1-3. Chemical structures of o -nitrotoluene analogues......................................................... 5 Figure 5-1. Urinary metabolites of o -nitrotoluene in rats and mice ............................................. 47 Figure 5-2. Formation and potenti al bioactivation routes for o -aminobenzyl alcohol from o -nitrotoluene ............................................................................................................ 54

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## 1 Introduction 1

o -Nitrotoluene is a nitro arom atic compound used as an important chemical intermediate in the synthesis of azo dyes. It is also used (either directly or as an intermediate) in the production of other dyes, ag ricultural chemicals, rubbe r chemicals, pesticides, petrochemicals, pharmaceuticals, and explosives. Based on data reported under U.S. EPA's Inventory Update Rule, production of o -nitrotoluene in the United States was in the range of 10 million to 50 million pounds pe r year from the mid 1980s until 2002 (the most recent available data) (EPA 2007). 2 3 4 5 6 7 8

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20 o -Nitrotoluene was nominated by the Nationa l Institute of Environmental Health Sciences for possible listing in the Report on Carcinogens based on the results of a National Toxicology Program (NTP) bi oassay (NTP 2002b), which reported clear evidence of carcinogenic activity , based on the occurrence of malignant tumors at a variety of tissue sites in male and female mice and rats.

1.1 Chemical identification 14

o -Nitrotoluene (also known as 2-nitrotoluene ) is a nitro aromatic compound with the structure illustrated in Figure 1-1. It is one of three isomers of nitrotoluene; the other two

are m -nitrotoluene (also known as 3-nitrotoluene) and p -nitrotoluene (also known as 4- nitrotoluene). The other two nitrotoluene isomers ( m - and p -nitrotoluene) and other nitrotoluene analogues are described in Section 1.3. Table 1-1 lists chemical identifying information for o -nitrotoluene.RoC Background Document for o -Nitrotoluene

2 6/20/08 Figure 1-1. Chemical structure of o -nitrotoluene

Table 1-1. Chemical identification of o -nitrotoluene
Characteristic Information CAS Registry number 88-72-2 Molecular formula C 7 H 7 NO 2 Synonyms 1-methyl-2-nitrobenzene 2-methylnitrobenzene 2-methyl-1-nitrobenzene 2-nitrotoluene 2-nitrotoluol benzene, 1-methyl-2-nitro o -mononitrotoluene o -nitrotoluol o -methylnitrobenzene ONT NSC 9577 RTECs No.: XT3150000 DOT/UN No.: 1664 Source: ChemIDplus 2007, CAS 2008
1.2 Physical-chemical properties 1

o -Nitrotoluene is a yellow liquid at room temper ature with an odor of bitter almonds. It is slightly soluble in water and soluble in acetone, benzene, chloroform, diethyl ether, ethanol, and petroleum ether. It has a flash point of 106°C (closed cup) and an autoignition temperature of 305°C (PTCL 2003) . It does not ignite easily; however, it may burn, and containers may explode wh en heated (HSDB 2008). The physical and chemical properties of o -nitrotoluene are summarized in Table 1-2. 2 3 4 5 6 7RoC Background Document for o -Nitrotoluene

6/20/08 3 Table 1-2. Physical and chemical properties of o -nitrotoluene Property Information Molecular weight 137.14 Melting point (°C) –9.5 (needles); –2.9 (crystals) Boiling point (°C) 222 at 760 mm Hg Critical temperature (°C) NA Specific gravity 1.162 at 19°C/15°C Solubility in water (at 30°C) 650 mg/L Octanol-water partition coefficient (log K ow ) 2.30 Dissociation constant (pK a ) NA Vapor pressure (mm Hg) 0.188 at 25°C Vapor density relative to air 4.73 Henry’s law constant 1.25 x 10 -5 atm-cu m/mole @ 25°C Source: HSDB 2008. NA = not available. 1.3 Identification of metabolites and analogues 1 Urinary metabolites in workers exposed to o -nitrotoluene include o -nitrobenzoic acid and

o -nitrobenzyl alcohol (Jones et al. 2005b, Sabbioni et al. 2006). The urinary metabolites of o -nitrotoluene identified following oral ad ministration to rats and mice include o nitrobenzoic acid, a sulfu r-containing conjugate of o -acetamidotoluene (tentatively identified as S -( o -acetamidobenzyl)- N -acetylcysteine), o -nitrobenzyl glucuronide, S -( o nitrobenzyl)- N -acetylcysteine ( o -nitrobenzyl mercapturic acid), a sulfur-containing conjugate of o -aminotoluene (tentatively identified as S -( o -aminobenzyl) glutathione), S

- ( o -nitrobenzyl) glutathione, o -aminobenzoic acid, o -nitrobenzyl sulfate, o -nitrobenzyl alcohol, o -aminobenzyl alcohol, and o -aminotoluene ( o -toluidine) (Chism et al. 1984, NTP 2002a). See Figure 1-2 for the structures of these metabolites and Section 5.1 for further discussion of metabolism. 2 3 4 5 6 7 8 9 10
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13 14 15 The structures of two other nitrotoluene isomers ( m - and p -nitrotoluene), o -toluidine ( o aminotoluene, prepar ed by reduction of o -nitrotoluene), and the is omers of dinitrotoluene (2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dinitr otoluene), are shown in Figure 1-3.RoC Background Document for o -Nitrotoluene

Figure 1-2. Chemical structures of urinary metabolites of o -nitrotoluene in rats and mice Chemical structures of urinary metabolites of o -nitrotoluene (see Table 5-3 for data on percent of dose excreted as these metabolites by F344 rats and B6C3F 1 mice) are illustrated above. The two bracketed structures indicate structures tentativel y identified by Chism and Rickert (1985). Cys(Ac) = acetylcysteine, G = glut athione, Gl = glucuronide.

a Tentatively identified by Chism and Rickert (1985) as the structure of the metabolite listed as a sulfurcontaining conjugate of o -acetamidotoluene in Table 5-3. b Tentatively identified by Chism and Rickert (1985) as the structure of the metabolite listed as a sulfurcontaining conjugate of o -aminotoluene in Table 5-3.

4 6/20/08RoC Background Document for o -Nitrotoluene

Figure 1-3. Chemical structures of o -nitrotoluene analogues

6/20/08 5RoC Background Document for o -Nitrotoluene

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## 2 Human Exposure
Exposure to o- nitrotoluene in the United States is primarily a result of occupational exposure during the production and use of this chemical. The United States produces large quantities of o -nitrotoluene (greater than 10 million pounds annually), which is used primarily in the production of important chemi cal intermediates used in the synthesis of dyes. Thus, there is the potent ial for significant exposure to o -nitrotoluene. This section discusses information related to human exposure, including uses, production, concentrations of o -nitrotoluene in the environment (e nvironmental occurrence) and in occupational facilities (occupational exposure), numbers of potentially exposed workers, biological indices of exposure, and regulati ons and guidelines to reduce exposure. [Note that for some studies, information was avai lable only in a foreign language publication, and the information reported here was extrac ted from an English translation of the abstract as noted below.] 1
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o -Nitrotoluene is primarily used in th e production of derivatives, including o -toluidine ( o aminotoluene), 2-amino-4-chlorotolu ene, 2-amino-6-chlorotoluene, and o -toluidine-4- sulfonic acid, which are intermediates in the production of various azo dyes (IARC 1996). It is used in the manufact ure (or manufacture of intermed iates) for other dyes such as magenta (which is produced by at least two companies in the United States) and various sulfur dyes for cotton, wool, silk, leather, and paper (HSDB 2008, IARC 1996). Other uses include as an intermediate in th e synthesis of (or synt hesis of intermediates for) explosives and of a variety of organic chemicals, including compounds used in the agricultural, petrochemical, pesticide, pharmaceutical, and rubb er industries (HSDB 2008).

2.2 Production 24 The nitrotoluenes are produced principally by th e nitration of toluene with a mixture of nitric acid and either sulfuric, aromatic sulfonic, or phosphoric acid (Kirk-Othmer 1996). Production can be either a batch or a continuous process. In a batch process, toluene is fed into a nitrator and cooled to about 25° C. The acid mixture is added slowly, and theRoC Background Document for o -Nitrotoluene

8 6/20/08 temperature of the reaction mixture is maintain ed at 25°C by adjustment of the acid feed rate and the amount of cooling. After the acids are added, the temperature is slowly raised to 35°C to 40°C. The reaction mixture is then put in a separator, where the acids are taken from the bottom, and the product is steam di stilled to remove excess toluene and then dried by distillation of the remaining traces of water. The isomers are separated by a combination of fractional distillation and crys tallization. The ratio of the isomers depends on the production conditions and the catalyst used, but generally is in the range of 45% to 62% o -nitrotoluene, 2% to 5% m -nitrotoluene, and 33% to 50% p -nitrotoluene. o Nitrotoluene is available commercially at a purity of 99.2% to 99.5% and typically contains the following impurities: m - and p -nitrotoluenes (0.8%), water (0.2%), and toluene (0.1%) (IARC 1996). 1
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27 28 29 Other processes that have been used to produce nitrotoluenes in clude (1) reaction of toluene with nitronium salts in the presence of crown ethers or polye thers and (2) reaction of toluene with nitric acid in the gas phase in the presence of solid silica-alumina catalysts (Kirk-Othmer 1996). The main advantage of these proce sses is that sulfuric acid is not used. U.S. production of o -nitrotoluene was calculated as 13 billion grams (29 million pounds) for 1981 (HSDB 2008). U.S. production of o - and p -nitrotoluene combined was estimated at 20 billion grams (44 million pounds) in 1983 (HSDB 2008), and production of o nitrotoluene only was estimated at 16,120 metric tons (35.5 million pounds) in 1993 (Kirk-Othmer 1996). o -Nitrotoluene is listed as a High Production Volume (HPV) chemical, and according to data submitted by companies under the Inventory Update Rule, U.S. production of o -nitrotoluene was between 10 million and 50 million pounds for every four-year reporting period from 1986 to 2002 (EPA 2007). SRI (2007) reported that in 2007 o -nitrotoluene was produced in only one U.S. facility. Eleven suppliers of o nitrotoluene were identified in the United States in 2007 (ChemSources 2007). No data specific for U.S. imports or exports of o -nitrotoluene were f ound. U.S. imports of nitrated benzene, nitrated toluene, and nitrated naphthalene were 270 million grams (602,000 pounds) in 1984 (HSDB 2008), and 95,000 kilograms (209,400 pounds) in 2005RoC Background Document for o -Nitrotoluene

6/20/08 9 (2005 data did not include p -nitrotoluene) (ITC 2007a). No imports of this group of compounds were reported in 2006. In 2006, the United States exported approximately 12.9 million kilograms (28 million pounds) of hydrocarbon derivatives containing only nitro or nitroso groups (excluding p -nitrotoluene and trinitrotoluene) (ITC 2007b). 1
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o -Nitrotoluene is expected to exist as a vapor in ambient air and has been observed to be completely degraded in aqueous sewage treatme nt systems, rivers, and streams. There are limited data on environmental levels of o -nitrotoluene. Some data show that o nitrotoluene has been released into the air and water, primarily from occupational settings, but it may also be formed from to luene vapor and nitrogen oxides in air (see Sections 2.3.1 and 2.3.2). o- Nitrotoluene also may be form ed through the degradation or combustion of military munitions (NAVFAC 2003, USACE 2002). In soil, o- nitrotoluene is expected to have moderate mobility and to volatilize slowly. o -Nitrotoluene is not listed in EPA’s Toxics Release Inventory (TR I), and no data were available from that source for its release into the environment. The National Response Center (NRC) serves as the sole national point of contact for reporting all oil, chemical, radiological, bi ological, and etiologi cal (i.e., biologically hazardous) spills into the environment anywhere in the United States and its territories ( http://www.nrc.uscg.mil/nrcback.html ). A query of NRC’s (2008) online database ( 19 http://www.nrc.uscg.mil/foia.html ) using the keyword “nitrot oluene” yielded 69 results. Of these 69 spills, 2 were reported as o -nitrotoluene (both in 1990), 1 was reported as nitrotoluene ( o -, p - and mixtures) (in 2000), and the rest were for other chemicals (the majority being for 2,4- or 2,6-dinitrotoluene). The level of information provided in the response to the query was not sufficient to estimate the extent of environmental contamination or the number of people exposed; however, it does suggest that the potential exists for environmental contam ination and general public exposure from inadvertent spills of o -nitrotoluene or chemical mixtures containing o -nitrotoluene.] 20
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10 6/20/08 2.3.1 Air 1 2 3 4 5 6 7 8 9

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26 27 28 29 Little information is available on concentrations of o -nitrotoluene in ambient air. Two ambient air samples collected in Boise, Idaho in the winter of 1986–1987 contained 0.03 and 0.29 ng/m 3 of o -nitrotoluene vapor (Nishioka and Lewtas 1992). o -Nitrotoluene was also found in ambient-air at a manufactur ing plant in New Jersey (IARC 1996) (see Section 2.5, Occupational Exposure). o -Nitrotoluene also has been found to sorb to snow (Roth et al. 2004). In a laboratory investigation, Atkinson et al. (1989) found that minor amounts of o -nitrotoluene were formed when to luene vapor and nitrogen oxides were mixed in air: m -nitrotoluene was the major isomer formed. According to a model of gas-particle part itioning of semivolatile organic compounds in the atmosphere, o -nitrotoluene released to air is exp ected to exist in the vapor phase and to be removed mainly by direct photolysis and reaction with photochemically generated hydroxyl radicals (HSDB 2008). The main photoproducts are expected to be 2-methyl-6- nitrophenol and 2-methyl-4-n itrophenol (Nojima and Kanno 1977) and a half-life of 42 days has been estimated for removal by r eaction with hydroxyl radicals (HSDB 2008).

2.3.2 Water

o -Nitrotoluene was detected in a paper-m ill waste-treatment lagoon (concentration and location not reported). o- Nitrotoluene, which can be form ed as a breakdown product of di- or trinitrotoluenes, has been detected in the effluent or wastewater of plants producing these chemicals. o -Nitrotoluene was detected at c oncentrations ranging from 320 to 16,000 μ g/L in effluent from a U.S. plant manufacturing 2,4,6-trinitrotoluene (IARC 1996, HSDB 2008). Other reports included co ncentrations ranging from 20 to 140 μ g/L in wastewater from a plant producing and purifying 2,4,6-trin itrotoluene, and 7,800 μ g/L in raw effluent from a plant manufacturing dinitrotoluene (geogr aphic locations not specified). The uses of di- and trinitrotoluenes include the production of commercial and military explosives. In the United States, o -nitrotoluene has been found in groundwater and surface water at munitions production facili ties, in groundwater at a military training facility, and in groundwater at a nuclear w eapons assembly/disassembly facility thatRoC Background Document for o -Nitrotoluene

6/20/08 11 previously had been a World War II m unitions factory (ATSDR 2007, Pantex 2004, 2006, WDHFS 2002). Maximum onsite groundwater levels seen at three munitions manufacturing facilities were 4,600 μ g/L for a plant in Texas, 21,000 μ g/L for a plant in Illinois, and 140,000 μ g/L for a plant in Tennessee (yea rs of analyses not provided) (ATSDR 2007). o -Nitrotoluene was found at a mean concentration of 42.6 mg/L (42,600 μ g/L) from one well at an army ammunition plant in Tennessee (Best et al. 2001), and Spain et al . reported “typical” o -nitrotoluene levels of 2.9 mg/L (2,900 μ g/L) in remediation studies of groundw ater highly contaminated with explosives and their impurities at the same Tennessee munitions arsenal. At a former munitions production site in Wisconsin between 1999 and 2002, o -nitrotoluene was detected in offsite, private well-water at a maximum concentration of 0.095 μ g/L. For the same facility and the same time period, o - and p -nitrotoluene combined was detected in 4 of 17 groundwater samples with levels ranging from 0.16 to 17.0 μ g/L (ATSDR 2007, WDHFS 2002). The maximum surface water concentration measured at a munitions manufacturing facility in Missouri was 0.12 μ g/L (years of analyses not provided), and the maximum groundwater level measured at a military training facility in Massachusetts was 25 μ g/L (ATSDR 2007). 1
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22 23 24 25 26 27 28 29 o -Nitrotoluene has been routin ely monitored in groundwater si nce 1995 at a facility in the Texas panhandle where conven tional munitions were produced for World War II and where nuclear-weapons assembly and disassemb ly activities have been performed since the mid-1970s. Testing has been performed on groundwater from both the Ogallala aquifer and from a perched aquifer above the Ogallala. Since 1999, o -nitrotoluene has been detected sporadically in water from both the Ogallala and the perched aquifer. Concentrations in water from the perched aquifer that exceeded the detection limit ranged from a minimum of 0.14 μ g/L in 2003 to a maximum of 5 μ g/L in 2004, while concentrations in water from the Ogalla la aquifer ranged from a minimum of 0.12 μ g/L to a maximum of 2.9 μ g/L, both in 2004 (Pantex 2003, 2004). o -Nitrotoluene was detected in a small percentage of sample s (between 1% and 7%) in 2001 to 2004 in the perched aquifer and in 1999 and 2002 in the Ogallala aquifer (Pantex 1996, 1997, 1998,RoC Background Document for o -Nitrotoluene

12 6/20/08 1999, 2000, 2001, 2002, 2003, 2004, 2005) but was not detected in 2005, the last year for which data were available (Pantex 2006). 1
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22 23 24 25 26 27 28 29 Nitrotoluene ( o - and p -nitrotoluene combined) was detected in the Netherlands (in the 1970s) in the Rhine River at a concentration of 10 μ g/L, in the River Waal at 4.5 μ g/L, and in the River Maas at 0.3 μ g/L (IARC 1996). In Germany, o -nitrotoluene was detected at 0.4 and 7.4 μ g/L in surface water near a former munitions plant and at 1.2 μ g/L in an adjacent river. Two ponds had concentrations of 0.4 and 22.0 μ g/L; these ponds fed into the River Oder, which had a concentration of < 0.01 μ g/L. The concentration of o nitrotoluene in three samples from the River Elbe ranged from 0.05 to 0.4 μ g/L. Concentrations detected in groundwater in France (in 1987) ranged from 90 to 165 μ g/L.
In water, o -nitrotoluene may undergo di rect or indirect photol ysis, volatilization, or aerobic biodegradation (HSDB 2008). It is not expected to adso rb to suspended solids or sediment. Half-lives for volatilization of o -nitrotoluene were estimated to be 56 hours from a model river (1 m deep, flowing 1 m/s, wind velocity of 3 m/s) and 30 days from a model lake (1 m deep, flowing 0.05 m/s, wi nd velocity of 0.5 m/s). The half-life for removal by indirect photolysis from a ri ver with a high concentration of humic substances was calculated to be 45 minutes. o -Nitrotoluene is not expected to bioaccumulate in aquatic organisms; a biocon centration factor of 12 was calculated, and experimentally determined bioconcentr ation factors in fish were low.

2.3.3 Soil A soil contaminated with 39,100 ppm of trinitr otoluene (TNT) at a historical testing ground in Idaho contained 1.4 ppm of o -nitrotoluene (Radtke et al. 2002a). o Nitrotoluene can result from anaerobic reduction of TNT (ACE 2004) and is a constituent of concern at former munitions sites. In December 2001, Congress passed the National Defense Authorization Act (for fiscal year 2002), which, in part, requires the Department of Defense (DoD) to develop an inventory of DoD sites that are no l onger in use but are known or suspected to contain military mun itions that will require clean-up. In a 2002 Annual Report, DoD provided a list of the 20 munitions constituents of greatest concern at contaminated military munitions sites due to their widespread use and potentialRoC Background Document for o -Nitrotoluene

6/20/08 13 environmental impacts, and o -nitrotoluene was included in that list. Development of the inventory of sites for clean-up does not requ ire any analytical measurements for the constituents of concern, and therefore, to-d ate, there are no data on the frequency or levels of o -nitrotoluene at these sites. However, there are over 15 million acres in the United States that are either known or susp ected to be contaminated with military munitions and “much of the land on which thes e sites are located has been or will be converted to nonmilitary uses such as farm ing, residential or commercial development, and recreation” (GAO 2002, 2003). A similar situ ation exists in other countries where munitions manufacture and ammunition testi ng have been done historically (Spiegel et al. 2005, Toze et al. 1999). 1
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22 23 24 25 26 27 28 29 o -Nitrotoluene is expected to be moderately mobile in soil, with an estimated soil adsorption coefficient (K oc ) of 420, and may volatilize from moist soil surfaces (HSDB 2008). Obernosterer et al . (2000) 1 used air stripping to remove 20% of o -nitrotoluene from quartz sand and loess loam. [This implies that o -nitrotoluene’s volatility could be an important transport and remediation mechanism.] Under aerobic conditions, o nitrotoluene persisted for more than 64 da ys in a silt loam inoculum (HSDB 2008). Under anaerobic conditions, o -nitrotoluene in soil has been obser ved to degrade to toluidine. A review on sorption, mobility, and biodegradation of nitrotoluenes in soil is available (Toze et al. 1999).

