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 27709 
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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 assistance from other Federal health and 

regulatory agencies and nongovernmental institutions. 

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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 process with multiple opportunities for public 

comment. The scientific peer-review groups 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 sources. 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occurrence may come from published and/or unpublished sources.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-reviewed 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 private sectors with relevant expertise and 

knowledge selected by the NTP in accordance with the Federal Advisory Committee Act 

and HHS guidelines and regulations. This document has been finalized based on the peerreview recommendations of the expert panel and public comments received on the draft 

document. Any interpretive conclusions, comments, or statistical calculations made by 

the authors or peer reviewers of this document that are not contained in the original 

citation are identified in brackets [ ]. 
RoC Background Document for o-Nitrotoluene 

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A detailed description of the RoC nomination review process and a list of all substances 

under consideration for listing in or delisting from the RoC can be obtained by accessing 

the 12th RoC at 

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http://ntp.niehs.nih.gov/go/9732. The most recent RoC, the 11th Edition 

(2004), is available at 

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4 http://ntp.niehs.nih.gov/go/19914. 
RoC 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. Laboratory 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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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 & Health 
RoC 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 exposure 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 incidence of malignant and/or a combination of 

malignant and benign tumors (1) in multiple species 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 carcinogenicity 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 indicating it would likely cause cancer in 

humans. 

Conclusions regarding carcinogenicity in humans or experimental animals are based on 

scientific judgment, with consideration given to all relevant information. Relevant 

information includes, but is not limited to, dose response, route of exposure, chemical 

structure, metabolism, pharmacokinetics, sensitive 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 indicating that the agent acts through mechanisms 

which do not operate in humans and would therefore 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 cancer mechanism is operating in people. 

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Executive Summary 

1 Introduction 

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o-Nitrotoluene is a nitro aromatic 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 synthesis of dyes that are used in the textile, 

paper, and other industries and also in the synthesis of agricultural, rubber, and other 

chemicals. 

o-Nitrotoluene was nominated for possible listing in the Report on Carcinogens based on 

the results of a 2002 National Toxicology Program (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 sulfuric, aromatic sulfonic, or phosphoric acid. oNitrotoluene is a high production volume (HPV) chemical, and its U.S. production was 

10 to 50 million pounds per year for every four-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 and 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 groundwater 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 effluent or wastewater of plants 

producing these chemicals. The uses of di- and trinitrotoluenes include the production of 

commercial and military explosives, and o-nitrotoluene has been found in the 
RoC Background Document for o-Nitrotoluene 

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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. 

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Human Cancer Studies 

No studies on the relationship between human cancer and specific exposure to onitrotoluene 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 entails exposures that are carcinogenic. Their 

assessment was based on two cohort studies and a case-control study, all of which 

reported an excess risk of bladder cancer; 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 onitrotoluene in humans because the workers were also exposed to other chemicals, such 

as o-toluidine, 2,5-diaminotoluene, and 4,4'-methylenebis(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 incidences 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 considered 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 incidences 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

1 Absorption, Distribution, Metabolism, and Excretion 

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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 skin contact and inhalation. o-Nitrotoluene is absorbed after oral 

administration to rats and 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 metabolism, most likely leading to urinary 

excretion. o-Nitrotoluene or its metabolites are distributed to the liver, as indicated by 

recovery of radiolabel from o-nitrotoluene in the bile after oral administration and by its 

binding to hepatic DNA. No data documenting the distribution 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 onitrobenzyl glucuronide; however, other metabolites, such as S-(o-nitrobenzyl)-Nacetylcysteine 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)-Nacetylcysteine as did male rats. The major biliary metabolite of o-nitrotoluene was onitrobenzyl 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 toxicity in male rats than in female rats, 

and most toxic effects occurred at or greater than 2,500 ppm in a 13-week study. oNitrotoluene exposure resulted in toxic effects in the kidney (hyaline droplet nephropathy 
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and increased renal α2u-globulin levels) and liver (hepatocyte vacuolization, oval-cell 

hyperplasia, PGST+ foci, and increased serum 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 concentrations less than 2,500 ppm. 

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Genotoxicity and Mechanistic Data 

o-Nitrotoluene did not cause mutations in prokaryotic systems. In mammalian in vitro 

systems, it induced (1) sister chromatid exchange in Chinese hamster ovary (CHO) cells; 

(2) chromosomal aberrations in Chinese hamster lung cells (CHL) and human peripheral 

lymphocytes (but not in CHO cells); (3) micronuclei in CHL 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 normochromatic micronuclei in high-dose male mice 

(equivocal response) but did not induce micronuclei 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 mammalian and bacterial) and is sex specific. DNA 

repair was induced only in male rats with an intact intestinal microflora. Incubation of onitrotoluene in vitro with hepatocytes isolated from male rats failed to induce DNA 

repair. Biliary excretion is an important 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 alcohol, conjugation of o-nitrobenzyl 

alcohol with glucuronic acid and excretion in bile, deconjugation of o-nitrobenzyl 
RoC Background Document for o-Nitrotoluene 

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glucuronide and reduction of the nitro group by intestinal bacteria, and final activation of 

o-aminobenzyl alcohol by the formation of o-aminobenzyl sulfate. 

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o-Nitrotoluene caused significantly increased incidences of tumors in tissues other than 

the liver in both rats and mice, including mammary gland, skin, lung, large intestine, and 

hemangiosarcomas in various tissues. This suggests that there are other mechanisms of 

activation for o-nitrotoluene. 

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 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 guanine 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 that cellular and molecular events involved in 

the induction of mesotheliomas are similar in both experimental animals (rats exposed to 

o-nitrotoluene) and humans. Microarray analysis 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 expression in mouse liver, a dose-dependent loss of expression of 

the fragile histidine triad gene (Fhit) and the WW domain-containing oxidoreductase 
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(Wwox) gene was seen from o-nitrotoluene exposure. The authors noted that these genes 

are human tumor suppressor genes and are often lost together in many human cancers. 

Further, 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 transcription 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. 

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The NTP has conducted bioassay studies in experimental animals on another nitrotoluene 

isomer, p-nitrotoluene. Based on the results from this study of p-nitrotoluene, the NTP 

stated that there was equivocal evidence of carcinogenic activity in male rats and male 

mice, some evidence of carcinogenic activity 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 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 macromolecules, or (3) greater metabolism of onitrotoluene to the o-nitrobenzyl glucuronide, which gives rise to o-aminobenzyl sulfate, 

the proposed proximal reactive metabolite. 
RoC Background Document for o-Nitrotoluene 

Abbreviations 

ACGIH: American Conference of Governmental 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 

Koc: soil organic adsorption coefficient 

L: liter 

m3:

 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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RoC Background Document for o-Nitrotoluene 

ng: nanogram 

NTP: National Toxicology Program 

OSHA: Occupational Safety and 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 Effects of Chemical Substances 

SCE: sister chromatid exchange 

SIR: standardized incidence ratio 

SMR: standardized mortality ratio 

TNT: trinitrotoluene 

UDS: unscheduled DNA synthesis 

μg: 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 exposed 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, metabolism, 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 carcinogenicity 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, metabolism, 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 following 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 onitrotoluene for two years.......................................................................................... 32 

Table 4-2b. Neoplastic lesions identified in F344/N rats following dietary exposure to onitrotoluene for two years (continued) ...................................................................... 33 

Table 4-3a. Hemangiosarcomas in B6C3F1 mice following dietary exposure to onitrotoluene for two years.......................................................................................... 36 

Table 4-3b. Neoplastic lesions of the large intestine and liver in B6C3F1 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 onitrotoluene................................................................................................................ 37 

Table 5-1. Metabolism of nitrobenzyl alcohols by rat hepatic glucuronyltransferase and 

sulfotransferase.......................................................................................................... 43 

Table 5-2. Metabolites excreted in urine (% of dose) by male and female F344 rats and 

male B6C3F1 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 onitrotoluene................................................................................................................ 66 

Table 5-8. p53, β-catenin, and K-ras mutations in hemangiosarcomas (subcutaneous 

tissue, skeletal muscle, and mesentery) from B6C3F1 mice orally exposed to 

o-nitrotoluene ............................................................................................................ 69 

Table 5-9. p53, β-catenin, and K-ras mutations in cecal carcinomas from B6C3F1 mice 

orally exposed to o-nitrotoluene................................................................................ 70 

Table 5-10. Gene mutations and gene expression in o-nitrotoluene-induced tumors in 

rodents. ...................................................................................................................... 72 

Table 5-11. Neoplastic lesions identified in rats and mice exposed to o-nitrotoluene and 

p-nitrotoluene ............................................................................................................ 74 

Table 5-12. Treatment-related lesions in B6C3F1 mice following dietary exposure to onitrotoluene for 13 weeksa

......................................................................................... 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 potential bioactivation routes for o-aminobenzyl alcohol from 

o-nitrotoluene ............................................................................................................ 54 

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RoC Background Document for o-Nitrotoluene 

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1 1 Introduction 

o-Nitrotoluene is a nitro aromatic 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, agricultural chemicals, rubber 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 per year from the mid 1980s until 2002 (the 

most recent available data) (EPA 2007). 

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o-Nitrotoluene was nominated by the National Institute of Environmental Health 

Sciences for possible listing in the Report on Carcinogens based on the results of a 

National Toxicology Program (NTP) bioassay (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. 

14 1.1 Chemical identification 

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 

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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 C7H7NO2

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 1.2 Physical-chemical properties 

o-Nitrotoluene is a yellow liquid at room temperature 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 when heated (HSDB 2008). The physical and 

chemical properties of o-nitrotoluene are summarized in Table 1-2. 

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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 Kow) 

2.30 

Dissociation constant (pKa) 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 1.3 Identification of metabolites and analogues 

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 administration to rats and mice include onitrobenzoic acid, a sulfur-containing conjugate of o-acetamidotoluene (tentatively 

identified as S-(o-acetamidobenzyl)-N-acetylcysteine), o-nitrobenzyl glucuronide, S-(onitrobenzyl)-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. 

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The structures of two other nitrotoluene isomers (m- and p-nitrotoluene), o-toluidine (oaminotoluene, prepared by reduction of o-nitrotoluene), and the isomers of dinitrotoluene 

(2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dinitrotoluene), 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 B6C3F1 mice) are illustrated above. The two bracketed 

structures indicate structures tentatively identified by Chism and Rickert (1985). 

Cys(Ac) = acetylcysteine, G = glutathione, 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. 

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RoC Background Document for o-Nitrotoluene 

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

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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 chemical intermediates used in the synthesis of 

dyes. Thus, there is the potential for significant exposure to o-nitrotoluene. This section 

discusses information related to human exposure, including uses, production, 

concentrations of o-nitrotoluene in the environment (environmental occurrence) and in 

occupational facilities (occupational exposure), numbers of potentially exposed workers, 

biological indices of exposure, and regulations and guidelines to reduce exposure. [Note 

that for some studies, information was available only in a foreign language publication, 

and the information reported here was extracted from an English translation of the 

abstract as noted below.] 

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13 2.1 Use 

o-Nitrotoluene is primarily used in the production of derivatives, including o-toluidine (oaminotoluene), 2-amino-4-chlorotoluene, 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 manufacture (or manufacture of intermediates) 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 the synthesis of (or synthesis of intermediates 

for) explosives and of a variety of organic chemicals, including compounds used in the 

agricultural, petrochemical, pesticide, pharmaceutical, and rubber industries (HSDB 

2008). 

24 2.2 Production 

The nitrotoluenes are produced principally by the 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 the 
RoC Background Document for o-Nitrotoluene 

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temperature of the reaction mixture is maintained 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 distilled 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 crystallization. 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. oNitrotoluene 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). 

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Other processes that have been used to produce nitrotoluenes include (1) reaction of 

toluene with nitronium salts in the presence of crown ethers or polyethers 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 processes 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 onitrotoluene 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 onitrotoluene were identified in the United States in 2007 (ChemSources 2007). 

No data specific for U.S. imports or exports of o-nitrotoluene were found. 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 2005 
RoC Background Document for o-Nitrotoluene 

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(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). 

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5 2.3 Environmental occurrence and fate 

o-Nitrotoluene is expected to exist as a vapor in ambient air and has been observed to be 

completely degraded in aqueous sewage treatment systems, rivers, and streams. There are 

limited data on environmental levels of o-nitrotoluene. Some data show that onitrotoluene has been released into the air and water, primarily from occupational 

settings, but it may also be formed from toluene vapor and nitrogen oxides in air (see 

Sections 2.3.1 and 2.3.2). o-Nitrotoluene also may be formed 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 (TRI), 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, biological, and etiological (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 

(

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http://www.nrc.uscg.mil/foia.html) using the keyword “nitrotoluene” 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 contamination and general public exposure from 

inadvertent spills of o-nitrotoluene or chemical mixtures containing o-nitrotoluene.] 

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1 2.3.1 Air 

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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/m3

 of o-nitrotoluene vapor (Nishioka and Lewtas 1992). o-Nitrotoluene was 

also found in ambient-air at a manufacturing 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 toluene vapor and nitrogen oxides were 

mixed in air: m-nitrotoluene was the major isomer formed. 

According to a model of gas-particle partitioning of semivolatile organic compounds in 

the atmosphere, o-nitrotoluene released to air is expected 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-nitrophenol (Nojima and Kanno 1977) and a half-life of 42 

days has been estimated for removal by reaction with hydroxyl radicals (HSDB 2008). 

2.3.2 Water 

o-Nitrotoluene was detected in a paper-mill waste-treatment lagoon (concentration and 

location not reported). o-Nitrotoluene, which can be formed 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 concentrations ranging from 320 to 

16,000 μg/L in effluent from a U.S. plant manufacturing 2,4,6-trinitrotoluene (IARC 

1996, HSDB 2008). Other reports included concentrations ranging from 20 to 140 μg/L 

in wastewater from a plant producing and purifying 2,4,6-trinitrotoluene, and 7,800 μg/L 

in raw effluent from a plant manufacturing dinitrotoluene (geographic 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 facilities, in groundwater at a military training 

facility, and in groundwater at a nuclear weapons assembly/disassembly facility that 
RoC Background Document for o-Nitrotoluene 

6/20/08 11

previously had been a World War II munitions factory (ATSDR 2007, Pantex 2004, 

2006, WDHFS 2002). Maximum onsite groundwater levels seen at three munitions 

manufacturing facilities were 4,600 μg/L for a plant in Texas, 21,000 μg/L for a plant in 

Illinois, and 140,000 μg/L for a plant in Tennessee (years of analyses not provided) 

(ATSDR 2007). o-Nitrotoluene was found at a mean concentration of 42.6 mg/L (42,600 

μg/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 μg/L) in 

remediation studies of groundwater 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 μg/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 μg/L (ATSDR 2007, WDHFS 2002). The 

maximum surface water concentration measured at a munitions manufacturing facility in 

Missouri was 0.12 μg/L (years of analyses not provided), and the maximum groundwater 

level measured at a military training facility in Massachusetts was 25 μg/L (ATSDR 

2007). 

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o-Nitrotoluene has been routinely monitored in groundwater since 1995 at a facility in the 

Texas panhandle where conventional munitions were produced for World War II and 

where nuclear-weapons assembly and disassembly 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 μg/L in 2003 to a maximum of 5 μg/L in 2004, while 

concentrations in water from the Ogallala aquifer ranged from a minimum of 0.12 μg/L 

to a maximum of 2.9 μg/L, both in 2004 (Pantex 2003, 2004). o-Nitrotoluene was 

detected in a small percentage of samples (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 

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1999, 2000, 2001, 2002, 2003, 2004, 2005) but was not detected in 2005, the last year for 

which data were available (Pantex 2006). 