2.4 General population exposure 20 The general population may be exposed to o -nitrotoluene via inhalation of ambient air in the vicinity of production sites, and by oral ingestion, particul arly of contaminated water (HSDB 2008), as well as via skin contact with contamin ated substances. In one experiment in which 115 volatile organic ch emicals were measured in expired air samples from 54 healthy individuals from an urban population, 19.1% of 387 expired air samples contained o -nitrotoluene at a mean concen tration of 0.04 ng/L; [however, no effort was made to elucidate the source of exposure, i.e., ambient air was not monitored, and no other potential sources of ex posure were assessed] (Krotoszynski et al. 1979). Since o -nitrotoluene has a log bioconcentrati on factor (BCF) of 1.30 in fish (Guo et al.

1 Information extracted from an abstract in English available from CAplus.RoC Background Document for o -Nitrotoluene

14 6/20/08 2004), [ingestion of fish should not be a very important source of human exposure to o nitrotoluene]. Accumula tion and depletion of o -nitrotoluene in carp ( Cyprinus carpio ) has been modeled (Hou et al. 1997). 1
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24 25 26 27 28 29 o -Nitrotoluene has been reported to be pres ent in the mainstream smoke of an 85-mm U.S. nonfilter cigarette at a level of 21.4 ng/ cigarette (Hoffmann and Rathkamp 1970); however, no other report of o -nitrotoluene in cigarette smoke was identified. [As noted above in Section 2.3.2, o -nitrotoluene has been detected in private well-water, suggesting the potential for exposure vi a groundwater ingestion.]

2.5 Occupational exposure 9 Limited information is availa ble on occupational exposure to o -nitrotoluene via inhalation. As noted above, th e compound was detected in ambient air at a chemical manufacturing plant in New Jersey , where a concentration of 47 ng/m 3 (0.000047 mg/m 3 ) was reported (IARC 1996). It also was detected in the air at concentrations of up to 2.0 mg/m 3 in the nitrotoluene production area of a chemical plant producing pharmaceuticals and explosives. Jones et al. (2005b) reported a mean 8- h TWA exposure level for o nitrotoluene of 0.759 mg/m 3 (ranging from undetected to 4.29 mg/m 3 ) for a group of 98 workers in a Chinese factory (Lianing Provi nce) manufacturing di nitrotoluene and 2,4,6- trinitrotoluene. As noted above (Section 2.2), production of o -nitrotoluene was reportedly carried out in only one U.S. facility, which according to its website has 134 employees, 50 contractors, and 20 retirees (FirstChem 2008). No additi onal information was found for the number of workers exposed to o -nitrotoluene in th e production of chemi cal intermediates.

2.6 Biological indices of exposure 23

o -Nitrobenzoic acid and o -nitrobenzyl alcohol [probably present as o -nitrobenzyl glucuronide] have been detected in the urine of workers exposed to o -nitrotoluene and were considered to provide a good marker for recently absorbed doses (Jones et al. 2005b, Sabbioni et al. 2006). Three potential biomarkers of exposure to o -nitrotoluene have been assessed in rats and mice: the urinary metabolites o -nitrobenzylmercapturic acid ( S -( o -nitrobenzyl)- N -acetylcysteine), o -aminobenzoic acid, and o -nitrobenzoic acid.RoC Background Document for o -Nitrotoluene

6/20/08 15 In a two-year study of dietary exposure of rats and mice to o -nitrotoluene (NTP 2002b), the concentrations of o -nitrobenzylmercapturic acid and o -aminobenzoic acid in the urine of mice were below the limit of quantitati on at most time points, and the ratio of o nitrobenzoic acid to cr eatinine in the urine of rats at 2 weeks and at 3, 12, and 18 months was linearly related to o -nitrotoluene exposure levels . The NTP did not consider o aminobenzoic acid to be a good biomarker for exposure to o -nitrotoluene because o aminobenzoic acid is a product of catabolism of tryptophan (White et al. 1978) and is a relatively minor metabolite of o -nitrotoluene (Chism et al. 1984). 1
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22 23 24 25 26 27 28 Jones et al . (2005a) measured hemoglobin adducts in Chinese workers exposed to the nitrotoluenes in a trinitrotoluene factory and found that for the mononitrotoluenes, the hemoglobin adduct of o -nitrotoluene was present in th e highest concentrations. The authors concluded that quantitation of hemoglobin adducts provides an effective biomarker of exposure to the nitrotoluenes.

Jones et al. (2005b) measured o -nitrobenzoic acid and o -nitrobenzyl alcohol in the urine of Chinese workers manufacturing dinitrot oluene and 2,4,6-trinitrotoluene. Mean concentrations of o -nitrobenzoic acid increased from 1,070 nmol/g creatinine preshift to 2,952 nmol/g creatinine postshift , while concentrations of o -nitrobenzyl alcohol increased from 55 to 213 nmol/g creatinine between pr e- and post-shift samples. However, the authors noted that no si gnificant correlation ( r = 0.17) was found for ai r concentrations of

o -nitrotoluene and the urinary metabolites. The importance of skin exposure was unknown. The American Conference of Governmental Industrial Hygienists (ACGIH) considers

o -nitrotoluene to be an inducer of meth emoglobin and recommends that methemoglobin in blood be used as a biological index of exposure to o -nitrotoluene (and the other nitrotoluene isomers) (ACGIH 2003). French et al . (1995) reported that o -nitrotoluene caused methemoglobin in sheep erythrocytes in vitro both with and without an NADPbioactivation system; however, work in other animal species such as rabbit and rat has shown that o -nitrotoluene has low to no methem oglobin-activating potency (AkahoriRoC Background Document for o -Nitrotoluene

16 6/20/08 1954 2 , Mlynarczyk and Sadowski 2001, Vasilenko 1976 2 ). [Where exposures are to technical grade nitrotoluene, most of the me themoglobin activity will be related to the concentration of the para- and meta - isomers rather than the ortho - isomer.] 1
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4 2.7.1 Regulations U.S. Department of Homeland Security Minimum requirements have been established for the safe transport of o -nitrotoluene on barges U.S. Department of Transportation (DOT) Considered a hazardous material; special re quirements have been set for marking, labeling, and transporting Safety measures after spills or l eaks are prescribed in accordance with o -nitrotoluene being a combustible toxic hazardous material
U.S. EPA 3 Comprehensive Environmental Respons e, Compensation, and Liability Act Reportable quantity (RQ) = 1,000 lb Occupational Safety and Health Administration (OSHA) Permissible exposure limit (PEL) = 5 ppm (30 mg/m 3 ) [skin] 4

2.7.2 Guidelines ACGIH Threshold limit value–time-weighted av erage (TLV-TWA) limit = 2 ppm [skin] Biological Exposure Index (BEI): methem oglobin in blood not to exceed 1.5% of hemoglobin measured during or at the end of a shift

2 Information extracted from an abstract in English available from CAplus. 3 EPA has not carried out an Integrated Ri sk Information System (IRIS) assessment for o -nitrotoluene. 4 The [skin] designation indicates the potential for dermal absorption; skin exposure should be prevented as necessary through the use of good work practices and gloves, coveralls, goggles, and other appropriate equipment.RoC Background Document for o -Nitrotoluene

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2.8 Summary 4

o -Nitrotoluene is used primarily to produce intermediates, such as o -toluidine, for the manufacture of azo dyes. It is also used (either directly or as an intermediate) in the production of other dyes, ag ricultural chemicals, rubbe r chemicals, pesticides, petrochemicals, pharmaceuticals, and explosives. o -Nitrotoluene is a HPV chemical, and production in the United States was reporte d to be greater than 10 million pounds in 2002. Little information is availabl e on environmental occurrence of o -nitrotoluene or on human exposure; however, since o -nitrotoluene is produced at high levels and is used in the production of many importa nt chemicals, human exposure is expected to be significant. o- Nitrotoluene has been detected in wo rkplace and ambient air, surface water, groundwater, and soils. In the United States, it ha s been measured in ambient air in Idaho and New Jersey (at a manufacturing plant). It has been detected in surface water or groundwater in the United States, France, the Netherlands, and Germany. It also can be formed as a breakdown product of di- or trinitrotoluenes, and o -nitrotoluene has been detected in the effluent or wastewater of plants producing these chemicals. The uses of di- and trinitrotoluenes include the producti on of commercial and military explosives, and

o -nitrotoluene has been detected in groundwat er, private well water, surface water, and soil at or near munitions production facilitie s and/or military training grounds in the United States and other countries. Envir onmental contamination and human exposure could also result from spills of o -nitrotoluene–containing materials in the environment. oNitrotoluene also was detected in mainstream smoke of a U.S. nonf ilter cigarette in one study. Biological indi ces of exposure to o -nitrotoluene include o -nitrobenzylmercapturic acid ( S -( o -nitrobenzyl)- N -acetylcysteine), o -aminobenzoic acid (also a product of catabolism of tryptophan and therefore not very useful as a biomarker for o -nitrotoluene), and o -nitrobenzoic acid in urine, and methem oglobin in blood. Hemoglobin adducts are another potential biomarker of exposure. The U.S. Department of Homeland Security,RoC Background Document for o -Nitrotoluene

18 6/20/08 DOT, EPA, and OSHA have set regulations, and ACGIH and NIOSH have set guidelines for o -nitrotoluene. 1
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## 3 Human Cancer Studies No human studies on the relationship be tween cancer and spec ific exposure to o nitrotoluene were identified; however, o -nitrotoluene may be used to manufacture magenta (see Section 2), and thus magenta manufacturing workers may be exposed to o nitrotoluene. IARC (1987, 1993) reviewed ma genta manufacturing and concluded that there is sufficient evidence in humans that the manufacture of magenta entails exposures that are carcinogenic (Group 1). As part of their assessment, they reviewed a case-control study and two cohort studies, one of which specifically identified o -nitrotoluene as an intermediate in the chemical processes. [The utility of these studies for evaluating the carcinogenicity of o -nitrotoluene is limited because the workers were also exposed to other chemicals, such as o -toluidine, 2,5-diaminotolue ne, and 4,4'-methylenebis(2- methylaniline), and exposure to o -nitrotoluene was not specifi cally assessed.] Studies on magenta manufacturing workers are described in Section 3.1. Studies of workers exposed to dinitrotoluenes or o -toluidine are briefly reviewed in Section 3.2. (The bioactivation of 2,4-dinitrotoluene is similar to that of o -nitrotoluene, and o-toluidine is a metabolite of onitrotoluene [see Section 5.5]). Section 3.3 discusses and summarizes the overall findings. 1
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22 23 24 25 26 27 28 29 3.1 Studies of magenta ma nufacturing workers 17 This section describes the cohort study (Rubino et al. 1982) that specifically mentions exposure to o -nitrotoluene, and briefly reports th e findings from the other two studies (Case and Pearson 1954, Vineis and Magna ni 1985) that do not mention whether o nitrotoluene was used in the manufacture of magenta. Rubino et al. conducted a cohort study of 906 male dyestuff fact ory workers in northern Ital y. Workers were included in the study if they had worked at least 1 month any time between 1922 and 1970, and the numbers of deaths were obser ved from 1946 to 1976; expected numbers of deaths were calculated using national rates for 1951 to 1976. Exposure was assessed based on knowledge of chemical processes for each job li sted in personnel records. Workers ever exposed to benzidine and naphthylamines were excluded from the an alysis. A significant excess of mortality due to bladder cancer was observed among the 53 fuchsin (magenta) and safranine T manufacturing workers (s tandardized mortality ratio [SMR] = 62.5; P <RoC Background Document for o -Nitrotoluene

20 6/20/08 0.001; 5 deaths). Laryngeal, esophageal, and lung cancer also showed statistically significant elevations in the entire cohort, but the small number of subjects in the magenta/safranine sub-cohort did not permit ev aluation of these other cancer types. The authors stated that the type of fuchsin ma nufactured at the plant was New Fuchsin or New Magenta. The manufacture of fuchsin and safranine T was carried out in two sections, and workers in both sec tions were potentially exposed to o -nitrotoluene. The first section (intermediates manuf acture) involved the synthesis of o -toluidine and 4,4'- methylenebis(2-methylaniline); o -nitrotoluene was an intermed iate in the manufacture of

o -toluidine from toluene. In the second s ection (fuchsin and safranine T manufacture), a mixture of o -toluidine, 4,4'-methylenebis(2-methylaniline), and o -nitrotoluene was heated to obtain fuchsin, and a mixture of o- toluidine and 2,5-diaminot oluene was oxidized in the presence of aniline to obtain safranine T. [This study had an excellent follow-up rate (96%). However, like all occupational cohor t SMR-based studies, the effect estimates utilized an external comparison group (to determine expected di sease rates). Rubino et al. used expected death rates for all Italy, in or der to give more precise estimates, but the workers were males from Northern Italy. A strength of the study is the fact that the authors studied a group of workers who were specifically exposed to o -nitrotoluene, although also exposed to o -toluidine and 4,4'-methylenebis(2-methylaniline) (but not to naphthylamines or benzidines).] Decarli et al . (1985) and Piolatto et al . (1991) reported on additional follow-up of the Italian dyestuff wo rkers, but no additional cases of bladder cancer in workers exposed to fuchsin or safranine T were identified. 1
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22 23 24 25 26 27 28 29 30 Case and Pearson (1954) conducted a cohort st udy of men who had worked at least 6 months in the manufacture of auramine a nd magenta in the British chemical industry between 1910 and 1952; workers exposed to benzidine or α - or β -naphthylamine were excluded from the study. The authors observed an earlier age at diagno sis, as well as an excess of bladder-cancer morta lity, in the cohort compared with the general population. A significant excess of bladde r-cancer mortality was observed among the 85 magenta manufacturing workers that were not invol ved in auramine manufacturing (SMR = 23.8; P

< 0.005; 3 observed cases; statistics reported by IARC 1993). [No information regarding exposure levels or duration or potential confounders was provided. It isRoC Background Document for o -Nitrotoluene

6/20/08 21 unknown whether the workers were exposed to o -nitrotoluene in this study because the authors did not describe the ma genta manufacturing process.] 1
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22 23 24 25 26 27 28 29 30 The third study on magenta manufacturing was a hospital-based, case-control study conducted in Italy between 1978 and 1983 that in cluded 512 cases of bladder cancer in males and 596 hospital contro ls (Vineis and Magnani 1985) . Exposure to specific chemicals was assessed using j ob titles, job activities, and kno wledge of industrial use of chemicals from the published literature. Vine is and Magnani looked at 74 chemicals as part of a job exposure matrix. [No adjustment s were made for multiple comparisons.] An increased risk of bladder cancer was f ound among workers potentially exposed to magenta (relative risk [RR] = 1.8, 95% conf idence interval [CI] = 1.1 to 2.9, when calculated using industrial br anches and RR = 3.0, 95% CI = 0.4 to 20.0, when calculated from job titles). [It is not clear whether the workers exposed to magenta were only involved in the manufacturing of magenta or also included workers using the dye magenta (who probably would not be exposed to o -nitrotoluene).]

3.2 Studies of workers expose d to dinitrotoluenes or o -toluidine 15 3.2.1 Dinitrotoluenes Several studies have investigated the potenti al for dinitrotoluenes to cause cancer in human subjects. In the first of these (Levine et al. 1986), the cause of death in 457 workers at two ammunition plants who had been exposed to dinitrot oluenes (76% to 98% 2,4-dinitrotoluene and 1% to 19% 2,6-dinitrotoluene, dependi ng on the process stage) for at least a month over a 10-year period was investigated. No significant increases in cancer mortality at specific sites were obs erved; however, the numbers of expected deaths were small. The authors did obse rve a non-significant excess of colon cancer (SMR = 186). A later study (Stayner et al. 1993), which examined nearly 5,000 munitions factory workers (< 10% of whom were part of the Levine et al. study) exposed to technical-grade dinitrotoluenes (76% 2,4- dinitrotoluene, 19% 2,6- dinitrotoluene, and 5% of the remaining four isomers), demonstr ated an excess of hepatobiliary cancer in exposed workers (standardized rate ratio = 3.88, 95% CI = 1.04 to 14.41) compared with 7,500 unexposed workers. No exposure-response relationship was demonstrated in this study; the authors noted a lack of workers with long durations of exposure and a lack ofRoC Background Document for o -Nitrotoluene

22 6/20/08 quantitative exposure information as possibl e reasons for this. Potential exposure to carcinogens other than dinitrotoluenes was a concern. A smaller study (Brüning et al. 1999, Brüning et al. 2002) of 500 underground miners e xposed to dinitrotoluenes through inhalation and direct skin contact with th e explosive Donarit, which contains 30% technical-grade dinitrotoluenes (75% 2,4-di nitrotoluene and 20% 2,6-dinitrotoluene), found a 4.5-fold increased incidence of urot helial cancer (6 cases) and a 14.3-fold increased incidence of renal-cell cancer ( 14 cases). [Confidence intervals were not provided.] Harth (2005) reported a cluster of 3 urothelial cancers among 60 workers exposed to 2,4-dinitrotoluene, with a calcu lated standardized incidence ratio (SIR) = 15.9. [Confidence intervals were not provided.] These data suggest the possibility of carcinogenicity of dinitr otoluenes in humans. 1
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22 23 24 25 26 27 28 29 30 31 3.2.2 o -Toluidine hydrochloride In an epidemiological study, Ward et al . (1991) evaluated the inci dence of bladder cancer in a chemical plant where the workers were e xposed to two potential bladder carcinogens,

o -toluidine and aniline. An excess of bl adder cancer cases was observed (7 cases observed versus 1.08 expected ) among all the 1,749 worker s in the plant (SIR = 3.60, 90% CI = 2.13 to 5.73). The SIR for bladde r cancer among the definitely exposed was 6.48 (95% CI = 3.0 to 12.2), which increased to 27.2 (95% CI = 11.8 to 53.7) for workers who were employed in the department with o -toluidine and aniline exposure for ≥ 10 years. Increased risk of bladder cancer was st rongly associated with increased length of employment in the departments where o -toluidine and aniline were used. The authors collected smoking history on a 5% sample of workers using compa ny medical records, and used modeling techniques to estimate that only a small proportion of the elevated SIR (1.05) could be attributed to higher smoking in the cohort ve rsus the general population. [This study has the advantage of calculating the SIRs (using incidence rates) rather than SMRs (using death certificates ).] The authors concluded that o -toluidine was more likely than aniline to be responsi ble for the bladder cancer exce ss, in view of the greater carcinogenic potency of o -toluidine than aniline in an imals. Stasik (1988) found excess bladder cancer incidence (SIR = 72.7, 95% CI = 31.4 to 153.3) among workers exposed to 4-chloro- o -toluidine, o -toluidine and two ot her monocyclic amines. o -Toluidine and its hydrochloride salt are listed in the Report on Carcinogens as reasonably anticipated to beRoC Background Document for o -Nitrotoluene

6/20/08 23 a human carcinogen based on limited evidence in huma ns and sufficient evidence in animals (NTP 2004), and o -toluidine is classified as carcinogenic to humans (Group 1) by IARC (Baan et al. 2008). 1
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4 Two cohort studies and one cas e-control study have reported excess risk of bladder cancer among magenta manufacturing workers. Efforts were made to exclude workers exposed to benzidine and α - and β -naphthylamine in the cohort studies, but there were limited exposure data (e.g., levels, durati ons, etc.), limited information on potential exposure to other agents, and small numb ers of exposed workers. Most of the aforementioned studies did not address othe r potential confounders such as cigarette smoking or genetic acetylato r phenotype (e.g., NAT1, NAT2 genotypes). However, the relative risks associated with smoking and genetic susceptibility as well as other risk factors are small (RR = 2 to 4) relative to those associated with dye manufacture. However, the risk estimates in the cohorts were very high, and the IARC working group concluded that the manufacture of magenta entails exposures that are carcinogenic (Group 1). Suspected substances used in th e manufacturing process and thought to cause cancer include o- nitrotoluene, magenta, o -toluidine, and 4,4'-methylenebis(2- methylaniline) (Siemiatycki et al. 2004). These studies are li mited for the evaluation of the carcinogenicity of o -nitrotoluene in humans. While o -nitrotoluene was specifically mentioned in the Italian cohort study, it was not specifically mentioned in the British chemical workers or the case-control study. Ev en in the Italian c ohort study, in which it was known that the workers were exposed to o -nitrotoluene, the workers were also exposed to other suspected human carcinogens, such as o -toluidine, and 4,4'- methylenebis(2-methylaniline), so it is not pos sible to evaluate whether specific exposure to o -nitrotoluene contributed to the increased risk of bladder cancer in these workers. o Toluidine is classified by IARC as carcinogenic to humans (Group 1) and listed in the Report on Carcinogens as reasonably anticipated to be a human carcinogen. 4,4'- Methylenebis(2-methylaniline) is classified by IARC as possibly carcinogenic to humans (Group 2B).RoC Background Document for o -Nitrotoluene

24 6/20/08 Limited epidemiologic studies on dinitrotoluene s are available and suggest elevations in hepatobiliary, urothe lial, renal, and possibly co lon cancer. Two studies on o -toluidine reported excess bladder cancer incidence.RoC Background Document for o -Nitrotoluene

6/20/08 25 

## 4 Studies of Cancer in Experimental Animals 1 The results of publicly available, peer-re viewed studies of the carcinogenicity of

o -nitrotoluene in experimental animals are summarized in this section. No studies of exposure by routes other than oral admi nistration were found. IARC (1996) evaluated o nitrotoluene (2-nitrotolu ene) and considered it not classifiable as to its carcinogenicity to humans (Group 3) based on inadequate evidence in humans and limited evidence in experimental animals. At the time of th e IARC review, no chronic bioassays were available (this was prior to the publication of the NTP two-year bioassay), and IARC’s conclusions were based on NTP’s sub-chr onic study (13 weeks), which reported rare mesotheliomas in male rats receiving o -nitrotoluene. 2 3 4 5 6 7 8 9 10

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27 28 29 Section 4.1 summarizes the studie s with rats, and S ection 4.2 summarizes the studies with mice. Findings in experimental animals are summarized in Section 4.3. The carcinogenicity of the other nitrotoluene is omers and related compounds is discussed in Section 5.5.