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Nitrotoluene (o- and p-nitrotoluene combined) was detected in the Netherlands (in the 

1970s) in the Rhine River at a concentration of 10 μg/L, in the River Waal at 4.5 μg/L, 

and in the River Maas at 0.3 μg/L (IARC 1996). In Germany, o-nitrotoluene was detected 

at 0.4 and 7.4 μg/L in surface water near a former munitions plant and at 1.2 μg/L in an 

adjacent river. Two ponds had concentrations of 0.4 and 22.0 μg/L; these ponds fed into 

the River Oder, which had a concentration of < 0.01 μg/L. The concentration of onitrotoluene in three samples from the River Elbe ranged from 0.05 to 0.4 μg/L. 

Concentrations detected in groundwater in France (in 1987) ranged from 90 to 165 μg/L. 

In water, o-nitrotoluene may undergo direct or indirect photolysis, volatilization, or 

aerobic biodegradation (HSDB 2008). It is not expected to adsorb 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, wind velocity of 0.5 m/s). The half-life for 

removal by indirect photolysis from a river with a high concentration of humic 

substances was calculated to be 45 minutes. o-Nitrotoluene is not expected to 

bioaccumulate in aquatic organisms; a bioconcentration factor of 12 was calculated, and 

experimentally determined bioconcentration factors in fish were low. 

2.3.3 Soil 

A soil contaminated with 39,100 ppm of trinitrotoluene (TNT) at a historical testing 

ground in Idaho contained 1.4 ppm of o-nitrotoluene (Radtke et al. 2002a). oNitrotoluene 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 longer in use but are 

known or suspected to contain military munitions 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 potential 
RoC Background Document for o-Nitrotoluene 

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environmental impacts, and o-nitrotoluene was included in that list. Development of the 

inventory of sites for clean-up does not require any analytical measurements for the 

constituents of concern, and therefore, to-date, 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 suspected to be contaminated with military 

munitions and “much of the land on which these sites are located has been or will be 

converted to nonmilitary uses such as farming, residential or commercial development, 

and recreation” (GAO 2002, 2003). A similar situation exists in other countries where 

munitions manufacture and ammunition testing have been done historically (Spiegel et al.

2005, Toze et al. 1999). 

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o-Nitrotoluene is expected to be moderately mobile in soil, with an estimated soil 

adsorption coefficient (Koc) 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, onitrotoluene persisted for more than 64 days in a silt loam inoculum (HSDB 2008). Under 

anaerobic conditions, o-nitrotoluene in soil has been observed to degrade to toluidine. A 

review on sorption, mobility, and biodegradation of nitrotoluenes in soil is available 

(Toze et al. 1999). 

20 2.4 General population exposure 

The general population may be exposed to o-nitrotoluene via inhalation of ambient air in 

the vicinity of production sites, and by oral ingestion, particularly of contaminated water 

(HSDB 2008), as well as via skin contact with contaminated substances. In one 

experiment in which 115 volatile organic chemicals 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 concentration 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 exposure were assessed] (Krotoszynski et al. 1979). 

Since o-nitrotoluene has a log bioconcentration factor (BCF) of 1.30 in fish (Guo et al.

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

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2004), [ingestion of fish should not be a very important source of human exposure to onitrotoluene]. Accumulation and depletion of o-nitrotoluene in carp (Cyprinus carpio) 

has been modeled (Hou et al. 1997). 

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o-Nitrotoluene has been reported to be present 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 via groundwater ingestion.] 

9 2.5 Occupational exposure 

Limited information is available on occupational exposure to o-nitrotoluene via 

inhalation. As noted above, the compound was detected in ambient air at a chemical 

manufacturing plant in New Jersey, where a concentration of 47 ng/m3

 (0.000047 mg/m3) 

was reported (IARC 1996). It also was detected in the air at concentrations of up to 2.0 

mg/m3 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 onitrotoluene of 0.759 mg/m3

 (ranging from undetected to 4.29 mg/m3) for a group of 98 

workers in a Chinese factory (Lianing Province) manufacturing dinitrotoluene 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 additional information was found for the number of 

workers exposed to o-nitrotoluene in the production of chemical intermediates. 

23 2.6 Biological indices of exposure 

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 quantitation at most time points, and the ratio of onitrobenzoic acid to creatinine 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 oaminobenzoic acid to be a good biomarker for exposure to o-nitrotoluene because oaminobenzoic 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). 

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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 the 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 dinitrotoluene 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 pre- and post-shift samples. However, the 

authors noted that no significant correlation (r = 0.17) was found for air 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 methemoglobin 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 methemoglobin-activating potency (Akahori 
RoC Background Document for o-Nitrotoluene 

16 6/20/08

19542, Mlynarczyk and Sadowski 2001, Vasilenko 19762). [Where exposures are to 

technical grade nitrotoluene, most of the methemoglobin activity will be related to the 

concentration of the para- and meta- isomers rather than the ortho- isomer.] 

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4 2.7 Regulations and guidelines 

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 requirements have been set for marking, 

labeling, and transporting 

Safety measures after spills or leaks are prescribed in accordance with o-nitrotoluene 

being a combustible toxic hazardous material 

U.S. EPA3

Comprehensive Environmental Response, Compensation, and Liability Act 

Reportable quantity (RQ) = 1,000 lb 

Occupational Safety and Health Administration (OSHA) 

Permissible exposure limit (PEL) = 5 ppm (30 mg/m3) [skin]4

2.7.2 Guidelines 

ACGIH 

Threshold limit value–time-weighted average (TLV-TWA) limit = 2 ppm [skin] 

Biological Exposure Index (BEI): methemoglobin 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 Risk 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 

6/20/08 17

1 NIOSH 

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Immediately dangerous to life and health (IDLH) = 200 ppm 

Recommended exposure limit (REL) = 2 ppm (11 mg/m3

) [skin] 

4 2.8 Summary 

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, agricultural chemicals, rubber chemicals, pesticides, 

petrochemicals, pharmaceuticals, and explosives. o-Nitrotoluene is a HPV chemical, and 

production in the United States was reported to be greater than 10 million pounds in 

2002. Little information is available 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 important chemicals, human exposure is expected to be 

significant. o-Nitrotoluene has been detected in workplace and ambient air, surface water, 

groundwater, and soils. In the United States, it has 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 production of commercial and military explosives, and 

o-nitrotoluene has been detected in groundwater, private well water, surface water, and 

soil at or near munitions production facilities and/or military training grounds in the 

United States and other countries. Environmental 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. nonfilter cigarette in one 

study. Biological indices 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 methemoglobin 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. 

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RoC Background Document for o-Nitrotoluene 

6/20/08 19

3 Human Cancer Studies 

No human studies on the relationship between cancer and specific exposure to onitrotoluene were identified; however, o-nitrotoluene may be used to manufacture 

magenta (see Section 2), and thus magenta manufacturing workers may be exposed to onitrotoluene. IARC (1987, 1993) reviewed magenta 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-diaminotoluene, and 4,4'-methylenebis(2-

methylaniline), and exposure to o-nitrotoluene was not specifically 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. 

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17 3.1 Studies of magenta manufacturing workers 

This section describes the cohort study (Rubino et al. 1982) that specifically mentions 

exposure to o-nitrotoluene, and briefly reports the findings from the other two studies 

(Case and Pearson 1954, Vineis and Magnani 1985) that do not mention whether onitrotoluene was used in the manufacture of magenta. Rubino et al. conducted a cohort 

study of 906 male dyestuff factory workers in northern Italy. 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 observed 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 listed in personnel records. Workers ever 

exposed to benzidine and naphthylamines were excluded from the analysis. A significant 

excess of mortality due to bladder cancer was observed among the 53 fuchsin (magenta) 

and safranine T manufacturing workers (standardized 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 evaluation of these other cancer types. The 

authors stated that the type of fuchsin manufactured 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 sections were potentially exposed to o-nitrotoluene. The 

first section (intermediates manufacture) involved the synthesis of o-toluidine and 4,4'-

methylenebis(2-methylaniline); o-nitrotoluene was an intermediate in the manufacture of 

o-toluidine from toluene. In the second section (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-diaminotoluene was oxidized in 

the presence of aniline to obtain safranine T. [This study had an excellent follow-up rate 

(96%). However, like all occupational cohort SMR-based studies, the effect estimates 

utilized an external comparison group (to determine expected disease rates). Rubino et al.

used expected death rates for all Italy, in order 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 workers, but no additional cases of bladder 

cancer in workers exposed to fuchsin or safranine T were identified. 