4.1 Rats 15 The NTP conducted 13-week studies with F344/N rats exposed to o -, m -, and p nitrotoluene to compare c linical toxicity, histopat hology, and reproductive system toxicity among the isomers (NTP 1992). A follow-up subchronic exposure study was conducted (NTP 1996) to confirm carcinogenic effects observed in the 1992 study and to compare the carcinogenicity and toxicity of o -nitrotoluene and o -toluidine hydrochloride (Section 4.1.1). Finally, NTP conducted a tw o-year toxicology and carcinogenesis bioassay of o -nitrotoluene in the diets of male and female F344/N rats (NTP 2002a) (Section 4.1.2). [The NTP chronic bioassays are conducted under FDA Good Laboratory Practice regulations and represent a high qual ity data set for making conclusions about the potential carcinogenicity of o -nitrotoluene.] The findings of these studies related to the carcinogenicity of o -nitrotoluene are summar ized in this section.

4.1.1 Subchronic exposure In 13-week studies, groups of 10 male a nd 10 female F344/N rats per treatment group received diets ad libitum containing o -nitrotoluene (> 96% purity) at concentrations of 0,RoC Background Document for o -Nitrotoluene

26 6/20/08 625, 1,250, 2,500, 5,000, or 10,000 ppm [approx. 40 to 700 mg/kg per day] (Dunnick et al. 1994, NTP 1992). The animals were 6 to 8 weeks old at the beginning of the study. All animals survived to the end of the studies and there were no treatment-related clinical signs of toxicity. Final body weights of tr eated males and females were significantly reduced in a dose-related fashion [1,250 ppm, P ≤ 0.05; 2,500 ppm and greater doses, P ≤ 0.01]. No treatment-related lesions were found in female rats. Two male rats in the highdose group had mesothelial- cell hyperplasia of the tunica vaginalis on the surface of the epididymis (which was considered to be a preneoplastic lesion), and mesothelioma occurred at the same anatomic location in three male rats in the 5,000-ppm group. No other treatment-related lesions were found in male rats in the other dose groups. At the time of this study, mesothelioma had not previo usly been identified in exposed or control rats from any of the 13-week toxicity st udies conducted by NTP (with any substance). The authors concluded that o -nitrotoluene was carcinogenic in male rats, based on the occurrence of mesothelioma a nd mesothelial hyperplasia. 1
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26 27 28 In a subsequent study (NTP 1996), male rats (45 days old at the beginning of the study) received o -nitrotoluene (99% purity) in feed for either 13 or 26 weeks at a concentration of 5,000 ppm [292 to 296 mg/kg per day]. Th e dose was based on the results of the 1992 study, in which o -nitrotoluene in the diet at 5,000 pp m caused mesothelioma but had little effect on survival or body weight. o -Nitrotoluene was administ ered to 60 rats, and the control group consisted of 20 rats. After 13 weeks of o -nitrotoluene exposure, 10 controlgroup rats and 20 treated rats were killed for a 13-week interim evaluation. Following the 13-week evaluation, administration of o -nitrotoluene was discontinued for 20 rats for the remaining 13 weeks (stop-exposure group), a nd the remaining 20 rats continued to receive o -nitrotoluene (26-week exposure). After 26 weeks, all remaining rats were killed, and necropsies and histopa thologic exams were performed.

All rats survived until the scheduled evaluations. Mean body- weight gain was lower in all exposed groups than in controls. No other c linical effects of exposure were observed in either group.RoC Background Document for o -Nitrotoluene

6/20/08 27 Mesothelial-cell hyperplasia (described as a potential preneoplastic lesion) or mesothelioma occurred on the mesothelial surface of the tunica vaginalis of the testis or epididymis of rats in both the 13-week stop-exposure and the 26-week-exposure groups. No tumors were observed after 13 weeks; however, by 26 weeks, mesothelioma in the epididymis was seen in 7 of 20 rats in the continuous-exposure group and in 5 of 20 in the stop-exposure group (epididymis and te stis combined). At 26 weeks, focal hyperplasia of the epididymal mesothelium also was seen in 4 rats from these two groups. Significant increases in the number of rats w ith mesothelioma of the epididymis were noted in the 26-week exposure group. At 26 weeks, 3 cholangiocarcinomas were observed (2 from the stop-exposure group and 1 from the 26-week-exposure group). The authors concluded that these studies confirmed the carcinogenicity of o -nitrotoluene, based on the high incidence of mesothelioma and occurrence of cholangiocarcinoma in male rats after short-term exposure. The results are shown in Table 4-1. 1
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22 23 24 25 26 27 28 29 The NTP (1996) also conducted 13-week and stop-exposure studies using rats with altered flora to assess the pot ential role of metabolism by gastrointestinal (GI) flora in o nitrotoluene-induced toxicity. Rats in the altered-flora groups received a single gavage dose of an antibiotic mixture in water daily for 6 days before the start of the study and daily for 13 weeks thereafter. Exposure conditio ns were similar to those described above (normal flora) except that there was no 26- week-exposure group. All rats survived until the scheduled evaluations. A consistent finding in rats with normal or altered intestinal flora was mesothelial-cell hyperplasia or me sothelioma. These lesions occurred on the mesothelial surface of the tunica vaginalis of the testis or epidi dymis of rats in 25% to 40% of the 13-week stop-exposure groups (Table 4-1). Mesothelioma also occurred in 2 of 20 rats with altered intestin al flora after 13 weeks. In cont rast to the study using rats with normal intestinal flora, no cholangi ocarcinomas were observed. The NTP was not able to draw any conclusions about the involvement of GI flora in the pathogenesis of o nitrotoluene-induced lesions because of the lo wer effectiveness of the antibiotic mixture against obligate anaerobic bacteria and the possible development of resistant aerobic bacteria after one week of antibiotic administration.RoC Background Document for o -Nitrotoluene

28 6/20/08 Table 4-1. Neoplastic lesions identified in F344/N male rats following dietary exposure to o -nitrotoluene for 13 or 26 weeks Tumor incidence (%) Mesothelioma Liver Exposure/GI flora status Conc. (ppm) N Testis Epididymis Combined Cholangiocarcinoma Normal flora 0 10 0 (0) 0 (0) 0 (0) 0 (0) 13 weeks 5,000 20 0 (0) 0 (0) 0 (0) 0 (0) 0 b 10 0 (0) 0 (0) 0 (0) 0 (0) Stop exposure a 5,000 20 2 (10) 4 (20) 5 (25) 2 (10) 0 b 10 0 (0) 0 (0) 0 (0) 0 (0) 26 weeks 5,000 20 2 (10) 7 (35)* 7 (35)* 1 (5) Altered flora c 0 10 0 (0) 0 (0) 0 (0) 0 (0) 13 weeks 5,000 20 0 (0) 2(10) 2(10) 0 (0) 0 10 0 (0) 0 (0) 0 (0) 0 (0) Stop exposure a 5,000 20 4 (20) 8 (40)* 8 (40)* 0 (0) Source: NTP 1996. * P ≤ 0.05 (compared with the control group by Fisher’s exact test).
a Rats were exposed to o -nitrotoluene in feed for 13 weeks and allowed to recover for 13 weeks. b The group of 10 control rats with normal GI flora k illed at 26 weeks served as the control group for both

the stop-exposure and 26-week groups.
c Rats in the altered-flora groups received a single gavage dose of an antibiotic mixture in water daily for 6

days before the start of the study and daily for 13 weeks thereafter. After 13 weeks, liver weight wa s greater in rats exposed to o -nitrotoluene than in controls [ P ≤ 0.01], and oval-cell hyperplasia was observed. Liver weight continued to increase and oval-cell hyperplasia persisted in bot h the stop-exposure and 26-week-exposure groups [ P ≤ 0.01]. Placental glutathione S -transferase positive (PGST+) foci (preneoplastic lesions) in the liver were sign ificantly increased in exposed groups. At the end of the 13-week recovery period, the numbe rs of foci observed in the stop-exposure groups (with both normal and altered flora) were less than in the 13-week-exposure groups but significantly greater [ P ≤ 0.01] than in controls (NTP 1996). In a separate analysis of the data on PGST+ foci in the rats with normal flora (Ton et al. 1995), the 26- week continuous-exposure group had more and la rger foci than the 13-week continuousexposure group [ P < 0.05]. Although the stop-exposure group at 26 weeks had fewer foci, the mean volume of foci was larger than in the 13-week continuous-exposure group. 1
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6/20/08 29 4.1.2 Chronic exposure 1 2 3 4 5 6 7 8 9 10
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27 28 29 NTP (2002b) performed a two-year study to investigate the chronic toxicity and carcinogenicity of o -nitrotoluene in the diet of rats. o -Nitrotoluene concentrations were based on the results of the subchronic toxi city studies (NTP 1992). In the core study, groups of 60 male and 60 female F 344/N rats were fed diets containing o -nitrotoluene (> 99% purity) at a concentration of 625, 1,250, or 2,000 ppm for 105 weeks. These dietary concentrations were equivalent to averag e daily doses of approximately 25, 50, or 90 mg/kg body weight (b.w.) for males and 30, 60, or 100 mg/kg b.w. for females. In a stopexposure study, groups of 70 male rats were fed diets containing o -nitrotoluene at a concentration of 2,000 or 5,000 ppm (equivalent to an aver age daily dose of roughly 125 or 315 mg/kg b.w.) for 13 weeks, after which o -nitrotoluene administration was discontinued for the remainder of the study (t wo years). Control groups consisted of 70 males and 60 females. All animals were 6 to 7 weeks old at the beginning of the study. After 13 weeks, 10 males from each stop-exposure group and 10 control males were killed for evaluation. In the stop-exposure study, all rats in th e 5,000-ppm group died before the end of the study, and only 11 rats in the 2,000-ppm group surv ived. Early deaths were attributed to the development of neoplasms (Dunnick et al. 2003). Absolute and relative liver weights of the rats in the 5,000-ppm group were significantly greater th an those of the controls [ P ≤ 0.01]. In the two-year chronic-e xposure study, survival was re duced in all exposure groups among males and in the highest exposure group among females because of the development of neoplasms. Feed consumpti on was similar between exposed and control groups; however, mean body weights were lowe r in all exposed gr oups (except the 625- ppm group of males). Non-neoplas tic lesions included alveolar epithelial hyperplasia in males and females and mammary hyperplasia in females. As shown in Tables 4-2a and b, similar tumor profiles were observed in males in the stop-exposure study and in the two-year chr onic-exposure study. Significantly increased incidences of malignant mesothelioma (most were associated with the tunica vaginalis ofRoC Background Document for o -Nitrotoluene

30 6/20/08 the testis or epididymis, but some were observed on the abdominal wall or surface of abdominal organs) and skin subcutaneous neoplasia (both lipoma and fibroma or fibrosarcoma) were observed in all exposure groups; mammary-gland fibroadenomas also were increased in all exposure groups ot her than high-dose males in the two-year chronic-exposure study. High incidences of s ubcutaneous skin fibroma or fibrosarcoma and malignant mesothelioma were obser ved in the high-dose groups (over 70%, compared with less than 10% in controls). Significantly increased incidences of hepatocellular adenoma or carcinoma (combine d) were observed in the high-dose groups in both the chronic and stop-exposure studi es, and increased incidences of liver cholangiocarcinoma and alveolar /bronchiolar lung tumors we re observed in the high-dose (5,000-ppm) group in the stop-exposure study. He patocholangiocarcinoma occurred in two male rats, one exposed to 625 ppm a nd the other to 2,000 ppm. Even though the incidence was low, the NTP considered these to be exposure related. No hepatocholangiocarcinomas or cholangiocarci nomas had been observed in male controls fed either the NTP-2000 or NI H-07 diet in two-year bi oassays. The incidence of hemangioma or hemangiosarcoma (combined) was significantly increased in the highdose males in the stop-exposure study, but th ese tumors were not considered by the authors to be exposure related. 1
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22 23 24 25 26 27 28 29 In females, a significantly increased in cidence of mammary-gl and fibroadenoma was observed in all exposure groups, and the inci dence of subcutaneous skin fibroma or fibrosarcoma was significantly increased at th e two highest dose levels. The incidence of hepatocellular adenoma in females was si gnificantly increased only in the high-dose (2,000-ppm) group. The incidence of mononuc lear-cell leukemia was significantly reduced in all exposure groups of males and females in the two-year chronic exposure study and in the males in th e stop-exposure study. In the stop-exposure study, the combined incidence of bilateral or unilateral interstitial-cell adenoma of the testis also was significantly decreased in the high-dos e (5,000-ppm) group. The NTP suggested that these decreased tumor incidences were associated with toxicity at the tissue site (spleen or testis).RoC Background Document for o -Nitrotoluene

6/20/08 31 The NTP concluded that there was clear evidence of carcinogenic activity of o nitrotoluene in male and female rats, ba sed on increased incidences of malignant mesothelioma (in males only), subcut aneous skin neoplasia, mammary-gland fibroadenoma, and liver neopl asia (in males only). The in creased incidences of lung neoplasia in male rats and hepa tocellular adenoma in female ra ts also were considered to be exposure related. 1
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.RoC Background Document foro-NitrotolueneTable 4-2a. Neoplastic lesions identified inF344/N rats following dietary exposure too-nitrotoluene for two yearsTumor incidence (%)aMammary glandSkin subcutaneousSexNConc.(ppm)MalignantmesotheliomaFibroadenomaLipomaFibroma/fibrosarcomaChronic exposure6002 (3.7)0 (0)0 (0)5 (9.3)6062520 (40.6)***7 (15.6)**4 (8.9)*47 (86.3)***601,25029 (62.4)***10 (26.2)***13 (33)***55 (98.7)***602,00044 (87.1)***2 (9.0)13 (44.8)***59 (99.8)***609HC23 (3.7)26 (3.8)8 (1.5)41 (6.4)trend testbP< 0.001P< 0.001P< 0.001P< 0.001Stop exposure602,00044 (80.3)***13 (31.2)***10 (24.6)***47 (89)***605,00054 (95.1)***20 (61.1)***12 (44.7)***53 (97.8)***cMtrend testbP< 0.001P< 0.001P< 0.001P< 0.001Chronic exposure6000 (0)23 (40)0 (0)3 (5.3)606250 (0)47 (82.8)***0 (0)3 (5.4)601,2500 (0)52 (91.7)***0 (0)21 (37.6)***602,0000 (0)56 (96.2)***0 (0)22 (40.6)***659HCNR284 (41.1)NR18 (2.6)Ftrend testb–P< 0.001–P< 0.001Sources: Dunnicket al.2003, NTP 2002b.*P≤0.05, **P< 0.01, ***P< 0.001 (compared with the concurrent control group by the Poly-3 test).HC = historical controls – incidences include data from all NTP contract laboratories for all F344/N rat control groups given the NTP-2000 diet (1995 to the present).NR = historical incidence data not reported in NTP 2002b.aPoly-3 estimated incidence after adjustment for intercurrent mortality.bPoly-3 test used to test for significant dose-related trend.cIncludes one sarcoma in an animal that also had a fibroma.326/20/08RoC Background Document foro-Nitrotoluene6/20/0833Table 4-2b. Neoplastic lesions identified inF344/N rats following dietary exposure too-nitrotoluene for two years (continued)Tumor incidence (%)aLiverLungSexNConc(ppm)HepatocellularadenomaHepatocellularadenoma/carcinomacCholangiocarcinomaHepatocholangiocarcinomaAlveolar/bronchiolaradenoma/carcinomaChronic exposure6002 (3.7)3 (5.6)0 (0)0 (0)2 (3.7)606253 (6.8)3 (6.8)0 (0)1 (2)5 (11.2)601,2503 (8.4)3 (8.4)0 (0)0 (0)1 (2.9)602,0007 (27.1)**8 (30.2)**0 (0)1 (2)2 (8.7)609HC5 (0.8)10 (1.8)0 (0)0 (0)26 (4.5)trend testbP= 0.007P= 0.009–NRP= 0.39Stop-exposure602,0003 (7.6)3 (7.6)0 (0)0 (0)3 (7.6)605,0004 (18.4)*6 (25.9)*3 (13.9)*0 (0)11 (42)***Mtrend testbP= 0.062P= 0.03–P< 0.001Chronic exposure6001 (1.8)1 (1.8)0 (0)0 (0)1 (1.8)606250 (0)0 (0)0 (0)0 (0)2 (3.6)601,2501 (1.9)1 (1.9)0 (0)0 (0)0 (0)602,0006 (11.2)*6 (11.2)*0 (0)0 (0)4 (7.5)659HC4 (0.7)NRNRNRNRFtrend testbP= 0.005P= 0.005––P= 0.126Sources: Dunnicket al.2003, NTP 2002b.*P≤0.05, **P< 0.01, ***P< 0.001 (compared with the concurrent control group by the Poly-3 test).HC = historical controls – incidences include data from all NTP contract laboratories for all F344/N rat control groups given the NTP-2000 diet (1995 to the present).NR = historical incidence data not reported in NTP 2002b.aPoly-3 estimated incidence after adjustment for intercurrent mortality (adjusted rates were not reportedfor hepatocholangiocarcinoma).bPoly-3 test used to test for significant dose-related trend.cNo hepatocellular carcinomas reported infemale mice; therefore, combined incidence is the same as adenoma incidence.RoC Background Document for o -Nitrotoluene

34 6/20/08 4.2 Mice 1 NTP (1992) conducted a 13-w eek subchronic study of o -nitrotoluene in B6C3F 1 mice. No tumors were reported; therefore, this study is summarized in Section 5.6. In a twoyear study to investigate the chroni c toxicity and carcinogenicity of o -nitrotoluene in B6C3F 1 mice (NTP 2002b), groups of 60 male and 60 female mice were fed diets containing o -nitrotoluene (> 99% purity) at a concentr ation of 0, 1,250, 2,500, or 5,000 ppm for 105 weeks. These concentrations we re based on the results of the subchronic exposure study and were equivalent to aver age daily doses of approximately 165, 360, or 700 mg/kg b.w. for males and 150, 320, or 710 mg /kg b.w. for females. The mice were 6 to 7 weeks old at the beginning of the study. In addition to the concurrent controls, NTP (2002b) also maintains a databa se of tumor incidences in untreated or control groups from all NTP-sponsored carcinogenicity studi es. The tumor incidences in historical controls were reported by NTP (2002b) for a ll studies that used the NTP-2000 diet and are included in data summary tables below. The NTP-2000 diet contains less protein and more fiber than the previous diet and was first used in 1995. 2 3 4 5 6 7 8 9 10
11
12 13 14 15
16 17 18 19 20
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22 23 24

25 26 27 28 29 30 All males in the two highest-exposure groups died before the end of the study. Survival of males in the 1,250-ppm group and female s in the 5,000-ppm group was significantly less than that of controls. Early developm ent of neoplasms was the primary cause of reduced survival in treated animals (Dunnick et al. 2003). Mean body weights were less for all exposed groups of males than for controls, and females in the 5,000-ppm group generally weighed less than controls. Non- neoplastic pathology findings in males and females included significantly increased inciden ces of eosinophilic and basophilic foci in the liver, renal tubule pigmentation, edema of the subcutaneous tissue, olfactory epithelial degeneration, and, in males only, chronic inflammation of the prostate gland. The neoplastic pathology findings for both male and female mice are summarized in Table 4-3a for hemangiosarcomas and Table 4- 3b for other significant neoplasms. All of the males and 50 out of 60 of the females in the high-dose (5,000-ppm) groups developed hemangiosarcoma, and the incidence was signifi cantly increased in all exposure groups of males and in the high-dose group of females (the tumor incidence rate in the females after adjustment for survival by the Poly -3 test was 90%). The hemangiosarcomasRoC Background Document for o -Nitrotoluene

6/20/08 35 occurred primarily in the mesentery, skeletal muscle, and subcutis of the skin. Incidences at each location were significantly increased in all male treatment groups and the highdose females (Table 4-3a). Some mice with multiple or large hemangiosarcomas had small hemangiosarcomas in other tissues, in cluding the lung, liver, spleen, uterus, or prostate. Most of these were considered to be metastases. A few male mice in the control group had hemangiosarcomas in the spleen or liver. The incidence of carcinoma of the large intestine (cecum) was significantly in creased in the low-dose (1,250-ppm) and middose (2,500-ppm) groups of males, and the in cidences of hepato cellular adenoma and hepatocellular adenoma or carcinoma (combined) were significantly increased in the middose (2,500-ppm) and high-dose (5,000-ppm) gr oups of females. The incidence of hepatocellular carcinoma was significantly incr eased in the high-dose females (16 out of 60 animals positive with a survival-adjusted ra te of 36.2%). Nonsignificantly increased incidences of carcinoma of the large intestin e (cecum) in females were considered by the NTP to be exposure related, because this neop lasm is extremely rare and had not been seen in females in the historical contro l groups. [The incidence of large-intestine carcinoma in both the 2,500-ppm and 5,000-ppm fe male mice was significantly increased ( P < 0.001) compared with historical controls by Fisher’s exact test.] These tumors were characterized by proliferati on of glandular structures composed of moderately pleomorphic mucosal epithelial ce lls that invaded the cell wall. 1
2 3 4 5 6 7 8 9 10
11
12 13 14 15 16 17 18 19

20
21
22 23 Based on these results, the NT P concluded that there was clear evidence of carcinogenic activity of o -nitrotoluene in male and female mi ce, based on increased incidences of hemangiosarcoma (in males and females), carc inoma of the large intestine (cecum) (in males), and hepatocellular neoplasia (in females).RoC Background Document for o -Nitrotoluene
Table 4-3a. Hemangiosarcomas in B6C3F 1 mice following dietary exposure to o nitrotoluene for two years Hemangiosarcoma incidence (%) a

Sex N Conc. (ppm) Mesentery Skeletal muscle Subcutis All organs b 60 0 0 (0) 0 (0) 0 (0) 4 (7) 60 1,250 8 (15.8)** 6 (11.7)* 4 (8)* 17 (32.7)*** 60 2,500 38 (83)*** 33 (79.1)*** 8 (29.4)*** 55 (97.9)*** 60 5,000 38 (92.8)*** 45 (95.3)*** 20 (77)*** 60 (100)*** Male

659 HC NR NR NR 37 (5.8) 60 0 0 (0) 0 (0) 0 (0) 0 (0) 60 1,250 0 (0) 0 (0) 0 (0) 2 (3.6) 60 2,500 0 (0) 0 (0) 2 (3.5) 3 (5.2) 60 5,000 32 (65.5)*** 16 (35.6)*** 19 (43.2)*** 50 (90.2)*** Female

659 HC NR NR NR 15 (2.6) Table 4-3b. Neoplastic lesions of th e large intestine and liver in B6C3F 1 mice following dietary exposure to o -nitrotoluene for two years Tumor incidence (%) a

Sex N Conc. (ppm) Large-intestine carcinoma Hepatocellular adenoma/carcinoma 60 0 0 (0) 27 (46.1) 60 1,250 12 (22.7)*** 28 (53.7) 60 2,500 9 (31.6)*** 7 (26.7) 60 5,000 0 (0) c 2 (18.5) Male
659 HC 1 (0.2) 304 (47.8) 60 0 0 (0) 9 (15.7) 60 1,250 1 (1.9) 9 (16.9) 60 2,500 4 (7.0) d 24 (42.1)*** 60 5,000 3 (7.4) d 39 (79.1)*** Female

655–659 HC 0 (0) 143 (22.8) Source: Dunnick et al. 2003, NTP 2002b. * P < 0.05, ** P < 0.01, *** P < 0.001 (compared with concurrent controls by the Poly-3 test). HC = historical controls – incidences include data from all NTP contract laboratories for all B6C3F 1 mouse

control groups given the NTP-2000 diet (1995 to the present ). NR = historical control incidence not reported in NTP 2002b.
a Poly-3 estimated percent incidence after adjustment for intercurrent mortality. b Includes hemangiosarcomas of the liver, lung, spleen, and prostate in males and the uterus in females.
c Dunnick et al. (2003) and NTP (2002b) suggested that the high-dose male mice did not survive long

enough for tumors to develop in the large intestine. d [Significantly different ( P

< 0.001) from historical controls (0/659) by Fisher’s exact test.]

36 6/20/08RoC Background Document for o -Nitrotoluene

6/20/08 37 4.3 Summary 1 The carcinogenicity of o -nitrotoluene was evaluated in feeding studies in rats and mice. Malignant mesothelioma was first observed in male rats administered o -nitrotoluene for 13 weeks. Tumors at multiple tissues sites were observed in a stop-exposure study in male rats and two-year chronic studi es in both sexes of rats and mice. o -Nitrotoluene caused tumors of the mammary gland, skin (s ubcutaneous), liver, lung, and mesothelium of the epididymis and testis in rats and of the large intestine (cecum), liver, and circulatory system (hemangiosarcoma) in mice. The findings are summarized in Table 4- 4. 2 3 4 5 6 7 8 9
Table 4-4. Neoplastic lesions identified in rats and mice following dietary exposure to o -nitrotoluene F344/N rats B6C3F 1 mice Organ or system Tumor type Male Female Male Female Mesothelium malignant mesothelioma lipoma Skin (subcutaneous) fibroma or fibrosarcoma Mammary gland fibroadenoma hepatocellular carcinoma hepatocellular adenoma or carcinoma a a
chlolangiocarcinoma Liver

hepatocholangiocarcinoma + b
Lung alveolar/bronchiolar adenoma or carcinoma Large intestine (cecum) carcinoma + c Circulatory system hemangiosarcoma

= Significantly greater incidence than in the concurrent control group ( P

< 0.05).
+ = Higher incidence than in the control group.

a Based on adenomas found in female F344/N rats. b Only 2 tumors were observed but they are very rare , and the NTP used them as part of their call.
c Not significantly different from concurrent controls, [but highly significant ( P

< 0.001) compared with NTP historical controls (0/659)].RoC Background Document for o -Nitrotoluene

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38 6/20/08RoC Background Document for o -Nitrotoluene

6/20/08 39 

## 5 Other Relevant Data 1
This section discusses releva nt mechanistic and other information needed to understand the toxicity and potential carcinogenicity of o -nitrotoluene. It includes information on absorption, distribution, meta bolism, and excretion of o -nitrotoluene (Section 5.1); bioactivation of o -nitrotoluene (Section 5.2); genetic da mage and related effects (Section 5.3); mechanistic studies a nd considerations (Section 5.4); carcinogenicity and genotoxicity of o -nitrotoluene metabolites and anal ogues and metabolites (Section 5.5); and other toxic effects of o -nitrotoluene (Section 5.6). Differences in metabolism among the three nitrotoluene isomers offer insight into possible mechanisms of carcinogenicity or mutagenicity. Therefore, the discussions of metabolism and mechanisms include data

on m - and p -nitrotoluene, in addition to o -nitrotoluene. The data in this section are summarized in Section 5.7. 2 3 4 5 6 7 8 9 10
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14 15 16 17 18 19 20
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23 24 25 26 27 28 29 5.1 Absorption, distribution, metabolism, and excretion 13 This section reviews the avai lable information for the absorption, distribution, and/or excretion of o -nitrotoluene in humans (Section 5.1.1) and in rodents (Section 5.1.2). This is followed by a description of the in vitro metabolism of the three nitrotoluene isomers (Section 5.1.3), the in vivo metabolism in rodents of o -nitrotoluene (Section 5.1.4) and the other two nitrotoluene isomers (Sec tion 5.1.5), a summary of the comparative metabolism data (Section 5.1.6), and a descript ion of biliary excretion (Section 5.1.7) of the three nitrotoluene isomers. Additional information on the in vitro and in vivo metabolism of nitrotoluenes can be found in reviews from the mid 1980s by Rickert et al

. (1986) and Rickert (1987).

5.1.1 Human data Limited data in humans were available. Information on absorption of o -nitrotoluene in humans is based on evidence that hemoglobin adducts in humans we re increased after exposure to o -nitrotoluene and/or 2,4- and 2,6-dinitrotoluene (Jones et al. 2005a). Jones et al. (2005b) also reported that the o -nitrotoluene metabolites o -nitrobenzoic acid and o nitrobenzyl alcohol [p robably present as o -nitrobenzyl glucuronide] were detected in the urine of 97% and 99%, respectively, of the workers occupationally exposed toRoC Background Document for o -Nitrotoluene

40 6/20/08 o -nitrotoluene in a factory in China (see S ection 2.6). The average concentration of the nitrobenzoic acid metabolite was more than 10-fold higher than the nitrobenzyl alcohol derivative. Post-shift urine samples contained about 3- to 4-fold higher concentrations of the metabolites than measured in pre-shift ur ine samples. Based on the lack of significant correlation between o -nitrotoluene detected in the air and the concentrations of urinary metabolites, the authors suggested that de rmal exposure makes up a large proportion of total exposure. Ahlborg et al. (1988) also reported that levels of diazo-positive compounds in the urine of workers expos ed to aromatic nitroamino compounds, including nitrotoluenes, were significantly higher after a wo rk shift than in unexposed workers, but no data on exposure specifically to o -nitrotoluene were reported. 1
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11 12 13 14 15 16 17 18 19 20
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22 23 24 25 26 27 28 29 30 5.1.2 Rodent data Absorption of o -nitrotoluene after oral administration has been studied in rats and mice. In the NTP (2002b) study male and female F344 rats and male B6C3F 1 mice received [ 14 C]- o -nitrotoluene by gavage at a dose of e ither 2 or 200 mg/kg b.w. The concentration of o -nitrotoluene in plasma peaked at 10,000 ng/g between 15 and 60 minutes postadministration of the 200 mg/kg b.w. dose in male rats but was not measurable at 24 hours post-administration. Male F344 rats excreted approximately 86% of the 200-mg/kg b.w. dose in urine within the first 24 hours wh ile urinary excretion in female rats was slightly higher at 92%. Male B6C3F 1 mice excreted approximately 66% of a 200-mg/kg b.w. dose during the first 24 hours. Excreti on of radioactivity (parent compound and metabolites) following a 2-mg/kg b.w. gavage dose of [ 14 C]- o -nitrotoluene was similar to that with the higher dose, with about 98% of the dose excreted in the urine of rats and 60% of the dose in mice in the first 24 hours after dosing. Fecal elimination accounted for 3% of the dose in male rats, 4% in female rats, and 9% in male mice. Absorption of

o -nitrotoluene also was reflected in the excretion of metabolites of o -nitrotoluene by male F344 rats after oral administration of [ 14 C]- o -nitrotoluene at a dose of 200 mg/kg b.w. More than 85% of the total oral dose was r ecovered in the urine within 24 hours, with lesser amounts in the feces (4.6% ) and expired air (0.1%) (Chism et al. 1984). These results indicate that most of the dose of o -nitrotoluene was absorbed and metabolized in rats and mice.RoC Background Document for o -Nitrotoluene

6/20/08 41 Another study in male and female F344 rats (Chism and Rickert 1985) demonstrated that 29% of an oral dose (200 mg/kg b.w.) was excreted in the bile in 12 hours; however, these metabolites are subject to reabsorption and further metabolism, most likely leading to urinary excreti on (see Section 5.1.7 for a discu ssion of biliary excretion of o nitrotoluene metabolites). 1
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16 17 18 19 20
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24 25 26 27 28 29 The half-life of o -nitrotoluene in plasma of F344 rats receiving a 200-mg/kg b.w. dose was calculated as 1.5 hours (NTP 2002b). o -Nitrotoluene or its metabolites are distributed to the liver, as indicated by r ecovery of radiolabel from o -nitrotoluene in the bile after oral administration (Chism and Rickert 1985) and by its binding to hepatic DNA (Rickert et al. 1984a). No data documenting the distribution of o -nitrotoluene to other tissues was found. However, it has been reported that o -nitrotoluene or its meta bolites may also form hemoglobin adducts in humans (Jones et al. 2005a) and rats (N TP 2002b, Sabbioni 1994, Sabbioni and Sepai 1995) implying distributi on to other tissues. The hemoglobin binding index for o -nitrotoluene in female Wistar rats was 0.72 ± 0.19 (mmol compound/mol Hb)/(mmol compound/kg b.w.).

5.1.3 In vitro metabolism of o-, m-, and p -nitrotoluene The metabolism of o- , m- , and p- nitrotoluene has been studie d in rat hepatic microsomes and in isolated rat hepatocytes (deBethizy and Rickert 1984). In rat hepatic microsomes, the only metabolites formed from the nitrot oluenes were the corre sponding nitrobenzyl alcohols. The formation was NADPH de pendent, and it was inhibited by carbon monoxide, suggesting that the reaction was catalyzed by cytochrome(s) P450.

o -Nitrobenzyl alcohol was formed at a ra te intermediate between the rates for m - and p -nitrobenzyl alcohol, but differences in th e initial rates of formation were small. There were no large differences in the rates of disappearance of th e nitrotoluenes from incubations with isolated rat hepatocyte s. However, there were quantitative and qualitative differences in the metabolites formed. o -Nitrotoluene was metabolized to

o -nitrobenzyl alcohol and the corres ponding glucuronide conjugate. These two metabolites accounted for approximately 80% of the metabolism of o -nitrotoluene. In addition, small amounts of o -nitrobenzoic acid and an uni dentified metabolite wereRoC Background Document for o -Nitrotoluene

42 6/20/08 formed. m -Nitrotoluene also was metabolized to the corresponding nitrobenzyl alcohol and its glucuronide, but those two metabol ites accounted for only about 43% of the m nitrotoluene metabolized. The major metabolite of m -nitrotoluene was m -nitrobenzoic acid, which accounted for approximately 56% of the m -nitrotoluene metabolized. p -Nitrotoluene was converted to p -nitrobenzyl alcohol and it s glucuronide conjugate, but only about 14% of the p -nitrotoluene metabolized followe d this pathway. About 2% was converted to p -nitrobenzoic acid, and the incubati on mixture also contained a small amount of p -nitrobenzyl sulfate (4% of the metabolized p -nitrotoluene). The major metabolite of p -nitrotoluene was S -(

p -nitrobenzyl) glutathione. This metabolite apparently arose from a glutathione S -transferase–catalyzed reaction between p -nitrobenzyl sulfate and glutathione. The r eaction did not proceed without glutathione S

-transferase, suggesting that un like some arylmethyl sulfates, p -nitrobenzyl sulfate is stable (deBethizy and Rickert 1983). 1
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25 26 27 28 29 30 Rat liver microsomal cytochromes P450 oxidized the isomeric aminobenzyl alcohols, metabolites of the nitrotoluenes, to h ydroxylamine and/or aminophenol metabolites (Kedderis and Rickert 1985). The metabolites were extracta ble into ethyl acetate and capable of reducing ferric iron, consistent with the formation of hydroxylamines and/or aminophenols. The oxidation rates of the ami nobenzyl alcohols were one-fifth those of the aminonitrobenzyl alcohols (metabolites of dinitrotoluenes). The rate of aminobenzyl alcohol oxidation varied as 2- ( o -) > 3- ( m -) > 4- ( p -). Thus the o -aminobenzyl alcohol metabolite of o -nitrotoluene (0.5 mM) was oxidized at a greater rate (0.29 ± 0.01 nmol formed/min per mg protein) than th e aminobenzyl alcohol metabolites of m -nitrotoluene (0.18 ± 0.00) or p -nitrotoluene (0.04 ± 0.00). These resu lts are consistent with the toxic potency of the mononitrotoluene isomers.

Examination of the metabolism of the nitrobenz yl alcohols by glucur onyl transferase, rat hepatic alcohol dehydrogenase, and sulfotra nsferase demonstrated that, based on the second-order rate constants for binding and catalysis (V/K) (see Table 5-1), o -nitrobenzyl alcohol was the best substrate of the th ree for glucuronyl transferase (V/K = 11.28),

m -nitrobenzyl alcohol was the best for alcohol dehydrogenase (V/K = 3.15), and p -nitrobenzyl alcohol was the best for sulfotransferase (V/K = 37.21) (Rickert et al.RoC Background Document for o -Nitrotoluene

6/20/08 43 1985), further supporting the predominance of glucuronidation in metabolism of o nitrotoluene. 1
2
Table 5-1. Metabolism of nitrobenzyl alcoho ls by rat hepatic glucuronyltransferase and sulfotransferase V/K (10 3

⋅ nmoles/min/mg/ μ M) Substrate Glucuronyltransferase Alcohol dehydrogenase Sulfotransferase

o -nitrobenzyl alcohol 11.28 ± 3.08 NM 4.87 ± 0.10

m -nitrobenzyl alcohol 5.25 ± 0.23 3.15 ± 0.38 4.23 ± 0.13
p -nitrobenzyl alcohol 6.00 ± 0.76 1.87 ± 0.54 37.21 ± 6.09 Source: Rickert et al . 1985. NM = no metabolism observed; V/K = second order rate constant for binding and catalysis. Note: Values are means (± S.E.) for 4 to 6 experiments. 5.1.4 In vivo metabolism of o -nitrotoluene in rodents 3 4 5 6 7 8 9 10
11
12 13 14 15 16 17 18

19 20 Male and female F344 rats and male B6C3F 1 mice excreted urine containing several metabolites of o -nitrotoluene after administration of single oral doses of 2 or 200 mg/kg b.w. (NTP 2002b). Major metabolites in male and female rats and male mice were

o -nitrobenzoic acid and o -nitrobenzyl glucuronide. The male and female rats also excreted S -( o -nitrobenzyl)- N -acetylcysteine and o -aminobenzyl alcohol. [Minor metabolites can be converted into major metabolites (see Figure 5-1).] Similar metabolites in similar percentages of the dose were seen in rats after either dose at 24 hours after administration (see Ta ble 5-2). Although the percentages of the dose excreted in 24 hours as o -nitrobenzoic acid (30.6% vs. 19.1%) and o -nitrobenzyl glucuronide (28% vs. 15.3%) were a little higher at the low dose than the high dose, there did not seem to be a dose-dependent change in metabolism between 2 and 200 mg/kg b.w. in male rats. The percentage of the dose excreted in urine was similar for male and female rats, as was the metabolite profile at both dos es. However, female rats excreted less than half as much of the dose as o -aminobenzyl alcohol or as S -( o -nitrobenzyl)- N

acetylcysteine as did male rats.