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Case and Pearson (1954) conducted a cohort study of men who had worked at least 6 

months in the manufacture of auramine and 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 diagnosis, as well as an 

excess of bladder-cancer mortality, in the cohort compared with the general population. A 

significant excess of bladder-cancer mortality was observed among the 85 magenta 

manufacturing workers that were not involved 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 is 
RoC 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 magenta manufacturing process.] 

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The third study on magenta manufacturing was a hospital-based, case-control study 

conducted in Italy between 1978 and 1983 that included 512 cases of bladder cancer in 

males and 596 hospital controls (Vineis and Magnani 1985). Exposure to specific 

chemicals was assessed using job titles, job activities, and knowledge of industrial use of 

chemicals from the published literature. Vineis and Magnani looked at 74 chemicals as 

part of a job exposure matrix. [No adjustments were made for multiple comparisons.] An 

increased risk of bladder cancer was found among workers potentially exposed to 

magenta (relative risk [RR] = 1.8, 95% confidence interval [CI] = 1.1 to 2.9, when 

calculated using industrial branches 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).] 

15 3.2 Studies of workers exposed to dinitrotoluenes or o-toluidine 

3.2.1 Dinitrotoluenes 

Several studies have investigated the potential 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 dinitrotoluenes (76% to 98% 

2,4-dinitrotoluene and 1% to 19% 2,6-dinitrotoluene, depending 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 observed; however, the numbers of expected 

deaths were small. The authors did observe 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), demonstrated 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 of 
RoC Background Document for o-Nitrotoluene 

22 6/20/08

quantitative exposure information as possible 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 exposed to dinitrotoluenes through 

inhalation and direct skin contact with the explosive Donarit, which contains 30% 

technical-grade dinitrotoluenes (75% 2,4-dinitrotoluene and 20% 2,6-dinitrotoluene), 

found a 4.5-fold increased incidence of urothelial 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 calculated standardized incidence ratio (SIR) = 

15.9. [Confidence intervals were not provided.] These data suggest the possibility of 

carcinogenicity of dinitrotoluenes in humans. 

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3.2.2 o-Toluidine hydrochloride 

In an epidemiological study, Ward et al. (1991) evaluated the incidence of bladder cancer 

in a chemical plant where the workers were exposed to two potential bladder carcinogens, 

o-toluidine and aniline. An excess of bladder cancer cases was observed (7 cases 

observed versus 1.08 expected) among all the 1,749 workers in the plant (SIR = 3.60, 

90% CI = 2.13 to 5.73). The SIR for bladder 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 strongly 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 company 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 versus 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 responsible for the bladder cancer excess, in view of the greater 

carcinogenic potency of o-toluidine than aniline in animals. 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 other monocyclic amines. o-Toluidine and its 

hydrochloride salt are listed in the Report on Carcinogens as reasonably anticipated to be 
RoC Background Document for o-Nitrotoluene 

6/20/08 23

a human carcinogen based on limited evidence in humans and sufficient evidence in 

animals (NTP 2004), and o-toluidine is classified as carcinogenic to humans (Group 1) by 

IARC (Baan et al. 2008). 

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4 3.3 Discussion and summary 

Two cohort studies and one case-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, durations, etc.), limited information on potential 

exposure to other agents, and small numbers of exposed workers. Most of the 

aforementioned studies did not address other potential confounders such as cigarette 

smoking or genetic acetylator 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 the 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 limited 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. Even in the Italian cohort 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 possible to evaluate whether specific exposure 

to o-nitrotoluene contributed to the increased risk of bladder cancer in these workers. oToluidine 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 dinitrotoluenes are available and suggest elevations in 

hepatobiliary, urothelial, renal, and possibly colon cancer. Two studies on o-toluidine 

reported excess bladder cancer incidence. 
RoC Background Document for o-Nitrotoluene 

6/20/08 25

1 4 Studies of Cancer in Experimental Animals 

The results of publicly available, peer-reviewed studies of the carcinogenicity of 

o-nitrotoluene in experimental animals are summarized in this section. No studies of 

exposure by routes other than oral administration were found. IARC (1996) evaluated onitrotoluene (2-nitrotoluene) 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 the 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-chronic study (13 weeks), which reported rare 

mesotheliomas in male rats receiving o-nitrotoluene. 

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Section 4.1 summarizes the studies with rats, and Section 4.2 summarizes the studies with 

mice. Findings in experimental animals are summarized in Section 4.3. The 

carcinogenicity of the other nitrotoluene isomers and related compounds is discussed in 

Section 5.5. 

15 4.1 Rats 

The NTP conducted 13-week studies with F344/N rats exposed to o-, m-, and pnitrotoluene to compare clinical toxicity, histopathology, 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 two-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 quality 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 summarized in this section. 

4.1.1 Subchronic exposure 

In 13-week studies, groups of 10 male and 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 treated 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 previously been identified in exposed or control 

rats from any of the 13-week toxicity studies conducted by NTP (with any substance). 

The authors concluded that o-nitrotoluene was carcinogenic in male rats, based on the 

occurrence of mesothelioma and mesothelial hyperplasia. 

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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]. The dose was based on the results of the 1992 

study, in which o-nitrotoluene in the diet at 5,000 ppm caused mesothelioma but had little 

effect on survival or body weight. o-Nitrotoluene was administered 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), and the remaining 20 rats continued to 

receive o-nitrotoluene (26-week exposure). After 26 weeks, all remaining rats were 

killed, and necropsies and histopathologic 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 clinical effects of exposure were observed in 

either group. 
RoC Background Document for o-Nitrotoluene 

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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 testis 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 with 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. 

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The NTP (1996) also conducted 13-week and stop-exposure studies using rats with 

altered flora to assess the potential role of metabolism by gastrointestinal (GI) flora in onitrotoluene-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 conditions 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 mesothelioma. These lesions occurred on the 

mesothelial surface of the tunica vaginalis of the testis or epididymis 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 intestinal flora after 13 weeks. In contrast to the study using rats 

with normal intestinal flora, no cholangiocarcinomas were observed. The NTP was not 

able to draw any conclusions about the involvement of GI flora in the pathogenesis of onitrotoluene-induced lesions because of the lower 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 

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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) 

0b 10 0 (0) 0 (0) 0 (0) 0 (0) Stop exposurea

5,000 20 2 (10) 4 (20) 5 (25) 2 (10) 

0b 10 0 (0) 0 (0) 0 (0) 0 (0) 26 weeks 

5,000 20 2 (10) 7 (35)* 7 (35)* 1 (5) 

Altered florac

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 exposurea

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 killed 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 was 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 both the stop-exposure and 26-week-exposure 

groups [P ≤ 0.01]. Placental glutathione S-transferase positive (PGST+) foci 

(preneoplastic lesions) in the liver were significantly increased in exposed groups. At the 

end of the 13-week recovery period, the numbers 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 larger 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. 

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RoC Background Document for o-Nitrotoluene 

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1 4.1.2 Chronic exposure 

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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 toxicity studies (NTP 1992). In the core study, 

groups of 60 male and 60 female F344/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 average 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 average 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 (two 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 the 5,000-ppm group died before the end of the 

study, and only 11 rats in the 2,000-ppm group survived. 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 than those of the controls [P

≤ 0.01]. 