Male B6C3F 1 mice administered a single gavage dose of 200 mg/kg b.w. o -nitrotoluene excreted o -nitrobenzoic acid and o -nitrobenzyl glucuronide in the 24-hour period afterRoC Background Document for o -Nitrotoluene

44 6/20/08 administration (NTP 2002b). The specific metabo lites and their percen tages were similar after a dose of 2 mg/kg b.w., except that the perc entages excreted as o -nitrobenzoic acid and o -nitrobenzyl glucuronide were somewhat higher than after the 200-mg/kg b.w. dose (Table 5-2). 1
2 3 4
Table 5-2. Metabolites excreted in urine (% of dose) by male and female F344 rats and male B6C3F 1 mice 24 hours after an oral dose of o -nitrotoluene F344 rats B6C3F 1 mice Dose (mg/kg b.w.) Dose (mg/kg b.w.) Metabolite Sex 200 2 200 2

o -nitrobenzoic acid M F 19.1 21.8 30.6 43.9 38.2 NT 20.1 NT

o -nitrobenzyl glucuronide M F 15.3 22.1 28.0 26.5 23.9 NT 27.9 NT S -( o -nitrobenzyl)- N

acetylcysteine M F 9.9 3.7 12.4 4.9 – NT – NT

o -nitrobenzyl alcohol M F 1.8 0.8 1.9 1.0 – NT – NT

o- aminobenzyl alcohol M F 17.0 7.9 11.0 4.4 – NT – NT

o- aminotoluene ( o -toluidine) M F 1.1 1.4 –

– – NT – NT Source: NTP 2002b. – = metabolite not found; NT = not tested. Exposure concentrations were compared w ith levels of the urinary metabolites o nitrobenzoic acid and o -nitrobenzyl mercapturic acid ( S -( o -nitrobenzyl)- N -acetylcysteine) at different time points in rats and mi ce from the NTP bioassay (see Section 2.6 for details related to use of these metabolites as potential biomar kers) (NTP 2002b). The ratios of urinary o- nitrobenzoic acid to creatinine dete rmined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure co ncentration in male and female rats and mice, while the ratio of o -nitrobenzyl mercapturic acid to creatinine was related to exposure in male and female ra ts only; the concentration of o- nitrobenzyl mercapturic acid in urine of mice was generally below the limit of quantitation. 5 6 7 8 9 10
11
12 13

14 15 The metabolites of o -nitrotoluene identified in urin e after a single oral dose are summarized in Figure 5-1. There were some quantitative and qualitative differencesRoC Background Document for o -Nitrotoluene

6/20/08 45 among rats and mice in the metabolites ex creted in urine, however, the relative proportions of the urinary metabol ites did not appear to be dose dependent between 2 and 200 mg/kg b.w. 1
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4 5 6 7 8 9

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22 23 24 25
26 27 28 29 When male F344 rats were give n 11 daily doses of unlabeled o -nitrotoluene (200 mg/kg b.w.) followed on the 12th day by a dose of [ 14 C]- o -nitrotoluene and then by two more daily doses of unlabeled com pound, excretion of radi oactivity was sim ilar to that found after a single dose of the compound (NTP 2002b). However, the percentage of the radiolabeled dose excreted as S -( o -nitrobenzyl)- N -acetylcysteine ( o -nitrobenzyl mercapturic acid) in this study was only half that seen after a singl e radiolabeled dose. When rats were pretreated with buthionine sulfoxamine, an inhi bitor of glutathione synthesis, about half as much of the radi oactive dose was excreted in the urine in 24 hours as in untreated rats (NTP 2002b). Excretion of S -( o -nitrobenzyl)- N

acetylcysteine was half that seen in untreated rats, while excretion of o -nitrobenzyl alcohol tripled. Excretion of the other identified metabolites was not greatly altered. This study also investigated th e urinary excretion of [ 14 C]- o -nitrotoluene after pretreatment of male rats with pentachlorophenol, an inhibi tor of sulfotransferase. Pretreated rats excreted significantly less of the dose in the urine in 24 hours than did untreated rats. The percentage of the dose excreted as S -( o -nitrobenzyl)- N -acetylcysteine was about 15% of that seen in untreated rats. The authors sugge sted that nitrobenzyl alcohol was converted by O -sulfation to a metabolite that could react with glutathione, resulting in the decreased excretion of S -( o -nitrobenzyl)- N -acetylcysteine after pretreatment with buthionine sulfoxamine or pentachlorophenol. After pent achlorophenol pretreat ment, excretion of o nitrobenzyl glucuronide wa s decreased by about half, as was excretion of o -aminobenzyl alcohol; the authors attributed thes e decreases to competition between o -nitrobenzyl alcohol and pentachlorophenol fo r glucuronyl transferase. An earlier report by Chism et al. (1984) described similar re sults for urinary metabolites for male F344 rats given an oral dose of [ 14 C]- o -nitrotoluene (200 mg/kg b.w.); however, there were some differences (see Table 5-3). The NT P (2002b) identified o -aminobenzyl alcohol as a major metabolite, whereas Chism et al. (1984) identified the sulfur-RoC Background Document for o -Nitrotoluene

46 6/20/08 containing conjugates of o -acetamidotoluene and aminotol uene as major metabolites. Section 5.1.5 describes the metabolites of m - and p -nitrotoluene identified in these studies. 1
2 3

4 5 6 7 8 9 Excretion rates for the metabol ites that had not undergone nitro-group reduction peaked within the first 4 hours after administra tion, while those for metabolites that had undergone nitro-group reduction peaked betw een 4 and 12 hours after administration (Chism et al. 1984). These findings are in contrast to the in vitro results discussed in Section 5.1.4, which showed smalle r percentages metabolized to o -nitrobenzoic acid and higher percentages metabolized to o -nitrobenzyl alcohol and o -nitrobenzyl glucuronide.RoC Background Document for o -Nitrotoluene

Figure 5-1. Urinary metabolites of o -nitrotoluene in rats and mice Source: NTP 2002b. The urinary metabolites found in rats and mice are show n above. The letters in parentheses after the name of the molecule denote whether the metabolite is a major or minor metabolite in a particular species. Note that two sulfur-containing metabolites for which qu antitative data are included in Table 5-2 are not illustrated above. The sulfur-containing conjugates of o -acetamidotoluene and o -aminotoluene have been tentatively identified by Chism and Rickert (1985) (see Figure 1-2), but the metabolic pathways giving rise to these urinary products have not been established.

6/20/08 47RoC Background Document for o -Nitrotoluene

48 6/20/08 5.1.5 In vivo metabolism of m - and p -nitrotoluene in rodents 1 2 3 4 5 6 7
8 9 10
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24 25 26 27 28 29 30 The in vivo disposition of m - and p -nitrotoluene after a 200-mg/ kg b.w. oral dose also has been studied in male F344 rats (Chism et al. 1984). The major urinary metabolites of m nitrotoluene were m -nitrohippuric acid, m -nitrobenzoic acid, and m -acetamidobenzoic acid, and the major urinary metabolites of p -nitrotoluene were p -nitrobenzoic acid, p -acetamidobenzoic acid, p -nitrohippuric acid, and S -(

p -nitrobenzyl)- N -acetylcysteine (Table 5-3). The NTP (2002a) studied the in vivo metabolism of p -nitrotoluene in male and female F344 rats and male and female B6C3F 1 mice after oral doses of 2 or 200 mg/kg b.w. At the higher dose, the major metabolites in male rats were p -nitrobenzoic acid, p acetamidobenzoic acid, p -nitrohippuric acid, and p -nitrobenzylmercapturic acid ( S -(

p nitrobenzyl)- N -acetylcysteine) along with other unid entified but more polar metabolites that accounted for approximately 5% of the dose; the results reported by NTP and by Chism et al. (1984) were generally consistent (Table 5-3). The metabolites excreted by female rats were similar, except th at females excreted somewhat more p -nitrobenzoic acid and somewhat less S -(

p -nitrobenzyl)- N -acetylcysteine. The pa tterns of excretion in both male and female rats were similar afte r a 2-mg/kg b.w. dose (d ata not shown). Male mice given a 200-mg/kg b.w. dose of p -nitrotoluene excreted the following major metabolites in urine: p -nitrohippuric acid, 2-me thyl-5-nitrophenyl su lfate, 2-methyl-5- nitrophenyl glucuronide, p -nitrobenzoic acid, and p -aetamidobenzoic acid (Table 5-3). Female mice excreted somewhat less of the dose as p -nitrohippuric acid or 2-methyl-5- nitrophenyl sulfate and somewhat more of the dose as 2-methyl-5-nitrophenyl glucuronide, p -nitrobenzoic acid, or p -acetamidobenzoic acid. The NTP (2002a) also studied the metabolism of p -nitrotoluene after multiple 200-mg/kg b.w. doses. After a single radiolabeled dose on day 9, the major urinary metabolite in the urine of male F344 rats was p -nitrobenzoic acid; smaller portions of the dose were excreted as p -acetamidobenzoic acid, p -nitrohippuric acid, and S -(

p -nitrobenzyl)- N

acetylcysteine. The half-life of total radioactivity in plas ma after an oral dose of radiolabeled p -nitrotoluene (200 mg/kg b.w.) to rats was about 1 hour (NTP 2002a), similar to that for o -nitrotoluene (NTP 2002b).RoC Background Document for o -Nitrotoluene

6/20/08 49 5.1.6 Comparison of o-, m-, and p -nitrotoluene metabolism in rodents 1

2 3 4 5 6 7 8 9 10
11
12 Taken together, the in vitro and in vivo studies on nitrotoluene metabolism demonstrated some differences in the excretion of o -, m - and p -nitrotoluene metabolites and across species and sex for o - and p -nitrotoluene metabolites (the results of the in vivo studies for all three isomers are summarized in Table 5-3) . The first step in nitrotoluene metabolism is conversion of the methyl group to an alcoho l. In contrast to what is observed in isolated rat hepatocytes, the main me tabolic pathway for each of the isomers in vivo is oxidation of the alcohol group to an acid moie ty. [Whereas very little of a dose of m - or p -nitrotoluene is converted to a nitrobenzyl gl ucuronide, this is an important pathway for

o -nitrotoluene. Furthermore, the appearance of reduced metabolites in the in vivo studies, but not in studies with microsomes or isol ated hepatocytes, suggests that nitro-group reduction occurs at a site other than the liver. ]RoC Background Document foro-NitrotolueneTable 5-3. Metabolites excreted in urine (% of dose) by rats and mice after an oraldose of 200 mg/kg b.w. ofo-,m-, orpnitrotolueneo-nitrotoluenem-nitrotoluenep-nitrotolueneF344 ratsB6C3F1miceF344 ratsF344 ratsB6C3F1miceMetaboliteSexCh.NTPNTPCh.Ch.NTPNTPx-nitrobenzoic acidaMF28.6NT19.121.838.2NT21.1NT28.0NT36.245.05.510.3sulfur-containing conjugateofx-acetamidotolueneMF15.9NT–––NT–NT–NT––––x-nitrobenzyl glucuronideMF14.1NT15.322.123.9NT2.0NT1.4NT––––S-(x-nitrobenzyl)-NacetylcysteineMF11.6NT9.93.7–NT–NT3.7NT7.11.2––sulfur-containing conjugateofx-aminotolueneMF6.0NT–––NT–NT–NT––––S-(x-nitrobenzyl)glutathioneMF3.9NT–––NT1.3NT–NT––––x-aminobenzoic acidMF1.8NT––NT1.2NT0.8NT––––x-nitrobenzyl sulfateMF0.5NT–––NT–NT–NT––––x-nitrobenzyl alcoholMF0.4NT1.80.8–NT–NT–NT––––x-aminobenzyl alcoholMF–NT17.07.9–NT–NT–NT––––506/20/08RoC Background Document foro-Nitrotoluene6/20/0851o-nitrotoluenem-nitrotoluenep-nitrotolueneF344 ratsB6C3F1miceF344 ratsF344 ratsB6C3F1miceMetaboliteSexCh.NTPNTPCh.Ch.NTPNTPx-nitrohippuric acidMF–NT–––NT23.6NT13.0NT10.38.720.514.7x-acetamidobenzoic acidMF–NT–––NT11.6NT27.1NT16.119.35.27.0x-aminotolueneMF–NT1.11.4–NT–NT–NT–NT––5-methyl-2-nitrophenylglucuronideMF–NT–––NT–NT0.3NT–NT––5-methyl-2-nitrophenylsulfateMF–NT–––NT–NT0.2NT–NT––2-methyl-5-nitrophenylglucuronideMF–NT–––NT–NT–NT–NT12.718.72-methyl-5-nitrophenylsulfateMF–NT–––NT–NT–NT–NT19.012.0Sources: Ch. = Chismet al.1984 (72-h data), NTP = NTP 2002b (24-h data) foro-nitrotoluene and NTP 2002a (24-h data) forp-nitrotoluene.– = metabolite not found; NT = not tested.ax-representso-,m-, orp-nitrotoluene metabolites.RoC Background Document for o -Nitrotoluene

52 6/20/08 5.1.7 Biliary excretion of o-, m-, and p -nitrotoluene in rats 1 2 3 4 5 6 7 8 9 10
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23 24 25 26 27 In F344 rats given an oral dose (200 mg/kg b.w.) of radiolabeled o -nitrotoluene, bile-duct cannulation decreased urinary ex cretion of radiolabel by about one-quarter in males and by about one-half in females after 12 hours, compared with sham-operated animals (Chism et al. 1984). Urinary excretion of radiolabel from m - or p- nitrotoluene in bileduct–cannulated F344 rats was also decreased by one-quarter to one-half. In the studies in male rats, 28.6% of an o- nitrotoluene dose was eliminated in the bile, while in female rats only 9.6% was eliminated in the bile. Biliary excretion of radiolabel dose due to m - or pnitrotoluene accounted for about 10% of the dos e in male rats and from 1.3% to 4.3% of the dose in female rats. The major biliary metabolite of o- nitrotoluene was o -nitrobenzyl glucuronide, which accounted for 22.1% of the dose in males and 8.3% of the dose in females. The next most abundant metabolite was S -( o -nitrobenzyl) glutathione, which accounted for 4.9% of the dose in males and 0.4% in females. The nitrobenzyl glucuronides formed from m- or p- nitrotoluene accounted for only 0.1% of the dose in males and 2.8% in females. The major biliary metabolite of m- nitrotoluene was m nitrobenzoic acid (3.4% of the dose in males and 1.7% in females). p- Nitrotoluene was excreted in the bile primarily as p -nitrobenzoic acid and S -(

p -nitrobenzyl) glutathione; each accounted for 2.8% of the dose in male rats. In another study (NTP 2002a), biliary excr etion accounted for 7.7% of a 200-mg/kg b.w. oral dose of [ 14 C]- p- nitrotoluene in male rats in 6 hours; the major metabolite was S -(

p nitrobenzyl) glutathione ( 4.4% of the dose), followed by p -nitrobenzoic acid (2.5%) and p -nitrobenzyl glucuronide (0.4%).

These results indicate differences in the bi liary excretion of the three nitrotoluene isomers; o- nitrotoluene metabolites are excreted via this route in approximately twice the amount as m- or p -nitrotoluene metabolites. There is also a sex difference in biliary excretion of o -nitrotoluene; males excrete about three times as much as o -nitrobenzyl glucuronide in the bile as females.RoC Background Document for o -Nitrotoluene

6/20/08 53 5.2 Bioactivation of o -nitrotoluene 1 Biliary excretion is an important step in the activation of o- nitrotoluene (see Figure 5-2). Interruption of bile flow into the intestine by cannulation of the bile duct decreased the covalent binding of o -nitrotoluene-related material at 12 hours post-administration to 7% (in males) or 22% (in females) of that seen in sham-operated animals (Chism and Rickert 1985). In intact rats, females tended to excrete o -nitrobenzyl glucuronide in the urine to a greater extent than male ra ts; in bile duct–cannulated an imals, similar amounts were excreted in the urine of both sexes. 2 3 4 5 6 7 8

9 10
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22 23 24 25 26 27 28 29 30 When covalent binding to hepatic macromolecu les was used as an indicator of potential genotoxicity in male F344 rats, 2 to 6 times as much radiolabel from o- nitrotoluene was bound to hepatic macromolecules as from m - or p -nitrotoluene (Rickert et al. 1984a). Only o -nitrotoluene was bound to hepatic DNA at c oncentrations above the assay’s limit of quantitation. When sulfotransfe rase activity was inhibited by pretreatment of rats with pentachlorophenol or 2, 6-dichloro-4-nitrophe nol, the binding of o -nitrotoluene–related material to total hepatic macromolecules was decr eased to less than half that seen with no pretreatment, and the binding of o -nitrotoluene–related mate rial to hepatic DNA was below the limit of quantitation. The binding of m - or p- nitrotoluene–related material to total hepatic macromolecules was unaffected by pretreatment. These results suggest that sulfotransferase is an important enzyme in the activation of an o- nitrotoluene metabolite to a compound capable of reacting with DNA. [All the above data suggest that, like the ac tivation of 2,6-dinitrot oluene (reviewed in Rickert et al. 1984b), the activation of o -nitrotoluene to a compound capable of covalently interacting with DNA requires metabolism to a benzyl alcohol, conjugation with glucuronic acid, elimination in bile, deconjugation, reduction of the nitro group, and reabsorption for delivery to the liver. On ce in the liver, the final activation of

o -aminobenzyl alcohol requires the action of sulfotransferase. The involvement of sulfotransferase is suggested by the experime nts measuring covalent binding to hepatic DNA in rats pretreated with pentachlorophe nol or 2,6-dichloro-4 -nitrophenol (Rickert et al. 1984a). It is also supported by in vitro studies of the binding of o -aminobenzyl alcohol to calf thymus DNA (Chism and Rick ert 1989). In those experiments, o -aminobenzylRoC Background Document for o -Nitrotoluene
Figure 5-2. Formation and potent ial bioactivation routes for o -aminobenzyl alcohol from o -nitrotoluene Source: Adapted from Ch ism and Rickert (1985).

o -Nitrobenzyl glucuronide formed from o -nitrotoluene in the liver (uppermost box on opposite page) is excreted via the bile into the small intestine (mi ddle box) where intestinal bacteria deconjugate the glucuronide and reduce the nitro group to an amine, forming o -aminobenzyl alcohol. o -Aminobenzyl alcohol is reabsorbed from the inte stine and carried by the portal circ ulation to the liver (lowermost box) where three potential pathways may be followed for fu rther metabolism culminating in the two nitrenium ions and one carbonium ion illustrated. As noted in the text, the pathway through the o -aminobenzyl alcohol sulfate (heavier arrows on the right side of the lowermost box) is considered the predominant pathway for bioactivation. (A) o -nitrotoluene (B) o -nitrobenzyl alcohol (C) o -nitrobenzyl alcohol glucuronide (D) o -aminobenzyl alcohol (E) o -( N

-hydroxylamino)benzyl alcohol (F) o -( N

-acetylamino)benzyl alcohol (G) o -aminobenzyl alcohol sulfate (H) o -( N

-hydroxylamino- N -sulfoxyl)benzyl alcohol (I) o -( N

-hydroxy- N -acetylamino)benzyl alcohol (J) o - N -acetoxy benzyl alcohol (K) o -acetamido( N

-sulfoxyl)benzyl alcohol (L) nitrenium ion (M) nitrenium ion (N) carbonium ion

54 6/20/08RoC Background Document for o -Nitrotoluene

6/20/08 55RoC Background Document for o -Nitrotoluene

56 6/20/08 alcohol-related material became bound to cal f thymus DNA when incubated with rat hepatic cytosol and 3'-phosphoadenosine 5'- phosphosulfate (PAPS), a source of sulfate for sulfotransferase. No binding was seen when the sulfotransferase inhibitor 2,6-dichloro-4-nitrophenol was added to th e incubation mixture. The final reactive intermediate does not appear to be the N,O -sulfate, as addition of NADPH and microsomes (necessary to produce the precursor hydroxylamine) did not increase covalent binding to calf thymus DNA above that seen in incubations containing cytosol and PAPS. The addition of acetyl coenzy me A did not result in covalent binding, suggesting that the proximal reactive meta bolite was not an acetoxyarylamine. Although binding to macromolecules through an N -hydroxy intermediate cannot be ruled out, binding in the presence of NA DPH and microsomes was much lower than binding in the presence of PAPS and cytosol. This suggest s that the proximal reactive metabolite is

o -aminobenzyl sulfate (see Figure 5-2). This pathway only describes th e situation in the liver because other tissues may not have th e necessary enzymes to activate aminobenzyl alcohol. Similar structures are proposed to be the reactive interm ediates formed from 7,12-dimethylbenz[ a ]anthracene (Watabe et al. 1982) and 1'-hydroxysafrole (Boberg et al. 1983).] 1
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22 23 24 DNA adducts are formed when o -aminotoluene is incubate d with calf thymus DNA; however, no hepatic DNA adducts were detected in rats treated with o -aminotoluene (Jones et al. 2003, Jones and Sabbioni 2003). Furtherm ore, the adducts formed when o aminotoluene was incubated with calf thymus DNA were different from those present in hepatic DNA after o- nitrotoluene was admini stered to rats (Jones et al. 2003). [These data add support to the possibility that an o- nitrotoluene metabol ite(s) modifies DNA through the methyl group, rather than an amino group.]RoC Background Document for o -Nitrotoluene

6/20/08 57 5.3 Genetic damage and related effects 1

o -Nitrotoluene has been tested for genotoxicity in a number of in vitro and in vivo test systems. The International Agency for Re search on Cancer (IARC 1996) reviewed the available literature and concluded that o -nitrotoluene was not genot oxic to bacteria but induced sister chromatid exchange (SCE) in cultured mammalian cells, and formed DNA adducts and induced unscheduled DNA synthesis in vivo in rats. This section reviews the available genotoxicity studies of o -nitrotoluene in prokaryotic and mammalian in vitro and in vivo systems, including those cited in the IARC review and studies published subsequently. Studies of mutations and gene expression in oncogenes and tumor suppressor genes are discussed in Section 5.4.2. Genotoxicity studies of nitrotoluene isomers and related compounds are discussed in Section 5.5. 2 3 4 5 6 7 8 9 10
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22 23 24 25 26 27 28 5.3.1 Prokaryotic systems

o -Nitrotoluene has been tested for the ability to induce reverse mutati on in several strains of Salmonella typhimurium and for differential toxicity in Bacillus subtilis . Table 5-4 summarizes the results of tests in prokaryotic systems.