In the two-year chronic-exposure study, survival was reduced in all exposure groups 

among males and in the highest exposure group among females because of the 

development of neoplasms. Feed consumption was similar between exposed and control 

groups; however, mean body weights were lower in all exposed groups (except the 625-

ppm group of males). Non-neoplastic 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 chronic-exposure study. Significantly increased 

incidences of malignant mesothelioma (most were associated with the tunica vaginalis of 
RoC Background Document for o-Nitrotoluene 

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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 other than high-dose males in the two-year 

chronic-exposure study. High incidences of subcutaneous skin fibroma or fibrosarcoma 

and malignant mesothelioma were observed in the high-dose groups (over 70%, 

compared with less than 10% in controls). Significantly increased incidences of 

hepatocellular adenoma or carcinoma (combined) were observed in the high-dose groups 

in both the chronic and stop-exposure studies, and increased incidences of liver 

cholangiocarcinoma and alveolar/bronchiolar lung tumors were observed in the high-dose 

(5,000-ppm) group in the stop-exposure study. Hepatocholangiocarcinoma occurred in 

two male rats, one exposed to 625 ppm and the other to 2,000 ppm. Even though the 

incidence was low, the NTP considered these to be exposure related. No 

hepatocholangiocarcinomas or cholangiocarcinomas had been observed in male controls 

fed either the NTP-2000 or NIH-07 diet in two-year bioassays. The incidence of 

hemangioma or hemangiosarcoma (combined) was significantly increased in the highdose males in the stop-exposure study, but these tumors were not considered by the 

authors to be exposure related. 

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In females, a significantly increased incidence of mammary-gland fibroadenoma was 

observed in all exposure groups, and the incidence of subcutaneous skin fibroma or 

fibrosarcoma was significantly increased at the two highest dose levels. The incidence of 

hepatocellular adenoma in females was significantly increased only in the high-dose 

(2,000-ppm) group. The incidence of mononuclear-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 the 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-dose (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 

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The NTP concluded that there was clear evidence of carcinogenic activity of onitrotoluene in male and female rats, based on increased incidences of malignant 

mesothelioma (in males only), subcutaneous skin neoplasia, mammary-gland 

fibroadenoma, and liver neoplasia (in males only). The increased incidences of lung 

neoplasia in male rats and hepatocellular adenoma in female rats also were considered to 

be exposure related. 

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. 
RoC Background Document for o-Nitrotoluene 

Table 4-2a. Neoplastic lesions identified in F344/N rats following dietary exposure to o-nitrotoluene for tw

Tumor incidence (%)a

Mammary gland Skin subcutaneous 

Sex N

Conc. (ppm)

Malignant 

mesothelioma Fibroadenoma Lipoma Fibroma/fibrosarc

Chronic exposure 

60 0 2 (3.7) 0 (0) 0 (0) 5 (9.3) 60 625 20 (40.6)*** 7 (15.6)** 4 (8.9)* 47 (86.3)*** 60 1,250 29 (62.4)*** 10 (26.2)*** 13 (33)*** 55 (98.7)*** 60 2,000 44 (87.1)*** 2 (9.0) 13 (44.8)*** 59 (99.8)*** 609 HC 23 (3.7) 26 (3.8) 8 (1.5) 41 (6.4) 

trend testb P < 0.001 P < 0.001 P < 0.001 P < 0.001 Stop exposure 

60 2,000 44 (80.3)*** 13 (31.2)*** 10 (24.6)*** 47 (89)*** 60 5,000 54 (95.1)*** 20 (61.1)*** 12 (44.7)*** 53 (97.8)***c

M 

trend testb P < 0.001 P < 0.001 P < 0.001 P < 0.001 

Chronic exposure

60 0 0 (0) 23 (40) 0 (0) 3 (5.3) 60 625 0 (0) 47 (82.8)*** 0 (0) 3 (5.4) 60 1,250 0 (0) 52 (91.7)*** 0 (0) 21 (37.6)*** 60 2,000 0 (0) 56 (96.2)*** 0 (0) 22 (40.6)*** 659 HC NR 284 (41.1) NR 18 (2.6) 

F 

trend testb – P < 0.001 – P < 0.001 

Sources: Dunnick et 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 

NR = historical incidence data not reported in NTP 2002b. 

a Poly-3 estimated incidence after adjustment for intercurrent mortality. b Poly-3 test used to test for significant dose-related trend. c Includes one sarcoma in an animal that also had a fibroma. 

32 
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Table 4-2b. Neoplastic lesions identified in F344/N rats following dietary exposure to o-nitrotoluene for two ye

Tumor incidence (%)a

Liver 

Sex N

Conc 

(ppm)

Hepatocellular 

adenoma

Hepatocellular 

adenoma/carcinomac Cholangiocarcinoma

Hepatocholangiocarcinoma Alv

ade

Chronic exposure 

60 0 2 (3.7) 3 (5.6) 0 (0) 0 (0) 60 625 3 (6.8) 3 (6.8) 0 (0) 1 (2) 60 1,250 3 (8.4) 3 (8.4) 0 (0) 0 (0) 60 2,000 7 (27.1)** 8 (30.2)** 0 (0) 1 (2) 609 HC 5 (0.8) 10 (1.8) 0 (0) 0 (0) trend testb P = 0.007 P = 0.009 – NR 

Stop-exposure 

60 2,000 3 (7.6) 3 (7.6) 0 (0) 0 (0) 60 5,000 4 (18.4)* 6 (25.9)* 3 (13.9)* 0 (0) 

M 

trend testb P = 0.062 P = 0.03 – 

Chronic exposure

60 0 1 (1.8) 1 (1.8) 0 (0) 0 (0) 60 625 0 (0) 0 (0) 0 (0) 0 (0) 60 1,250 1 (1.9) 1 (1.9) 0 (0) 0 (0) 60 2,000 6 (11.2)* 6 (11.2)* 0 (0) 0 (0) 659 HC 4 (0.7) NR NR NR 

F 

trend testb P = 0.005 P = 0.005 – – 

Sources: Dunnick et 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 

NR = historical incidence data not reported in NTP 2002b. a Poly-3 estimated incidence after adjustment for intercurrent mortality (adjusted 

for hepatocholangiocarcinoma). 

b Poly-3 test used to test for significant dose-related trend. c No hepatocellular carcinomas reported in female mice; therefore, combined incidence is the same as adenoma incidence. 
RoC Background Document for o-Nitrotoluene 

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1 4.2 Mice 

NTP (1992) conducted a 13-week subchronic study of o-nitrotoluene in B6C3F1 mice. 

No tumors were reported; therefore, this study is summarized in Section 5.6. In a twoyear study to investigate the chronic toxicity and carcinogenicity of o-nitrotoluene in 

B6C3F1 mice (NTP 2002b), groups of 60 male and 60 female mice were fed diets 

containing o-nitrotoluene (> 99% purity) at a concentration of 0, 1,250, 2,500, or 5,000 

ppm for 105 weeks. These concentrations were based on the results of the subchronic 

exposure study and were equivalent to average 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 database of tumor incidences in untreated or control groups 

from all NTP-sponsored carcinogenicity studies. The tumor incidences in historical 

controls were reported by NTP (2002b) for all 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. 

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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 females in the 5,000-ppm group was significantly 

less than that of controls. Early development 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 incidences 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 significantly 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 hemangiosarcomas 
RoC Background Document for o-Nitrotoluene 

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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, including 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 increased in the low-dose (1,250-ppm) and middose (2,500-ppm) groups of males, and the incidences of hepatocellular adenoma and 

hepatocellular adenoma or carcinoma (combined) were significantly increased in the middose (2,500-ppm) and high-dose (5,000-ppm) groups of females. The incidence of 

hepatocellular carcinoma was significantly increased in the high-dose females (16 out of 

60 animals positive with a survival-adjusted rate of 36.2%). Nonsignificantly increased 

incidences of carcinoma of the large intestine (cecum) in females were considered by the 

NTP to be exposure related, because this neoplasm is extremely rare and had not been 

seen in females in the historical control groups. [The incidence of large-intestine 

carcinoma in both the 2,500-ppm and 5,000-ppm female mice was significantly increased 

(P < 0.001) compared with historical controls by Fisher’s exact test.] These tumors were 

characterized by proliferation of glandular structures composed of moderately 

pleomorphic mucosal epithelial cells that invaded the cell wall. 

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Based on these results, the NTP concluded that there was clear evidence of carcinogenic 

activity of o-nitrotoluene in male and female mice, based on increased incidences of 

hemangiosarcoma (in males and females), carcinoma of the large intestine (cecum) (in 

males), and hepatocellular neoplasia (in females). 
RoC Background Document for o-Nitrotoluene 

Table 4-3a. Hemangiosarcomas in B6C3F1 mice following dietary exposure to onitrotoluene for two years 

Hemangiosarcoma incidence (%)a

Sex N 

Conc. 

(ppm) Mesentery 

Skeletal 

muscle Subcutis All organsb

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 the large intestine and liver in B6C3F1 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 B6C3F1 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.] 

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1 4.3 Summary 

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. o-Nitrotoluene 

caused tumors of the mammary gland, skin (subcutaneous), 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. 

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Table 4-4. Neoplastic lesions identified in rats and mice following dietary exposure 

to o-nitrotoluene 

F344/N rats B6C3F1 mice 

Organ or system Tumor type Male Female Male Female 

Mesothelium malignant mesothelioma 

Skin (subcutaneous) lipoma 

fibroma or fibrosarcoma 

Mammary gland fibroadenoma 

hepatocellular carcinoma 

hepatocellular adenoma or 

carcinomaa

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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1 5 Other Relevant Data 

This section discusses relevant mechanistic and other information needed to understand 

the toxicity and potential carcinogenicity of o-nitrotoluene. It includes information on 

absorption, distribution, metabolism, and excretion of o-nitrotoluene (Section 5.1); 

bioactivation of o-nitrotoluene (Section 5.2); genetic damage and related effects (Section 

5.3); mechanistic studies and considerations (Section 5.4); carcinogenicity and 

genotoxicity of o-nitrotoluene metabolites and analogues 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. 

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13 5.1 Absorption, distribution, metabolism, and excretion 

This section reviews the available 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 (Section 5.1.5), a summary of the comparative 

metabolism data (Section 5.1.6), and a description 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 were 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 onitrobenzyl alcohol [probably present as o-nitrobenzyl glucuronide] were detected in the 

urine of 97% and 99%, respectively, of the workers occupationally exposed to 
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o-nitrotoluene in a factory in China (see Section 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 urine 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 dermal 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 exposed to aromatic nitroamino compounds, 

including nitrotoluenes, were significantly higher after a work shift than in unexposed 

workers, but no data on exposure specifically to o-nitrotoluene were reported. 

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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 B6C3F1 mice received 

[

14C]-o-nitrotoluene by gavage at a dose of either 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 while urinary excretion in female rats was 

slightly higher at 92%. Male B6C3F1 mice excreted approximately 66% of a 200-mg/kg 

b.w. dose during the first 24 hours. Excretion of radioactivity (parent compound and 

metabolites) following a 2-mg/kg b.w. gavage dose of [14C]-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 [14C]-o-nitrotoluene at a dose of 200 mg/kg b.w. 

More than 85% of the total oral dose was recovered 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. 
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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 excretion (see Section 5.1.7 for a discussion of biliary excretion of onitrotoluene metabolites). 

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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 recovery 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 metabolites may also form 

hemoglobin adducts in humans (Jones et al. 2005a) and rats (NTP 2002b, Sabbioni 1994, 

Sabbioni and Sepai 1995) implying distribution 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 studied in rat hepatic microsomes 

and in isolated rat hepatocytes (deBethizy and Rickert 1984). In rat hepatic microsomes, 

the only metabolites formed from the nitrotoluenes were the corresponding nitrobenzyl 

alcohols. The formation was NADPH dependent, and it was inhibited by carbon 

monoxide, suggesting that the reaction was catalyzed by cytochrome(s) P450. 

o-Nitrobenzyl alcohol was formed at a rate intermediate between the rates for m- and 

p-nitrobenzyl alcohol, but differences in the initial rates of formation were small. 

There were no large differences in the rates of disappearance of the nitrotoluenes from 

incubations with isolated rat hepatocytes. However, there were quantitative and 

qualitative differences in the metabolites formed. o-Nitrotoluene was metabolized to 

o-nitrobenzyl alcohol and the corresponding 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 unidentified metabolite were 
RoC 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 metabolites accounted for only about 43% of the mnitrotoluene 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 its glucuronide conjugate, but 

only about 14% of the p-nitrotoluene metabolized followed this pathway. About 2% was 

converted to p-nitrobenzoic acid, and the incubation 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 reaction did not proceed without glutathione 

S-transferase, suggesting that unlike some arylmethyl sulfates, p-nitrobenzyl sulfate is 

stable (deBethizy and Rickert 1983). 

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 

Rat liver microsomal cytochromes P450 oxidized the isomeric aminobenzyl alcohols, 

metabolites of the nitrotoluenes, to hydroxylamine and/or aminophenol metabolites 

(Kedderis and Rickert 1985). The metabolites were extractable into ethyl acetate and 

capable of reducing ferric iron, consistent with the formation of hydroxylamines and/or 

aminophenols. The oxidation rates of the aminobenzyl 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 the aminobenzyl alcohol metabolites of m-nitrotoluene 

(0.18 ± 0.00) or p-nitrotoluene (0.04 ± 0.00). These results are consistent with the toxic 

potency of the mononitrotoluene isomers. 

Examination of the metabolism of the nitrobenzyl alcohols by glucuronyl transferase, rat 

hepatic alcohol dehydrogenase, and sulfotransferase 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 three 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 onitrotoluene. 1 

2 

Table 5-1. Metabolism of nitrobenzyl alcohols by rat hepatic glucuronyltransferase 

and sulfotransferase 

V/K (103 ⋅ nmoles/min/mg/μM) 

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. 

3 5.1.4 In vivo metabolism of o-nitrotoluene in rodents 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

Male and female F344 rats and male B6C3F1 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 Table 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 doses. However, female rats excreted less than 

half as much of the dose as o-aminobenzyl alcohol or as S-(o-nitrobenzyl)-Nacetylcysteine as did male rats. 

Male B6C3F1 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 after 
RoC Background Document for o-Nitrotoluene 

44 6/20/08

administration (NTP 2002b). The specific metabolites and their percentages were similar 

after a dose of 2 mg/kg b.w., except that the percentages 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 B6C3F1 mice 24 hours after an oral dose of o-nitrotoluene 

F344 rats B6C3F1 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)-Nacetylcysteine 

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 with levels of the urinary metabolites onitrobenzoic acid and o-nitrobenzyl mercapturic acid (S-(o-nitrobenzyl)-N-acetylcysteine) 

at different time points in rats and mice from the NTP bioassay (see Section 2.6 for 

details related to use of these metabolites as potential biomarkers) (NTP 2002b). The 

ratios of urinary o-nitrobenzoic acid to creatinine determined at 2 weeks and at 3, 12, and 

18 months were linearly related to exposure concentration 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 rats 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 urine after a single oral dose are 

summarized in Figure 5-1. There were some quantitative and qualitative differences 
RoC Background Document for o-Nitrotoluene 

6/20/08 45

among rats and mice in the metabolites excreted in urine, however, the relative 

proportions of the urinary metabolites did not appear to be dose dependent between 2 and 

200 mg/kg b.w. 

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 

When male F344 rats were given 11 daily doses of unlabeled o-nitrotoluene (200 mg/kg 

b.w.) followed on the 12th day by a dose of [14C]-o-nitrotoluene and then by two more 

daily doses of unlabeled compound, excretion of radioactivity was similar 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 single radiolabeled dose. 