Reverse mutation in Salmonella typhimurium In studies with S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 (see Table 5-4 for the concentrations tested), o -nitrotoluene did not induce reverse mutation, either with or without ex ogenous metabolic activation (Chiu et al. 1978, Haworth et al. 1983, Shimizu and Yano 1986, Spanggord et al. 1982a, Spanggord et al. 1982b, Suzuki et al. 1983, Tokiwa et al. 1981). In the presence of norharman (200 μ g/plate), a co-mutagen found in tobacco tar and in pyrolysat e of tryptophan, and with S9 metabolic activation, o -nitrotoluene (at concentrations of 100 to 300 μg/plate) induced mutations in S. typhimurium strain TA98 but not in TA100 (Suzuki et al. 1983). Norharman may alter metabolic activation by the S9 mixture or increase the susceptibility of DNA to damage by intercalation into DNA. The lack of positive results with TA100 may indicate that norharman was effective as a co-mutagen only for the frameshift mutations detectable in strain TA98.RoC Background Document for o -Nitrotoluene

58 6/20/08 Differential toxicity in Bacillus subtilis 1 2 3 Shimizu and Yano (1986) repor ted negative results for o -nitrotoluene (concentration not reported) in the rec assay in B. subtilis strains H17 and M45 wit hout metabolic activation.
Table 5-4. Results of ge notoxicity testing of o -nitrotoluene in prokaryotic systems Results Test system End point (concentration) with S9 without S9 Reference S. typhimurium TA98, TA100 reverse mutation (0.1–10 μ mol) – – Chiu et al. 1978 S. typhimurium TA98, TA100 reverse mutation (NR) – – Tokiwa et al. 1981 S. typhimurium TA98, TA100 reverse mutation (100–300 μ g/plate) – – Suzuki et al. 1983 S. typhimurium TA98 with norharman reverse mutation (100–300 μ g/plate) + NT Suzuki et al. 1983 S. typhimurium TA100 with norharman reverse mutation (100–300 μ g/plate) – NT Suzuki et al. 1983 S. typhimurium TA98, TA1537, TA1538 reverse mutation (0.01–5 μ L/plate) – – Shimizu and Yano 1986 S. typhimurium TA98, TA100, TA1535, TA1537 reverse mutation (3–333 μ g/plate) – – Haworth et al. 1983

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 reverse mutation (10–5,000 μ g/plate) – – Spanggord et al. 1982a, 1982b S. typhimurium TA98, TA1535 reverse mutation (33–1,000 μ g/plate) – – Lee et al. 2007

B. subtilis H17, M45 differential toxicity (NR) NT – Shimizu and Yano 1986 NR = not reported; NT = not tested; + = positive; – = negative. 5.3.2 Mammalian systems 4 5 6 7 8 9 10 o -Nitrotoluene has been tested for ge notoxicity in a number of mammalian in vitro systems, including Chinese hamster ovary (CHO) and lung (CHL/IU) cells, rat and human hepatocytes, and rat pachytene spermatocytes and round spermatids. Mammalian in vivo tests of o -nitrotoluene included DNA adduct formation in rat livers, the unscheduled DNA synthesis (UDS) assay in ra t hepatocytes, and the acute micronucleus test in bone marrow in rats and mice.RoC Background Document for o -Nitrotoluene

6/20/08 59 In vitro systems 1 2 3 4 5
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22 23 24 25 26 27 Table 5-5 summarizes the results of tests in in vitro mammalian systems. Galloway et al. (1987) observed significantly in creased incidences of SCE w ith S9 metabolic activation at o -nitrotoluene concentrations from 355 to 423 μ g/mL and equivocal results without S9 at concentrations ranging from 117 to 282 μ g/mL in cultured CHO cells.
Chromosomal aberrations were evaluated in several studies. In cultured CHO cells, the incidence of chromosomal aberrations wa s not increased, eith er with S9 at o -nitrotoluene concentrations from 375 to 422 μ g/mL or without S9 at concentrations from 201 to 394 μ g/mL (Galloway et al 1987). o -Nitrotoluene did not induce structural chromosomal aberrations but did induce polyploi dy in CHL cells exposed to 250 μ g/mL for 48 h. Huang et al. (1996) reported an increase in chro mosomal aberrations in human peripheral lymphocytes exposed to 0.005 to 1 mM o -nitrotoluene for 24 h (Ishidate et al. 1988).

Matsushima et al. (1999) conducted a va lidation study of the in vitro micronucleus test for 66 chemicals using CHL/IU cells. CHL cells exposed to o -nitrotoluene at concentrations of 2 to 50 μ g/mL for 6 h followed by 18 h recovery had an increased frequency of micronuclei. However, Lee et al. (2007) did not find an increase in micronuclei (cytokinesis-block assa y) in CHO-K1 cells exposed to o -nitrotoluene at 50 to 200 μ g/mL with or without S9 for 4 h.

Lee et al. (2007) reported that o -nitrotoluene (50 to 200 μ g/mL for 2 h with and without S9) induced DNA damage (as measured by the Olive Tail Moment in the comet assay) in L5178Y mouse lymphoma cells. Nega tive results were reported for o -nitrotoluene in a number of tests measuring DNA repair as UDS (all of which used con centrations of 10 to 1,000 μ M), including tests in male and fema le F344/N rat hepatocytes (Doolittle et al. 1983), human hepatocy tes (Butterworth et al. 1989), and rat pachyt ene spermatocytes and round spermatids (Working and Butterworth 1984). Parton et al. (1995) reported that culturing rat hepatocytes in serum-free medi a for 24 hours before exposure increased the assay’s sensitivity; these investigators reported that o -nitrotoluene induced DNA repair.RoC Background Document for o -Nitrotoluene

60 6/20/08 Table 5-5. Results of ge notoxicity testing of o -nitrotoluene in in vitro mammalian systems Results Test system (tissue or cell type) End point (conc. or dose) with S9 without S9 Reference CHO cells SCE (355–423 μ g/mL) + NT Galloway et al. 1987 CHO cells SCE (117–282 μ g/mL) NT ± Galloway et al. 1987 CHO cells chromosomal aberrations (375–422 μ g/mL) – NT Galloway et al. 1987 CHO cells chromosomal aberrations (201–394 μ g/mL) NT – Galloway et al. 1987 CHL/IU cells chromosomal aberrations (250 μ g/mL) NT + a Ishidate et al. 1988 Human peripheral lymphocytres chromosomal aberrations (0.005–1.0 mM) NT + Huang et al. 1996 CHL/IU cells micronuclei (2–50 μ g/mL) NT + Matsushima et al. 1999 CHO-K1 cells micronuclei (50–200 μ g/mL) – – Lee et al. 2007
L5178Y mouse lymphomacells DNA damage (comet assay) (50–200 μ g/mL) + + Lee et al. 2007 F344/N rat hepatocytes DNA repair (10–1,000 μ M) NT – Doolittle et al. 1983 F344/N rat hepatocytes (serum-free media) DNA repair (0.1–500 μ g/mL) NT + Parton et al. 1995 Human hepatocytes DNA repair (10–1,000 μ M) NT – Butterworth et al. 1989 Rat spermatocytes and spermatids DNA repair (10–1,000 μ M) NT – Working and Butterworth 1984 NT = not tested; + = positive; – = negative; ± = equivocal.
a Positive for numerical aberrations (polyploidy) but not structural aberrations.
In vivo systems 1 2 3
4 5 6 7 The results of tests for DNA adduct forma tion, DNA repair, and micronucleus formation in in vivo mammalian systems are summarized below and in Table 5-6.

DNA adduct formation in rats Rickert et al. (1984a) measured covalent binding of the nitrotoluenes to hepatic macromolecules and DNA in male F344/N rats . A single oral dose (200 mg/kg b.w.) of

o -nitrotoluene resulted in its covalent bi nding to hepatic macromolecules and hepaticRoC Background Document for o -Nitrotoluene

6/20/08 61 DNA. Jones et al. (2003) reported dose-dependent formation of DNA and hemoglobin adducts in the livers of WELS-F ohm male rats administered o -nitrotoluene at a daily oral dose of 40 to 250 mg/kg b.w. for 12 weeks. In another study (Jones and Sabbioni 2003), DNA adduct formation was not observed in the liv ers of female Wistar rats administered

o -nitrotoluene (0.1 mL/100 g b.w.) by gavage; however, an exposure marker, hydrolyzable hemoglobin adducts, was detected. 1
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25 26 27 28 29 30 DNA repair in rats The effects of o -nitrotoluene and intestinal bacteria on UDS were assessed in male and female F344 rats, germ-free male F344 rats, a nd germ-free male F344 rats treated with Charles River Altered Schaedler Flora (CRASF), a mixture of eight bacterial strains used to simulate the autochthonous (native) GI flora (Doolittle et al. 1983). o -Nitrotoluene was administered via gavage (200 to 500 mg/kg b.w.), and DNA repair was assessed 12 hours later by the in vivo

– in vitro rat hepatocyte UDS assay in primary cultures of hepatocytes isolated from exposed animals. o -Nitrotoluene induced a dose- dependent increase in both the percentage of hepatocytes undergoing DNA repair and the exte nt of the repair in male rats, but not in female rats. Increases in DNA repair were observed in CRASF rats (albeit to a lesser extent than in rats with conventi onal flora) but not in th e germ-free rats. [The inability of o -nitrotoluene to induce DNA repair in germ-free rats and the results of previous studies showing that it did not induce DNA repair in rat hepatocytes in vitro led the authors to suggest that intestinal bacter ia are needed for the metabolic activation of

o -nitrotoluene to a hepatic genotoxicant.] Se x differences in the DNA repair responses did not appear to be due to intestinal bacteria, because males and females had nearly identical types of intestinal flora. The aut hors suggested that differences between sexes were probably in the hepatic metabolism and/or disposition of o -nitrotoluene.

Micronucleus formation in rats and mice The acute micronucleus test, measuring the frequency of micronucleated polychromatic erythrocytes (PCEs) in bone marrow followi ng a single intraperiton eal (i.p.) dose of

o -nitrotoluene dissolved in corn oil, was carried out in male F344/N rats according to two protocols: (1) a dose of 625, 1,250, or 2,500 mg/kg b.w. and bone marrow analysis 24 hours post-injection and (2) a dose of 625 or 2,500 mg/kg b.w. and bone marrowRoC Background Document for o -Nitrotoluene

62 6/20/08 analysis 48 hours post-injection. Negative results were report ed for both protocols (NTP 2002b). 1
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3 4 5 6 7 8 9 10
11 Male B6C3F 1 mice injected i.p. with o -nitrotoluene dissolved in corn oil (100 to 400 mg/kg b.w.) three times at 24-hour interval s showed no significant increase in the frequency of micronucleated PCEs in bone marrow, although small increases were observed at all exposure levels. In a study of mice administered o -nitrotoluene in the feed for 13 weeks at concentrations of 625 to 10,000 ppm, no increase in the frequency of micronucleated normochromatic erythrocytes (N CEs) was seen in the peripheral blood of females. In males, the frequency of micronuc leated NCEs was sligh tly increased at the highest exposure level (10,000 ppm); this re sult was judged to be equivocal (NTP 2002b).RoC Background Document for o -Nitrotoluene

Table 5-6. Results of ge notoxicity testing of o -nitrotoluene in in vivo mammalian systems Test system (tissue or cell type) End point (conc. or dose) Results Reference Male F344 rats (liver, cell type not specified) Covalent binding to hepatic macromolecules (200 mg/kg b.w.) + Rickert et al. 1984a

Male F344 rats (liver, cell type not specified) Covalent binding to DNA (200 mg/kg b.w.) + Rickert et al. 1984a

Male WELS-Fohm rats (whole liver, cell type not specified) DNA adducts (40–250 mg/kg b.w.) + Jones et al. 2003

Female Wistar rats (whole liver, cell type not specified) DNA adducts (0.1 mL/100 g b.w.) – Jones and Sabbioni 2003 Male F344/N rats (hepatocytes) DNA repair (200–500 mg/kg b.w.) + Doolittle et al. 1983 Female F344/N rats (hepatocytes) DNA repair (200–500 mg/kg b.w.) – Doolittle et al. 1983 Germ-free male F344/N rats (hepatocytes) DNA repair (200–500 mg/kg b.w.) – Doolittle et al. 1983 Male F344/N rats raised germ-free but inoculated with CRASF (a mixture of 8 bacteria) 2 weeks pre-exposure (hepatocytes) DNA repair (200–500 mg/kg b.w.) + Doolittle et al. 1983

Male F344/N rats (bone marrow) micronucleated PCEs (625–2,500 mg/kg b.w.) – NTP 2002b Male B6C3F 1 mice (bone marrow) micronucleated PCEs (100–400 mg/kg b.w.) – NTP 2002b Male B6C3F 1 mice (bone marrow) micronucleated NCEs (625–10,000 ppm) ± NTP 2002b Female B6C3F 1 mice (bone marrow) micronucleated NCEs (625–10,000 ppm) – NTP 2002b + = positive; – = negative; ± = equivocal.

6/20/08 63RoC Background Document for o -Nitrotoluene

64 6/20/08 5.4 Mechanistic studies and considerations 1 This section discusses potent ial mechanisms of carcinogenicity and genotoxicity of o nitrotoluene including mechanisms related to the bioactivation of o -nitrotoluene (discussed in Section 5.2) and studies in rodents evaluating mutations and gene expression from o -nitrotoluene–induced tumors. 2 3 4 5
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25 26 27 28 5.4.1 Potential mechanisms of carcinogenic ity and genotoxicity related to the bioactivation of o- nitrotoluene The genotoxicity of the nitrotoluenes as measured by the in vivo–in vitro DNA repair assay in rats depends on isomer, sex, and intestinal microflora (Doolittle et al. 1983) (see Section 5.3.2). Of the three nitrotoluene isomers, DNA repair was induced only by onitrotoluene and only in male rats with an intact intestin al microflora. Incubation of o nitrotoluene in vitro with hepatocytes isolated from male rats failed to induce DNA repair. [These results suggest that DNA dama ge and subsequent repair is dependent on metabolism, both mammalian and bacterial.] [The activation scheme discussed in Section 5.2 and depicted in Fi gure 5-2 explains the observations on sex and isomer differences in the covalent binding of nitrotoluene-related material in hepatic DNA, and it also explai ns the sex, isomer, and intestinal flora dependency of the in vivo

– in vitro DNA repair assay. The significantly increased incidences of tumors in othe r tissues of rats or mice, including mammary gland, skin, lung, large intestine, and hemangiosarcomas in various tissues, support the concept that other activation pathways may exist.]

5.4.2 Gene expression and mutation studies This section discusses gene expression st udies in cultured mouse cells or non-tumor tissue from mice exposed to o -nitrotoluene and gene expression and mutation studies using o -nitrotoluene–induced tumors.

Cultured mouse cells or non-tumor cells Kim et al. (2005) used cDNA microarra y analysis in L5178Y Tk +/- mouse lymphoma cells to evaluate gene expression profiles for o -nitrotoluene, which they considered a nongenotoxic carcinogen. Genes were consid ered differentially expressed whenRoC Background Document for o -Nitrotoluene

6/20/08 65 logarithmic gene expression ratios in four i ndependent hybridizations showed a two-fold difference in expression. Exposure to o -nitrotoluene consistently affected four genes. Cyp2j6 (an unstable cytochrome P450 isoform), S100a4 (S100 calcium binding protein A4), which is linked to the invasive a nd metastatic phenotype of cancer cells, and IL7 , which has antitumor properties, were upregulated. Akap10 , a protein kinase A anchoring protein, was downregulated. 1
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22 23 24 25 26 27 28 29 Lee et al. (2007) also performed cDNA microarray analysis in L5178Y cells exposed to 200 μ g/mL of o -nitrotoluene for 24 h. Data was anly sed by hierarchical clustering, and a few pathways (see Table 5-7) were identifie d in which gene expr ession was increased (greater than 2-fold log change) by o -nitrotoluene. The authors did not identify the specific genes or discuss their findings in details.

Iida et al . (2005) investigated early gene expression involved in mouse liver carcinogenesis. Mice were treated for 2 weeks with several animal carcinogens (including

o -nitrotoluene and oxazepam) and non-carcinogens (including p -nitrotoluene). Female mice were treated with 1,250- or 5,000-ppm o -nitrotoluene. Gene-expression changes from o -nitrotoluene occurred in only 26 ge nes from the 1,250-ppm dose and 33 genes from the 5,000-ppm dose; in comparison, expression of 221 genes was noted in male mice and 183 genes in female mice from oxazepam treatment. A dose-dependent loss of expression of the fragile histidine triad gene ( Fhit ) and the WW domain-containing oxidoreductase ( Wwox ) gene was seen from o -nitrotoluene exposure. The authors noted that these genes are human tumor suppressor ge nes and are often lost together in many human cancers. o -Nitrotoluene treatment also resulte d in strong up-regu lation of the cell cycle genes, cyclin G1 (Ccng1) and p21 ( Cdkn1a ), down-regulation of the epidermal growth factor ( Egfr ) gene, down-regulation of the tran scription factor early growth response 1 ( Egr1 ) gene at 5,000 ppm in males only, and down-regulation of inhibin β -A ( Inhba ), a member of the TGF- β superfamily, and Jun-B oncogene ( Junb ), a negative regulator of proliferation genes. There we re 20 gene expression changes that were common to both male and female mice treated with 5,000 ppm o -nitrotoluene. These included Fhit , Wwox , deoxyribonuclease II α ( Dnase2a ), and cytokine inducible SH2-RoC Background Document for o -Nitrotoluene

66 6/20/08 containing protein ( Cish ), all of which were down-regulated and have a role in promoting apoptosis. 1
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3 4 5 6 7 8 Iida et al. (2007) investigated the role of transforming growth factor- β (TGF- β )– stimulated clone 22 ( Tsc-22 ) in carcinogenesis in mouse liver. Previous studies had indicated that Tsc-22 was a putative tumor suppressor a nd transcriptional repressor and was down-regulated in B6C3F 1 mouse liver following treatm ent with carcinogenic doses of oxazepam or Wyeth-14,643. Tsc-22 expression was not changed in mice treated with 1,250 or 5,000 ppm o -nitrotoluene for 2 weeks. Table 5-7. Gene expression in cultured cells or tissues from mice exposed to o nitrotoluene Test system (tissue) Gene or Pathway Results Reference Cyp2j6 gene ↑ expression S100a4 gene ↑ expression IL7 gene ↑ expression L5178Y mouse lymphoma cells Akap10 gene ↓ expression Kim et al. 2005

Pathways:

Purine metabolism ↑ expression SNARE interactions in vesicular transport ↑ expression L5178Y mouse lymphoma cells MAPK signaling pathway ↑ expression Lee et al . 2007

fragile histidine triad gene ( Fhit ) ↓ expression WW domain-containing oxidoreductase gene ( Wwox ) ↓ expression cyclin G1 ( Ccng1 ) ↑ expression p21 (Cdkn1a) ↑ expression epidermal growth factor gene (Egr1) ↓ expression early growth response 1 gene (Egfr) ↓ expression a
inhibin β -A gene (Inhba) ↓ expression B6C3F 1 mice – in vivo (liver)

Jun-B gene (Junb) ↓ expression Iida et al . 2005

a In 5,000-ppm males only.RoC Background Document for o -Nitrotoluene

6/20/08 67 Gene expression and mutations in oncogenes, tumor suppressor genes, and other critical genes in rodents 1 2 3 4 5 6 7
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26 27 28 29 30 Several studies have examined the poten tial molecular mechanisms underlying the development of tumors in rodents exposed to o -nitrotoluene. Hong et al. (2003) and Sills et al. (2004) analyzed gene mutations in col on tumors from the same chronic study. Kim et al. (2006) characterized the gene expression profile in mesotheliomas collected from rats treated with o -nitrotoluene. In an attempt to evaluate the underlying molecular mechanisms for development of o nitrotoluene-induced tumors, Hong et al . (2003) analyzed mutations in the K- ras , p53, and β -catenin ( Catnb) genes and production of p53 and β -catenin protein in 15 hemangiosarcomas (from skeletal muscle, s ubcutaneous tissue, and mesentery) from B6C3F 1 mice exposed to o -nitrotoluene in the NTP two-y ear bioassay (see Section 4.2.2) and in 15 spontaneously occu rring subcutaneous hemangios arcomas. Protein production (indicating gene expression) was asse ssed by immunohistochemical staining, and mutations were detected by sequencing. All of the o -nitrotoluene-induced tumors tested positive for p53 protein, and 73% (11 of 15) of the tumors had missense mutations in the p53 gene while none of the spontaneous hemangiosarcomas showed these changes. The majority of the 15 identified p53 mutations (which included 4 double mutations) involved G
⋅ A base pairs. Deletions in the β -catenin gene (most in exon 2 splice sites) were identified in 47% (7 of 15) of the o -nitrotoluene–induced hemangiosarcomas, and a point mutation in the K- ras gene (in codon 61) was identif ied in 1 tumor. Production of β

catenin protein also was detected in 47% of the o -nitrotoluene–induced tumors (in 6 of the 7 tumors with a deletion and 1 tumor w ithout a deletion) but not in any of the spontaneous hemangiosarcomas (see Table 5-8) . The authors concluded that the p53 and β -catenin gene mutations were likely a result of the genotoxic effect of o -nitrotoluene.