When rats were pretreated with buthionine sulfoxamine, an inhibitor of glutathione 

synthesis, about half as much of the radioactive dose was excreted in the urine in 

24 hours as in untreated rats (NTP 2002b). Excretion of S-(o-nitrobenzyl)-Nacetylcysteine 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 the urinary excretion of [14C]-o-nitrotoluene after pretreatment of 

male rats with pentachlorophenol, an inhibitor 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 suggested 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 pentachlorophenol pretreatment, excretion of onitrobenzyl glucuronide was decreased by about half, as was excretion of o-aminobenzyl 

alcohol; the authors attributed these decreases to competition between o-nitrobenzyl 

alcohol and pentachlorophenol for glucuronyl transferase. 

An earlier report by Chism et al. (1984) described similar results for urinary metabolites 

for male F344 rats given an oral dose of [14C]-o-nitrotoluene (200 mg/kg b.w.); however, 

there were some differences (see Table 5-3). The NTP (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 aminotoluene 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 metabolites that had not undergone nitro-group reduction peaked 

within the first 4 hours after administration, while those for metabolites that had 

undergone nitro-group reduction peaked between 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 smaller 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 shown 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 quantitative 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 47
RoC Background Document for o-Nitrotoluene 

48 6/20/08

1 5.1.5 In vivo metabolism of m- and p-nitrotoluene in rodents 

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 

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 mnitrotoluene 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 B6C3F1 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, pacetamidobenzoic acid, p-nitrohippuric acid, and p-nitrobenzylmercapturic acid (S-(pnitrobenzyl)-N-acetylcysteine) along with other unidentified 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 that females excreted somewhat more p-nitrobenzoic 

acid and somewhat less S-(p-nitrobenzyl)-N-acetylcysteine. The patterns of excretion in 

both male and female rats were similar after a 2-mg/kg b.w. dose (data 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-methyl-5-nitrophenyl sulfate, 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)-Nacetylcysteine. The half-life of total radioactivity in plasma 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

1 5.1.6 Comparison of o-, m-, and p-nitrotoluene metabolism in rodents 

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 alcohol. In contrast to what is observed in 

isolated rat hepatocytes, the main metabolic pathway for each of the isomers in vivo is 

oxidation of the alcohol group to an acid moiety. [Whereas very little of a dose of m- or 

p-nitrotoluene is converted to a nitrobenzyl glucuronide, 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 isolated hepatocytes, suggests that nitro-group 

reduction occurs at a site other than the liver. ] 
RoC Background Document for o-Nitrotoluene 

Table 5-3. Metabolites excreted in urine (% of dose) by rats and mice after an oral dose of 200 mg/kg b.w. 

nitrotoluene 

o-nitrotoluene m-nitrotoluene p-nitr

F344 rats 

B6C3F1

mice F344 rats F344 rats 

Metabolite Sex Ch. NTP NTP Ch. Ch. Nx-nitrobenzoic acida M 

F 

28.6 

NT 

19.1 21.8 

38.2 

NT

21.1 

NT 

28.0 

NT 

34

sulfur-containing conjugate of x-acetamidotoluene 

M 

F 

15.9 

NT 

–

– 

–

NT 

–

NT 

–

NT 

x-nitrobenzyl glucuronide M 

F 

14.1 

NT 

15.3 22.1 

23.9 

NT 

2.0 

NT 

1.4 

NT 

S-(x-nitrobenzyl)-Nacetylcysteine M F 11.6 NT 9.9 3.7 –NT –NT 3.7 NT sulfur-containing conjugate of x-aminotoluene 

M 

F 

6.0 

NT 

– – 

–

NT 

–

NT 

–

NT 

S-(x-nitrobenzyl) glutathione 

M 

F 

3.9 

NT 

– – 

–

NT 

1.3 

NT 

– 

NT 

x-aminobenzoic acid M 

F 

1.8 

NT 

– –

NT 

1.2 

NT 

0.8 

NT 

x-nitrobenzyl sulfate M 

F 

0.5 

NT 

– – 

–

NT 

–

NT 

–

NT 

x-nitrobenzyl alcohol M 

F 

0.4 

NT 

1.8 0.8 

–

NT 

–

NT 

–

NT 

x-aminobenzyl alcohol M 

F 

– 

NT 

17.0 

7.9 

–

NT 

–

NT 

–

NT 

50 
RoC Background Document for o-Nitrotoluene 

6/20/08 

o-nitrotoluene m-nitrotoluene p-nitr

F344 rats 

B6C3F1

mice F344 rats F344 rats 

Metabolite Sex Ch. NTP NTP Ch. Ch. Nx-nitrohippuric acid M 

F 

– 

NT 

– – 

–

NT 

23.6 

NT 

13.0 

NT 

1

x-acetamidobenzoic acid M 

F 

–

NT 

–

– 

–

NT 

11.6 

NT 

27.1 

NT 

11

x-aminotoluene M 

F 

– 

NT 

1.1 1.4 

– 

NT 

– 

NT 

– 

NT 

N

5-methyl-2-nitrophenyl glucuronide 

M 

F 

– 

NT 

– – 

– 

NT 

– 

NT 

0.3 

NT 

N

5-methyl-2-nitrophenyl sulfate 

M 

F 

– 

NT 

– – 

– 

NT 

– 

NT 

0.2 

NT 

N

2-methyl-5-nitrophenyl glucuronide 

M 

F 

– 

NT 

– – 

– 

NT 

– 

NT 

– 

NT 

N

2-methyl-5-nitrophenyl sulfate 

M 

F 

– 

NT 

– – 

– 

NT 

– 

NT 

– 

NT 

N

Sources: Ch. = Chism et al. 1984 (72-h data), NTP = NTP 2002b (24-h data) for o-nitrotoluene and NTP 2002a (24-h data) for p-nitroto

– = metabolite not found; NT = not tested. 

a 

x- represents o-, m-, or p-nitrotoluene metabolites. 
RoC Background Document for o-Nitrotoluene 

52 6/20/08

1 5.1.7 Biliary excretion of o-, m-, and p-nitrotoluene in rats 

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 

In F344 rats given an oral dose (200 mg/kg b.w.) of radiolabeled o-nitrotoluene, bile-duct 

cannulation decreased urinary excretion 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 dose 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 mnitrobenzoic 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 excretion accounted for 7.7% of a 200-mg/kg b.w. 

oral dose of [14C]-p-nitrotoluene in male rats in 6 hours; the major metabolite was S-(pnitrobenzyl) 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 biliary 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

1 5.2 Bioactivation of o-nitrotoluene 

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 rats; in bile duct–cannulated animals, similar amounts were 

excreted in the urine of both sexes. 

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 

When covalent binding to hepatic macromolecules 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 concentrations above the assay’s limit 

of quantitation. When sulfotransferase activity was inhibited by pretreatment of rats with 

pentachlorophenol or 2,6-dichloro-4-nitrophenol, the binding of o-nitrotoluene–related 

material to total hepatic macromolecules was decreased to less than half that seen with no 

pretreatment, and the binding of o-nitrotoluene–related material 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 activation of 2,6-dinitrotoluene (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. Once in the liver, the final activation of 

o-aminobenzyl alcohol requires the action of sulfotransferase. The involvement of 

sulfotransferase is suggested by the experiments measuring covalent binding to hepatic 

DNA in rats pretreated with pentachlorophenol 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 Rickert 1989). In those experiments, o-aminobenzyl 
RoC Background Document for o-Nitrotoluene 

Figure 5-2. Formation and potential bioactivation routes for o-aminobenzyl alcohol 

from o-nitrotoluene 

Source: Adapted from Chism 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 (middle 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 intestine and carried by the portal circulation to the liver (lowermost box) 

where three potential pathways may be followed for further 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 6/20/08
RoC Background Document for o-Nitrotoluene 

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

56 6/20/08

alcohol-related material became bound to calf 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 the 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 coenzyme A did not result in covalent binding, 

suggesting that the proximal reactive metabolite was not an acetoxyarylamine. Although 

binding to macromolecules through an N-hydroxy intermediate cannot be ruled out, 

binding in the presence of NADPH and microsomes was much lower than binding in the 

presence of PAPS and cytosol. This suggests that the proximal reactive metabolite is 

o-aminobenzyl sulfate (see Figure 5-2). This pathway only describes the situation in the 

liver because other tissues may not have the necessary enzymes to activate aminobenzyl 

alcohol. Similar structures are proposed to be the reactive intermediates formed from 

7,12-dimethylbenz[a]anthracene (Watabe et al. 1982) and 1'-hydroxysafrole (Boberg et 

al. 1983).] 