Sills et al. (2004) characterized the molecular prof ile of oncogenes and tumor suppressor genes in carcinomas of the large intestine ( cecum) from the same NTP two-year bioassay of o -nitrotoluene. Eleven colon tumors from B6C3F 1 mice exposed to o -nitrotoluene were analyzed for mutations in the p53, K- ras, and β -catenin genes and for expression of these three proteins and the adenomatous polyposis coli (APC) protein, and comparedRoC Background Document for o -Nitrotoluene

68 6/20/08 with normal colon tissue. Most of the colon tumors had increased protein levels of p53, β -catenin, and cyclin D1, but no difference in protein expression was found for APC. Mutations in all three genes were identified in most of the o -nitrotoluene–induced tumors. All but one of the K- ras gene mutations were G → T transversions (Gly to Val) (see Table 5-9). 1
2 3 4 5RoC Background Document for o -Nitrotoluene

6/20/08 69 Table 5-8. p53, β -catenin, and K- ras mutations in hemangiosarcomas (subcutaneous tissue, skeletal muscle, and mesentery) from B6C3F 1 mice orally exposed to o nitrotoluene Treatment group, mutations p53 (exon 6-8), number (%) b Catnb a , number (%) b K-ras (codon 61), number (%) b Controls o- Nitrotoluene 0 15 0 8 0 1 Total mutations according to dose (ppm) 1,250 2,500 5,000 1 (6.7) 1 (6.7) 13 (86.7) 1 (12.5) 0 7 (87.5) 0 0 1 (100) Type of mutations identified Transitions (all) G
⋅ C → A
⋅ T A
⋅ T → G
⋅ C 12 (80) 9 (60) c 3 (20) 1 (12.5) 0 1 (12.5) 0 0 0 Transversions (all) G
⋅ C → T ⋅ A G
⋅ C → C
⋅ G A
⋅ T → C
⋅ G A
⋅ T → T ⋅ A 3 (20) 2 (13.3) 0 0 1 (6.7) 0 0 0 0 0 1 (100) 0 0 0 1 (100) Deletions 0 7 (87.5) 0 Source: Hong et al. 2003.
a 5 deletions in exon 2 splice site s and 2 deletions in other sites, 1 base substitution in codon 28. b Percent of total mutations identified, not percent of hemangiosarcomas with mutati ons. Fifteen mutations in p53 were

identified in 11 hemangiosarcomas (4 hemangiosar comas had double mutations), 8 mutations in C atnb were identified in 7 hemangiosarcomas (1 hemangiosarcoma had a double mutation), 1 mutation in K- ras was identified in 1 hemangiosarcoma.
c 6 mutations were G → A transitions and 3 mutations were C → T transitions. [The pattern of mutations in both of these st udies is consistent with targeting of guanine for adduct formation since mutations in the p53 gene in hemangiosarcomas (see Table 5- 7) mainly involved G
⋅ C → A
⋅ T transitions and almost all the mutations in the K- ras gene in cecal carcinomas (see Table 5-8) were G
⋅ C → T ⋅ A transversions.] 1
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Table 5-9. p53, β -catenin, and K- ras mutations in cecal carcinomas from B6C3F 1 mice orally exposed to o -nitrotoluene Treatment group, Mutations p53 (exon 7), number (%) b Catnb a , number (%) b K-ras (codon 10-13), number (%) b Controls c
o- nitrotoluene 0 12 0 14 0 10 Total mutations according to dose (ppm) 1,250 2,500 4 8 6 8 5 5 Type of mutation identified Transitions (all) G
⋅ C → A
⋅ T A
⋅ T → G
⋅ C 4 (33.3) 3 (25.0) 1 (8.3) 1 (7.1) 1 (7.1)

0 1 (10) 1 d

(10) 0 Transversions (all) G
⋅ C → T ⋅ A G
⋅ C → C
⋅ G A
⋅ T → C
⋅ G A
⋅ T → T ⋅ A 8 (66.7) 2 (16.7) 0 0 6 (50) 3 (21.4) 0 1 (7.1)

0 2 (14.3) 9 (90) 9 e (90) 0 0 0 Deletions 0 10 (71.4) 0 Source: Sills et al. 2004.
a 10 deletions occurred in exon 2 and/or 5, and 4 base-pai r substitutions occurred in codons 15, 25, 37, and 41. b Percent of total mutations identified, not percent of cecal carcinomas with mutations. Twelve mutations in p53 were

identified in 9 carcinomas (3 carcinomas had double mutations), 14 mutations in Catnb were identified in 11 carcinomas (3 carcinomas had doubl e mutations), 10 mutationS in K- ras were identified in 9 carcinomas (1 carcinoma had a double mutation).
c No cecal carcinomas occurred in control animals, so norma l colon tissue from non-treated mice or non-tumor regions

from mice exposed to o -nitrotoluene was used as controls. d Codon 13.
e 1 mutation in codon 10 and 8 mutations in codon 12.

70 6/20/08RoC Background Document for o -Nitrotoluene

6/20/08 71 Human colorectal cancers also have a high frequency of mutations in the K- ras and p53 genes, and the β -catenin and cyclin D1 genes are upr egulated. Therefore, both human and mouse colon tumors have alterations in the β -catenin/Wnt signaling pathway, ras /MAP kinase pathway, and cell-cycle checkpoint genes (e.g., the cyclin D1 and p53 genes). Specifically, these pathways may interact in the formation of large-intestine tumors to provide self-sufficiency in gr owth signaling through the K- ras oncogene, upregulation of growth and avoidance of apoptosis through the p53 gene, and increased cyclin D1 production through the combination of β -catenin and ras activation. Sills et al. (2004) concluded that the acquisition of similar genetic alterations in both human and mouse large-intestinal cells suggests that the chemically induced tumors in mice model the human cancer quite well, and the results are likely to be relevant to humans. 1
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22 Kim et al. (2006) investigated the genes involved in peritoneal mesotheliomas induced in male F344 rats by exposure to o -nitrotoluene or bromochlor oacetic acid. Mesotheliomas were collected from four rats, RNA was is olated, and gene expression analysis was conducted. The analysis identified 169 differe ntially expressed cancer-related genes that were categorized according to cancer-related function. The major carcinogenic pathways involved in peritoneal mesothelioma forma tion were identified as insulin-like growth factor 1 (IGF-1), p38 MAP kinase, Wnt/ β -catenin, and integrin signaling pathways. The authors noted that similar signaling pathwa ys were activated in studies of human mesotheliomas and mesothelioma cell lines. The results of the studies by Hong et al. (2003), Sills et al. (2004), and Kim et al . (2006) are summarized in Table 5-10.RoC Background Document for o -Nitrotoluene

Table 5-10. Gene mutations and gene expression in o -nitrotoluene-induced tumors in rodents. Test system (species and tumor type ) End point Results Reference p53 gene mutation + p53 protein + β -catenin gene mutation + β -catenin protein + B6C3F 1 mice (hemangiosarcomas)

K- ras gene mutation

– a Hong et al. 2003

p53 gene mutation + p53 protein + β -catenin gene mutation + β -catenin protein + K- ras gene mutation + cyclin D1 protein + B6C3F 1 mice (colon tumors)

APC protein – Sills et al. 2004

IGF-1 signaling pathway akt2, igf-1, igfb2, igfbp3, igfbp6, prkcz ↑ expression

fos ↓ expression P38 MAPK pathway mapkapk2 , stat1, tgfbr2 ↑ expression
tgfb2 , tgfb3 , tnfrsf6 ↓ expression Wnt/ β

-catenin pathway akt2 , ppp2r2 , tgfbr2, wnt4 ↑ expression
fzd2, gnaq, ilk, tgfb2, tgfb3 ↓ expression Integrin pathway itgb2, akt2 ↑ expression F344 rats (peritoneal mesotheliomas)

actn1, actn4, itga8, itgb1, actg2 , arf4, colla2 ↓ expression Kim et al. 2006

+ = positive; – = negative.
a one mutation was detected.

72 6/20/08RoC Background Document for o -Nitrotoluene

6/20/08 73 5.5 Carcinogenicity and genotoxicity of o -nitrotoluene analogues and metabolites 1 An NTP (1992) study included comparat ive toxicity evaluations of the o -, m -, and p nitrotoluene isomers in rats and mice. Th e NTP conducted a two-year carcinogenicity study of rats and mice exposed to p -nitrotoluene; however, no st udies describing chronic exposure to m -nitrotoluene were found. A great deal of research also has been done on the metabolism, mutagenicity, and carcinoge nicity of the dinitrotoluenes, and the bioactivation of 2,4-dinitrotolue ne is similar to that of o -nitrotoluene. The NTP has studied one metabolite of o -nitrotoluene, o -toluidine (as its hydrochloride), in a subchronic exposure study in male rats. This section briefly discusses the findings of these studies of o -nitrotoluene analogues and metabolites. 2 3 4 5 6 7 8 9 10

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22 23 5.5.1 p -Nitrotoluene Rats and mice exposed to p -nitrotoluene in the diet for two years had increased incidences of tumors in a number of or gans and systems (e.g., clitoral gland, skin, hematopoietic system or spleen, testis, a nd lung) (NTP 2002a). However, most of the increased incidences either were not si gnificantly higher than the incidences in concurrent or historical controls or were inconsistent among exposure groups. The NTP concluded that there was equivocal evidence of carcinogenic activity of p -nitrotoluene in male F344/N rats, based on increased incidenc es of subcutaneous skin neoplasia, and some evidence of carcinogenic activity in female F344/ N rats, based on increased incidences of clitoral-gland neoplasia (see Table 5-11). There was equivocal evidence of carcinogenic activity of p -nitrotoluene in male B6C3F 1 mice, based on increased incidences of alveolar/bronchiolar neoplas ia, and no evidence of carcinogenicity in female mice.RoC Background Document for o -Nitrotoluene

74 6/20/08 Table 5-11. Neoplastic lesions identi fied in rats and mice exposed to o -nitrotoluene and p -nitrotoluene

o -Nitrotoluene p -Nitrotoluene Organ or system F344/N rats B6C3F 1 mice F344/N rats B6C3F 1 mice Mesothelium Skin (subcutaneous) Mammary gland Liver Lung Large intestine (cecum) Circulatory system Clitoral gland Source: NTP 2002a, 2002b.
The NTP also concluded that o -nitrotoluene had greater carcinogenic potential than p -nitrotoluene ( o -nitrotoluene was tested in male and female rats at 625, 1,250, and 2,000 ppm in the diet, and p -nitrotoluene was tested at 1,250, 2,500, and 5,000 ppm; male and female mice were exposed to 1,250, 2,500, and 5,000 ppm in the diet for both isomers). The authors noted that this greater carcinogenic potentia l was predicted from studies showing that covalent binding of o -nitrotoluene to total rat hepatic macromolecules was 3.5 times higher than that of p -nitrotoluene and that o -nitrotoluene, but not p nitrotoluene, also bi nds to male F344 rat hepatic DNA (see Section 5.2). In addition, quantitative differences in metabolism of o -nitrotoluene and p -nitrotoluene have been observed, such as greater metabolism of o -nitrotoluene to the n itrobenzyl glucuronide, which is thought to give rise to o -aminobenzyl sulfate, the proximal reactive metabolite (see Section 5.2 and Figure 5-2) (NTP 2002a). 1
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13 14 15 16 17 18 19 Additionally, aromatic amine carcinogens upon metabolic activation yield electrophilic intermediates that bind to DNA yielding N -(deoxyguanosin-8-yl)arylamines (Marques et al. 1997). DNA binding studies have suggested that while o -, m -, and p -substituted arylamines all bind to DNA, the substitution in the ortho position yields a more stable DNA adduct (Marques et al. 1997). The Marques et al. study did not look at the comparative stability of the o -, m -, and p -nitrotoluene DNA adducts. However, the results suggest that further stud ies on the stability of o -, m -, and p -nitrotoluene DNA adducts areRoC Background Document for o -Nitrotoluene

6/20/08 75 warranted and may help to explain why o -nitrotoluene is a more potent chemical carcinogen than p -nitrotoluene. 1
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27 28 29 5.5.2 Dinitrotoluenes In 1984 Rickert et al. reviewed the metabolism, mutage nicity, and carcinogenicity of dinitrotoluenes. At the time of their review , three independent chronic bioassays, all of which were feeding studies, had been comple ted: (1) a National Cancer Institute (NCI) (1978) study of 2,4-dinitrotolue ne in F344 rats and B6C3F 1 mice, (2) a Chemical Industry Institute of Toxicology (CIIT) study of technical-grade dinitrotoluene in F344 rats, and (3) a U.S. Army-funded (conducted at Midwest Research In stitute) study of 2,4- dinitroluene in Sprague-Daw ley rats and Swiss mice. In the NCI study, 2,4-dinitrotol uene (95% pure, contamin ants not specified) was administered in the feed at time-weighted average dietary concentrations of 0.02% and 0.008% for 50 male and 50 female F344 rats and at 0.04% and 0.008% for 50 male and 50 female B6C3F 1 mice for 78 weeks followed by an additional 26 weeks of observation for rats and 13 weeks for mice. In male ra ts, a significantly increased incidence of fibroma of the skin and subcutaneous tissue occurred in both dose groups, and a statistically significant incide nce of fibroadenoma of the mammary gland occurred in the high-dose female rats. No tumors associated with 2,4-dinitrotoluene exposure were reported in mice. In the CIIT study, male and female F344 rats (10 per exposure group) were administered technical-grade dinitrotoluene (a mixt ure containing 2,4-, 2,6-, 3,4-, 2,3-, 2,5- and 3,5- dinitrotoluene at 76.4%, 18.8%, 2.4%, 1.5%, 0.7%, and < 0.1%, respectively) at 0, 3.5, 14, and 35 mg/kg per day for 2 years. High incidences of hepatic neoplasms (hepatocellular carci nomas or neoplastic nodules) were observed in high- and mid-dose males and females. Technical-grade dinitr otoluene also induced cholangiocellular carcinoma in both sexes. In the third study (U.S. Army funded) reviewed by Rickert et al. , a mixture of 98% 2,4- and 2% 2,6-dinitrotoluene was administered in the feed to Sprague-Dawley rats and Swiss mice for 2 years. In rats, the average intake of 2,4-dinitrotol uene (mg/kg per day)RoC Background Document for o -Nitrotoluene

76 6/20/08 was 0.575, 3.92, or 34.5 for males and 0.706, 5.14, or 45.3 for females. High-dose female rats had significantly increased incidences of hepatocellular carcinoma. In mice, the average intake of 2,4-dinitrotoluene (mg/ kg per day) was 13.3, 96.9, or 885 for males and 13.7, 93.8, or 911 for females. An increase in kidney tumors was reported in mid-dose males. It was noted that high-dose males had early deaths. 1
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26 27 28 29 Initiation-promotion studies in male F344 rats have demonstrated that technical-grade dinitrotoluene and 2,6- dinitrotoluene have initiating a nd promoting activ ity in the rat liver, whereas 2,4-dinitrotol uene had only promoting ac tivity (reviewed by Rickert et al. 1984b). Leonard et al . (1987) noted that 2,4-di nitrotoluene did not induce hepatic tumors in F344 rats in the NCI study. However, tec hnical-grade dinitrotoluene caused a 100% liver tumor incidence by 55 weeks in F 344 rats in the CIIT study. Leonard et al. noted that rats in the CIIT study r eceived a 5- to 10-times higher do se of 2,6-dinitrotoluene than the rats in the NCI study, which was base d on the relatively high 2,6-dinitrotoluene concentration in the CIIT study (~ 19%) vs. the lower concentration ( ≤ 5%) as a contaminant in the NCI study, and on the dosi ng rate of 5.6 or 14.0 mg/kg per day for the NCI study as compared with 3.5, 14, or 35 mg/kg per day for the CIIT study). Because of the contrasting results in the th ree chronic bioassays, Leonard et al . (1987) conducted a feeding study in F344 rats to compare the carcinogenic activity of technical-grade dinitrotoluene (a mixture of 76.5% 2,4-dinitr otoluene and 18% 2,6-dinitrotoluene), and the two purified isomers (2,4- and 2,6-dinitrotoluene). Rats were fe d diets containing the dinitrotoluenes resulting in average intakes (mg/kg per day) of 27 for 2,4-dinitrotoluene, 7 or 14 for 2,6-dinitrotoluene, and 35 for tec hnical-grade dini trotoluene for 52 weeks. At 52 weeks, hepatocellular carci nomas were observed in the 2,6-dinitroluene–treated animals (100% of high-dose and 85% of low-dose rats) and technical-grade dinitrotoluene–treated animals (47%) but not the 2,4-dinitrotoluene –treated animals. In addition to the chronic bioassays, 2,4- and 2,6- dinitrotoluene were tested in short-term bioassays for their ability to produce pulm onary tumors. Both compounds were inactive by oral or intraperitoneal admi nistration in strain A (Schut et al. 1983, Schut et al. 1982) and A/J mice (Stoner et al. 1984).RoC Background Document for o -Nitrotoluene

6/20/08 77 Mutagenicity studies of the di nitrotoluenes in bacterial and yeast systems showed activity for all the isomers (Rickert et al. 1984b). The increased activ ity of putative reduced metabolites of the dinitrotoluenes together with the inactivity in systems lacking nitroreductase suggests that ni troreduction is necessary for mutagenic activity. Studies in in vitro mammalian systems gave uniformly ne gative results for all isomers of dinitrotoluene, suggesting that extrahepatic metabolism was necessa ry for activation of these compounds. Studies in the in vivo

– in vitro rat hepatocyte DNA repair assay yielded results analogous to those obtained by Doolittle et al. (1983) for o- nitrotoluene (Section 5.3.2). The majority of the activity to induce DNA repair resided with 2,6-dinitrotoluene; the activity was greater in male than in female rats and depended upon an intact intestinal microflora. 1
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22 23 24 25 26 27 28 29 These studies showed that sulfotransferase inhibition in vivo decreased the covalent binding of the hepatocarcinogen 2,6-dinitr otoluene to hepatic DNA by > 95% and indicate that sulfation is involved in the bioactivation of 2,6-dinitrotoluene. These studies, along with the in vitro studies of N -oxidation cited above (see Section 5.1.3), helped clarify the complex bioactivation mechan isms of the dinitr otoluenes (Kedderis et al. 1984). Kedderis and Rickert (1985) noted that the rate of metabolism of 2-amino-6- nitrobenzyl alcohol to hydroxlyamines and aminophenols was significantly lower with microsomes from female rats compared with male rats. Thus, sex differences in the rate of metabolism of 2,6-dinitrotoluene may be re lated to sex differences in tumor formation.
The metabolism of 2,6-dinitrotoluene and rela ted isomers is also analogous to that of

o- nitrotoluene and related isomers (Rickert et al. 1984b). Activation of 2,6-dinitrotoluene is thought to involve oxidation of the methyl group to an alcohol, followed by conjugation with glucuronic acid and excretion in bile. As with o- nitrotoluene, male rats excrete more of a dose of 2,6-dinitrotoluene in the bile as 2,6-dini trobenzyl glucuronide than do females. Once in the intestine, the gl ucuronic acid moiety is cleaved, and one of the nitro groups is reduced before reab sorption. The final activation may involve sulfation at the benzyl alcohol group or via N -hydroxylamine formation (Chism and Rickert 1989).RoC Background Document for o -Nitrotoluene

78 6/20/08 Several studies have investigated the potenti al for dinitrotoluenes to cause cancer in human subjects (see Section 3.2.1). These data suggest the possibility of carcinogenicity of dinitrotoluenes in humans. 1
2 3

4 5 6 7 8 9 10
11
12

13 14 15 16 17 18 19 20
21

22 23 24 25 26

28 29 5.5.3 o -Toluidine hydrochloride

o- Toluidine ( o -aminotoluene) hydrochloride administered in feed to F344 rats and B6C3F 1 mice for 101 to 104 weeks (NCI 1979) incr eased the incidences of mesothelioma of the abdominal cavity or scro tum in male rats, transitional- cell carcinoma of the urinary bladder in female rats, and several types of sarcoma in both male and female rats. In mice, hemangiosarcoma was induced at various sites in males, and hepatocellular carcinoma or adenoma was induced in females. The NCI concluded that o -toluidine hydrochloride was carcinogenic in both rats a nd mice, producing a significantly increased incidence of one or more types of neoplasia. In a later study (NTP 1996), the toxicity of o -nitrotoluene and o -toluidine hydrochloride administered in feed at approximately equi molar doses (5,000 ppm) for 13 or 26 weeks to male F344/N rats was examined because of the structural similarity of these two molecules. Both o -nitrotoluene and o -toluidine hydrochloride caused mesothelial hyperplasia and mesothelioma in male rats after 13 or 26 weeks of dietary exposure. However, the incidence of mesothelioma was greater and the latency was less in rats administered o -nitrotoluene than in rats administered o -toluidine hydrochloride. The two chemicals had similar toxic effects on the spleen; however, their morphologic effects on the testis, epididymis, liver, kidne y, and urinary bladder differed. Epidemiological data indicate an increased risk of bladder cancer in workers exposed to

o -toluidine and anilin e (see Section 3.2.2). o -Toluidine and its hydr ochloride salt are listed in the Report on Carcinogens as reasonably anticipated to be a human carcinogen based on limited evidence in humans and sufficient evidence in animals, and o -toluidine is classified by IARC (Baan et al. 2008) as carcinogenic to humans (Group 1).

5.6 Toxicity 27 Groups of 10 male and 10 female F344/N rats per exposure group received diets ad libitum containing o -nitrotoluene at concentrations of 0, 625, 1,250, 2,500, 5,000, orRoC Background Document for o -Nitrotoluene

6/20/08 79 10,000 ppm (consumption of o -nitrotoluene was estimated by NTP to range from approximately 40 to 700 mg/kg b.w . per day) for 13 weeks (Dunnick et al. 1994, NTP 1992). All animals survived to the end of th e studies but males showed more treatmentrelated toxicity than females. Toxic effect s were graded on a scale of 1 to 4 where 1 represented minimal effects and 4 represented se vere effects. Most of the effects occurred at concentrations of 2,500 ppm or greater . The most severe lesions were liver vacuolization (grade 3 at 10,000 ppm), ki dney nephropathy (grade 2.8 at 5,000 ppm and 2.6 at 10,000 ppm), and testis degeneration (grade 4 at 10,000 ppm). In addition to hepatocyte vacuolization, other hepatotoxi c effects included oval-cell hyperplasia, PGST+ foci, and increased serum bile aci ds, sorbitol dehydrogenase, and alanine aminotransferase. Kidney pigmentation (grade 1.8 at 10,000 ppm) and spleen pigmentation (grade 2 at 5,000 and 10,000 ppm) we re the predominant effects in females.

o -Nitrotoluene also resulted in hematopoiesis and pigmentation in the spleen in male and female rats exposed for 2 years (NTP 2002b). No kidney or spleen lesions occurred in females at concentrations less than 2,500 pp m, and no liver lesions were observed in females at any concentration tested. 1
2 3 4 5 6 7 8 9 10
11
12 13 14 15 16

17 18 19 20

21
22 23 24 25 26 27 28 29 30 All three isomers of nitrotoluene have caused kidney toxicity in male rats, characterized by hyaline droplet nephropathy. Exposure to o - or p -nitrotoluene also increased renal α 2u globulin levels in male rats (the m -isomer was not tested) (Dunnick et al. 1994, NTP 1992, 1996). NTP (1992) conducted 13-week studies with B6C3F 1 mice exposed to o -nitrotoluene (< 96% purity), to compare c linical toxicity, histopat hology, and reproductive-system toxicity among the nitrotoluene isomers ( o -, m -, and p -). Each compound was administered separately in feed to groups of 10 males and 10 females per isomer per exposure group at a concentration of 0, 625, 1,250, 2,500, 5,000, or 10,000 ppm (consumption of o -nitrotoluene was estimated by NTP to range from approximately 100 to 1,700 mg/kg b.w. per day). The mice were 6 to 8 weeks old at the beginning of the studies and all animals survived to the end of the studies. Body-weight gain was lower in the two highest-exposure groups of males than in contro ls. The only treatment-related histopathological lesions were olfactory epithelium degene ration/metaplasia in both maleRoC Background Document for o -Nitrotoluene

80 6/20/08 and female mice (see Table 5-12). The observe d changes included m oderate thinning of the olfactory nuclear layer, decreased diamet er of the associated nerve fiber bundles in the lamina propria, replacement of the ol factory epithelium by cuboidal respiratory epithelium, and dilation of the submucosa underlying Bowman’s glands (Dunnick et al. 1994). 1
2 3 4 5

Table 5-12. Treatment-related lesions in B6C3F 1 mice following dietary exposure to o -nitrotoluene for 13 weeks a
Nose
Sex Concentration (ppm) Olfactory epithelium degeneration/metaplasia Male 0 625 1,250 2,500 5,000 10,000 0 0 1 (1.0) b 2 (1.0) 10 (2.0) 10 (3.0) Female 0 625 1,250 2,500 5,000 10,000 0 0 2 (1.5) 9 (1.0) 10 (1.9) 10 (2.9) Source: NTP 1992, Dunnick et al. 1994.
a Ten animals per treatment group/sex per species. b Numbers in parentheses represent average severity grade; 1 = minimal, 2 = mild, 3 = moderate, 4 = severe. 5.7 Summary 6 5.7.1 Absorption, distribution, metabolism, and excretion 7 8 9 10
11
12 13 14 15 16 17 18 o -Nitrotoluene has been shown to be absorbed after oral administration to rats and mice, and evidence suggests that absorption also occurs in humans exposed to o -nitrotoluene. Metabolites of o -nitrotoluene, which include o -nitrobenzoic acid and o -nitrobenzyl alcohol, have been detected in the urine of factory workers and indi cate that absorption occurs from skin contact a nd inhalation. The half-life of o -nitrotoluene in plasma of rats is fairly short, approximately 1.5 hours, and the primary route of excretion is urinary, occurring mainly in the firs t 24 hours after exposure, with lesser amounts in feces and expired air. o -Nitrobenzoic acid and o -nitrobenzyl glucuronide are major metabolites of

o -nitrotoluene in both rats and mice, but other metabolites, such as S -( o -nitrobenzyl)- N

acetylcysteine and o -aminobenzyl alcohol, are found only in rats. Female rats were found to excrete less than half as much of an oral dose of o- nitrotoluene as o -aminobenzylRoC Background Document for o -Nitrotoluene

6/20/08 81 alcohol or as S -( o -nitrobenzyl)- N -acetylcysteine as did male rats. The major biliary metabolite of o- nitrotoluene was o -nitrobenzyl glucuronide, which accounted for 22.1% of the dose in males and 8.3% of the dose in females. The next most abundant metabolite

was S -( o -nitrobenzyl) glutathione, which accounte d for 4.9% of the dose in males and 0.4% in females. Metabolites excreted thr ough the bile can be r eabsorbed and further metabolized. 1
2 3 4 5 6

7 8 9 10
11
12 13 14 15 16 17 18 19

20 21
22 23 24 25 26

27 28 29 5.7.2 Bioactivation of o -nitrotoluene Deleterious effects of o -nitrotoluene generally are more severe in male rats than in females and include changes in hepatic, rena l, or splenic histopat hology, tumor incidence, macromolecular covalent binding, or DNA repair. The toxicity of o -nitrotoluene, particularly in male rats, likely involves its metabolism by oxidation of the methyl group to an alcohol, conjugation of o -nitrobenzyl alcohol with gluc uronic acid and excretion in bile, deconjugation of o -nitrobenzyl glucuronide and reduction of the nitro group by intestinal bacteria, and final activation of o -aminobenzyl alcohol by the formation of

o -aminobenzyl sulfate. This activation scheme adequately explains the observations on sex and isomer differences in the covalent binding of nitrotoluene-r elated material in hepatic DNA, and it also explains the sex, isomer, and intestinal flora dependency of the in vivo

– in vitro DNA repair assay. In rats exposed to o -nitrotoluene in vivo , DNA adducts and increased DNA repair were detected in males but not females.

5.7.3 Genetic damage and related effects

o -Nitrotoluene did not cause mutations in prokaryotic systems. In mammalian in vitro systems, it induced (1) sister chromatid exch ange in Chinese hamster ovary (CHO) cells; (2) chromosomal aberrations in Chinese ha mster lung cells (CHL) and human peripheral lymphocytes (but not in CHO cells); (3) micronuclei in CH L cell line (but not in CHOK1 cells), and (4) DNA damage in L5178Y m ouse lymphoma cells. It did not induce DNA repair in rat or human hepatocytes.

In vivo genotoxicity studies in laboratory rodents indicated covalent binding to hepatic macromolecules and DNA in male but not female rats treated with o -nitrotoluene. Other studies indicated that o -nitrotoluene induced a dose-depen dent increase in the percentageRoC Background Document for o -Nitrotoluene

82 6/20/08 of hepatocytes undergoing DNA repair and the ex tent of DNA repair in male rats. Studies with germ-free male rats did not show an increase in DNA repair and suggest that intestinal bacteria are needed for metabo lic activation. Differen ces between males and females were likely due to differences in hepatic metabolism and/or disposition because there were no significant differences in the intestinal flora of male and female rats. Cytogenetic studies indicated that o -nitrotoluene did not cause an increase in micronucleus frequency in F344 rats or B6C3F 1 mice except for a slight increase in the frequency of micronucleated NCEs in male mice exposed to 10,000 ppm in one study. 1
2 3 4 5 6 7 8

9 10
11
12 13

14 15 16 17 18 19 20
21
22

23 24 25 26 27 28 29 Gene expression studie s with mouse L5178Y Tk +/- lymphoma cells in vitro identified four genes and a few pathways whose expression was consistently affected by o nitrotoluene. A study of early ch anges in gene expression in mouse liver after exposure to

o -nitrotoluene in vivo for 2 weeks identifie d changes in tumor s uppressor genes, cellcycle genes, and apoptosis-promoting genes.

5.7.4 Mechanistic studies and considerations [The activation of o -nitrotoluene to o -aminobenzyl sulfate by a combination of mammalian and bacterial metabolism appears ade quate to explain the carcinogenicity of

o -nitrotoluene in male rats; however, based on results of chronic bioassay studies in male and female rats and male and female mice, there appear to be other mechanisms of activation of o -nitrotoluene. The significantly increas ed incidences of tumors in other tissues of rats or mice, including mammary gland, skin, lung, large intestine, and hemangiosarcomas in various tissues support th e concept that other activation pathways may exist.] In gene expression studies on mouse liver, a dose-dependent loss of expression of the fragile histidine triad gene ( Fhit ) and the WW domain-cont aining oxidoreductase ( Wwox ) gene was observed with exposure to o -nitrotoluene. The authors noted that these genes are human tumor suppressor genes and are of ten lost together in many human cancers.

o -Nitrotoluene treatment also resulted in strong up-regulation of the cell-cycle genes, cyclin G1 (Ccng1) and p21 ( Cdkn1a ), down-regulation of the epidermal growth factor ( Egfr ) gene, down-regulation of the transcrip tion factor early growth response 1 ( Egr1 )RoC Background Document for o -Nitrotoluene

6/20/08 83 gene at 5,000 ppm in males only, and down-regulation of inhibin β -A ( Inhba ), a member of the TGF- β superfamily, and Jun-B oncogene ( Junb ), a negative regulator of proliferation genes. 1
2 3

4 5 6 7 8 9
10
11
12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30 Mutations in the p53 and β -catenin genes and production of these proteins in mice were detected in o -nitrotoluene–induced hemangiosarcomas and colon tumors but not in spontaneous hemangiosarcomas. K- ras gene mutations and cyc lin D1 protein production also were detected in the colon tumors. Mutations in p53, β -catenin, and K- ras genes may be a result of the genotoxic effects of o -nitrotoluene. The pattern of mutations is consistent with targeting of guanine for a dduct formation since mutations in the p53 gene in hemangiosarcomas mainly involved G
⋅ C → A
⋅ T transitions, and almost all the mutations in the K- ras gene in cecal carcinomas were G
⋅ C → T ⋅ A transversions. Human colorectal cancers also have a hi gh frequency of mutations in the K- ras and p53 genes, and the β -catenin and cyclin D1 genes are upreg ulated. As a result of these genetic effects, both human and mouse colon tumors have alterations in pathways that are considered important for the progression of cells from a normal state to cancer; these pathways include the β -catenin/Wnt signaling pathway, ras /MAP kinase pathway, and cell-cycle checkpoint genes (e.g., the cyclin D1 and p53 genes). Major carcinogenic pathways involved in peritoneal mesoth elioma formation in mice exposed to o nitrotoluene also were similar to pathways activated in human me sotheliomas, including IGF-1, p38 MAPK, Wnt/B-catenin, a nd integrin signaling pathways.

5.7.5 Carcinogenicity and genotoxicity of o -nitrotoluene analogues and metabolites The NTP also conducted cancer studies in expe rimental animals on another nitrotoluene isomer, p -nitrotoluene, and found equivocal evidence of carcinogenic a ctivity in male rats and male mice, some evidence of carcinogenicity in female rats and no evidence of carcinogenic activity in female mice. The NTP concluded that o -nitrotoluene had greater carcinogenic potential than p -nitrotoluene and that the differences in carcinogenicity potential between the two isomers may be due to (1) greater stability of the ortho adduct, (2) higher covalent binding of o -nitrotoluene to rat hepatic m acromolecules, or (3) greater metabolism of o -nitrotoluene to the n itrobenzyl glucuronide, which gives rise to o aminobenzyl sulfate, the proposed proximal reactive metabolite.RoC Background Document for o -Nitrotoluene

84 6/20/08 5.7.6 Toxicity 1 2 3 4 5 6 7 8 9 10 o -Nitrotoluene caused more treatment-related toxi city in male rats than in female rats, and most toxic effects occurred at or greater than 2,500 pp m in a 13-week study. o Nitrotoluene exposure resulted in toxic effect s in the kidney (hyalin e droplet nephropathy and increased renal α 2u -globulin levels) and liver (hep atocyte vacuolization, oval-cell hyperplasia, PGST+ foci, and increased seru m bile acids, sorbitol dehydrogenase, and alanine aminotransferase). Hematopoesis and pigmentation in the spleen were noted in male and female rats exposed to o- nitrotoluene for two years, but no liver lesions were noted in female rats at any concentration (up to 10,000 ppm tested). No kidney or splenic lesions occurred in females at conc entrations less than 2,500 ppm.RoC Background Document for o -Nitrotoluene


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. Cancer Res 43(6): 2836-2842. (Support not reported. Authors affiliated with Chemical Industry Institute of Toxicology.) 26. Dunnick JK, Elwell MR, Bucher JR . 1994. Comparative toxicities of o -, m -, and p nitrotoluene in 13-week feed studies in F344 rats and B6C3F 1 mice. Fundam Appl Toxicol 22(3): 411-421. (Support not reported. Au thors affiliated with NIEHS.) 27. Dunnick JK, Burka LT, Mahler J, Si lls R. 2003. Carcinogenic potential of o nitrotoluene and p -nitrotoluene. Toxicology 183(1-3): 221-234. (Support not reported. Authors affiliated with NIEHS.) 28. EPA. 2007. Inventory Update Rule . U.S. Environmental Protection Agency. http://www.epa.gov/opp t/iur/iur02/index.htm . Last accessed: 4/26/07. 29. FirstChem. 2008. First Chemical Corporation - Company Information . DuPont Corporation. http://www2.dupont.com/FirstChem/en_US /tech_info/profile/company_info.html

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## Glossary of Terms

Adenoma: An ordinarily benign neoplasm of epith elial tissue in which the tumor cells form glands or gland-like structures in the stroma. Autoignition temperature: The minimum temperature required to cause self-sustained combustion without any other source of heat. Boiling point: The boiling point of the anhydrous s ubstance at atmospheric pressure (101.3 kPa) unless a different pre ssure is stated. If the substance decomposes below or at the boiling point, this is noted (dec). The te mperature is rounded off to the nearest °C. Carcinoma: Any of the various types of malignant neoplasms derived from epithelial tissue in several sites. Cholangiocarcinoma: An adenocarcinoma, primarily in intrahepatic bile ducts, composed of ducts lined by cuboidal or co lumnar cells that do not contain bile. Covalent binding: A bond in which each atom of a bound pair contributes one electron to form a pair of electrons. Critical temperature: The temperature at and above wh ich a gas cannot be liquefied, no matter how much pressure is applied. Density: The density for solids and liquids is expressed in grams per cubic centimeter (g/cm 3 ) and is generally assumed to refer to te mperatures near room temperature unless otherwise stated. Values for gases are gene rally the calculated ideal gas densities in grams per liter at 25°C and 101.325 kPa. Diazo-positive compounds: Non-specific markers of exposure to aromatic amines. Fibroma: A benign neoplasm derived from fibrous connective tissue. Flash point: The lowest temperature at atmosphe ric pressure (101.3 kPa) at which a liquid gives off so much combustible vapor at the liquid surface that this vapor, when mixed intimately with air, can be ignited by a flame or spark. Half-life: The time required for one half of a given material to undergo chemical reactions.
Isomer: One of two or more chemical substances having the same elementary percentage composition and molecular weight but differing in structure. Hemangiosarcoma: A malignant tumor characterize d by rapidly proliferating cells derived from the blood vessels and li ning irregular blood-filled spaces.

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Henry’s Law constant at 25°C: The ratio of the aqueous-phase concentration of a chemical to its equilibrium partial pressure in the gas phase. The la rger the Henry’s law constant the less soluble it is (g reater tendency for vapor phase). Hepatocholangiocarcinoma: A malignant neoplasm containing cells resembling both hepatocytes and biliary epithelium. Hepatocyte: A parenchymal liver cell. Hyperplasia: An increase in the number of cells in a tissue or organ, excluding tumor formation. Inventory Update Rule (IUR): The purpose of the Inventory U pdate Rule is to assist EPA in keeping an inventory of chemical s ubstances in commerce in the United States. Initially, the rule required any company that produced or imported any chemical in the TSCA Chemical Substances Inventory List (TSCA Inventory) at a quantity of 10,000 pounds or more to report to EPA some basic information on that chemical. Reporting was required every four years. Amendments to th e rule enacted in 2003 raised the threshold limit to 25,000 pounds and increased the re porting period to every 5 years.
K oc : Soil organic adsorption coefficient, wh ich is calculated as the ratio of the concentration of a chemical adsorbed to the organic matter co mponent of soil or sediment to that in the aqueous phase at equilibrium. Lipoma: A benign neoplasm of adipose tissue, comprised of mature fat cells. Lipophilic: Having a strong affinity for fats. Log octanol-water partition coefficient (log K ow ): The ratio of concentrations of a substance in octanol and in water, when disso lved in a mixture of octanol and water. For convenience, the logarithm of K ow is used. The octanol/water partition coefficient of a substance is useful as a means to predic t soil adsorption, biologi cal uptake, lipophilic storage, and bioconcentration. Melting point: The melting point of the substance at atmospheric pressure (101.3 kPa). When there is a significant difference betw een the melting point and the freezing point, a range is given. In case of hydrated substances (i.e., those with crysta l water), the apparent melting point is given. If the substance decom poses at or below its melting point, this is noted (dec). The temperature is rounded off to the nearest °C. Mesothelioma: A neoplasm derived from the lining cel ls of the pleura and peritoneum that is composed of either epithe lial-like cells, spi ndle cells, or both. Methemoglobin: A compound formed from hemoglobin by oxidation of the iron atom from the ferrous (Fe 2+ ) to the ferric (Fe 3+ ) state with essentially ionic bonds, rendering it incapable of functioning reve rsibly as an oxygen carrier. Methemoglobin is present in small amounts in blood normally, but injury or toxic agents can increase the conversion.

100 6/20/08RoC Background Document for o -Nitrotoluene
Molecular weight: The molecular weight of a substa nce is the weight in atomic mass units of all the atoms in a given formula. The value is rounded to the nearest tenth. Neoplasm: Tumor. Negative log acid dissociation constant (pK a ): A measure of the degree to which an acid dissociates in water (a measurement of acid strength). The pKa is the negative logarithm (to the base 10) of the acid dissoci ation constant (Ka); the lower the pKa, the stronger the acid.
Norharman: A co-mutagen found in tobacco tar and in pyrolysate of tryptophan
. Perched aquifer: An aquifer that has a confining la yer below the groundwater and sits above the main water table. Physical state: Substances may either be gases, liquids, or solids according to their melting and boiling points. Solids may be de scribed variously as amorphous, powders, pellets, flakes, lumps, or crystalline; and the sh ape of the crystals is specified if available. Solids also may be described as hygro scopic or deliquescent depending upon their affinity for water. Pleomorphic: Occurring in various distinct forms. Poly-3 test: Poly-3 test: A survival-adjusted statistic al test that takes survival differences into account by modifying the denominator in the numerical (quantal) estimate of lesion incidence to reflect more closely the total numbe r of animal years at risk. For analysis of a given tumor site, each animal is assigned eith er (1) a risk weight of one if the animal had a lesion at that site or if it survived until terminal sacrifice or (2) a risk weight that is the fraction of the entire study tim e that it survived, raised to the 3rd power, if the animal died prior to terminal sacrifice and did not ha ve a lesion at that site . The resulting test is similar to the Cochran-Armitage trend test, with the adjusted tumor rates replacing the observed tumor rates in the test statistic (Bieler and Williams 1993, Portier and Bailer 1989). Specific gravity: the ratio of the density of a mate rial to the density of a standard material, such as water at a specified temper ature; when two temperatures are specified, the first is the temperature of the material and the second is the temperature of water. Subcutaneous: Beneath the skin. Tunica vaginalis : The serous membrane covering the front and sides of the testis and epididymis. Ubiquitination : Post-translational modification of a protein by covalent attachment of one or more ubiquitin monomers, which functio ns to mark the protein for proteolytic destruction.

6/20/08 101RoC Background Document for o -Nitrotoluene

102 6/20/08 Vapor density, relative: A value that indicates how many times a gas (or vapor) is heavier than air at the same temperature. If the substance is a li quid or solid, the value applies only to the vapor fo rmed from the boiling liquid. Vapor pressure: The pressure of the vapor over a liquid (and some solids) at equilibrium, usually expressed as mm Hg at a specific temperature (°C).