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DNA adducts are formed when o-aminotoluene is incubated 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). Furthermore, the adducts formed when oaminotoluene was incubated with calf thymus DNA were different from those present in 

hepatic DNA after o-nitrotoluene was administered to rats (Jones et al. 2003). [These 

data add support to the possibility that an o-nitrotoluene metabolite(s) modifies DNA 

through the methyl group, rather than an amino group.] 
RoC Background Document for o-Nitrotoluene 

6/20/08 57

1 5.3 Genetic damage and related effects 

o-Nitrotoluene has been tested for genotoxicity in a number of in vitro and in vivo test 

systems. The International Agency for Research on Cancer (IARC 1996) reviewed the 

available literature and concluded that o-nitrotoluene was not genotoxic 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. 

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5.3.1 Prokaryotic systems 

o-Nitrotoluene has been tested for the ability to induce reverse mutation 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 exogenous 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 μg/plate), a co-mutagen found in tobacco tar and in pyrolysate 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. 
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1 Differential toxicity in Bacillus subtilis 

2 

3 

Shimizu and Yano (1986) reported negative results for o-nitrotoluene (concentration not 

reported) in the rec assay in B. subtilis strains H17 and M45 without metabolic activation. 

Table 5-4. Results of genotoxicity 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 μmol) 

– – Chiu et al. 1978 

S. typhimurium

TA98, TA100 

reverse mutation 

(NR) 

– – Tokiwa et al. 1981 

S. typhimurium

TA98, TA100 

reverse mutation 

(100–300 μg/plate) 

– – Suzuki et al. 1983 

S. typhimurium 

TA98 with norharman 

reverse mutation 

(100–300 μg/plate) 

+ NT Suzuki et al. 1983 

S. typhimurium 

TA100 with norharman

reverse mutation 

(100–300 μg/plate) 

– NT Suzuki et al. 1983 

S. typhimurium

TA98, TA1537, TA1538 

reverse mutation 

(0.01–5 μL/plate) 

– – Shimizu and Yano 1986 

S. typhimurium

TA98, TA100, TA1535, 

TA1537 

reverse mutation 

(3–333 μg/plate) 

– – Haworth et al. 1983 

S. typhimurium

TA98, TA100, TA1535, 

TA1537, TA1538 

reverse mutation 

(10–5,000 μg/plate) 

– – Spanggord et al. 1982a, 

1982b 

S. typhimurium 

TA98, TA1535

reverse mutation 

(33–1,000 μg/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. 

4 5.3.2 Mammalian systems 

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8 

9 

10 

o-Nitrotoluene has been tested for genotoxicity 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 rat hepatocytes, and the acute micronucleus 

test in bone marrow in rats and mice. 
RoC Background Document for o-Nitrotoluene 

6/20/08 59

1 In vitro systems 

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Table 5-5 summarizes the results of tests in in vitro mammalian systems. Galloway et al.

(1987) observed significantly increased incidences of SCE with S9 metabolic activation 

at o-nitrotoluene concentrations from 355 to 423 μg/mL and equivocal results without S9 

at concentrations ranging from 117 to 282 μg/mL in cultured CHO cells. 

Chromosomal aberrations were evaluated in several studies. In cultured CHO cells, the 

incidence of chromosomal aberrations was not increased, either with S9 at o-nitrotoluene 

concentrations from 375 to 422 μg/mL or without S9 at concentrations from 201 to 

394 μg/mL (Galloway et al 1987). o-Nitrotoluene did not induce structural chromosomal 

aberrations but did induce polyploidy in CHL cells exposed to 250 μg/mL for 48 h. 

Huang et al. (1996) reported an increase in chromosomal 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 validation 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 μg/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 assay) in CHO-K1 cells exposed to o-nitrotoluene at 50 to 

200 μg/mL with or without S9 for 4 h. 

Lee et al. (2007) reported that o-nitrotoluene (50 to 200 μg/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. Negative results were reported for o-nitrotoluene in a 

number of tests measuring DNA repair as UDS (all of which used concentrations of 10 to 

1,000 μM), including tests in male and female F344/N rat hepatocytes (Doolittle et al.

1983), human hepatocytes (Butterworth et al. 1989), and rat pachytene spermatocytes and 

round spermatids (Working and Butterworth 1984). Parton et al. (1995) reported that 

culturing rat hepatocytes in serum-free media 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 genotoxicity 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 μg/mL) + NT Galloway et al. 1987 

CHO cells SCE 

(117–282 μg/mL) NT ± Galloway et al. 1987 

CHO cells chromosomal aberrations

(375–422 μg/mL) – NT Galloway et al. 1987 

CHO cells chromosomal aberrations

(201–394 μg/mL) NT – Galloway et al. 1987 

CHL/IU cells chromosomal aberrations

(250 μg/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 μg/mL) NT + Matsushima et al.

1999 

CHO-K1 cells micronuclei 

(50–200 μg/mL) – – Lee et al. 2007 

L5178Y mouse 

lymphomacells 

DNA damage (comet 

assay) 

(50–200 μg/mL) 

+ + Lee et al. 2007 

F344/N rat hepatocytes DNA repair 

(10–1,000 μM) NT – Doolittle et al. 1983 

F344/N rat hepatocytes 

(serum-free media) 

DNA repair 

(0.1–500 μg/mL) NT + Parton et al. 1995 

Human hepatocytes DNA repair 

(10–1,000 μM) NT – Butterworth et al. 1989 

Rat spermatocytes and 

spermatids 

DNA repair 

(10–1,000 μM) NT – Working and 

Butterworth 1984 

NT = not tested; + = positive; – = negative; ± = equivocal. 

a Positive for numerical aberrations (polyploidy) but not structural aberrations. 

1 In vivo systems 

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7 

The results of tests for DNA adduct formation, 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 binding to hepatic macromolecules and hepatic 
RoC 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-Fohm 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 livers 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. 

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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, and 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 extent 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 conventional flora) but not in the 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 bacteria are needed for the metabolic activation of 

o-nitrotoluene to a hepatic genotoxicant.] Sex 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 authors 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 following a single intraperitoneal (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 marrow 
RoC Background Document for o-Nitrotoluene 

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analysis 48 hours post-injection. Negative results were reported for both protocols (NTP 

2002b). 

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Male B6C3F1 mice injected i.p. with o-nitrotoluene dissolved in corn oil (100 to 400 

mg/kg b.w.) three times at 24-hour intervals 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 (NCEs) was seen in the peripheral blood of 

females. In males, the frequency of micronucleated NCEs was slightly increased at the 

highest exposure level (10,000 ppm); this result was judged to be equivocal (NTP 

2002b). 
RoC Background Document for o-Nitrotoluene 

Table 5-6. Results of genotoxicity 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 B6C3F1 mice 

(bone marrow) 

micronucleated PCEs 

(100–400 mg/kg b.w.) – NTP 2002b 

Male B6C3F1 mice 

(bone marrow) 

micronucleated NCEs 

(625–10,000 ppm) ± NTP 2002b 

Female B6C3F1 mice 

(bone marrow) 

micronucleated NCEs 

(625–10,000 ppm) – NTP 2002b 

+ = positive; – = negative; ± = equivocal. 

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

64 6/20/08

1 5.4 Mechanistic studies and considerations 

This section discusses potential mechanisms of carcinogenicity and genotoxicity of onitrotoluene 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. 

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5.4.1 Potential mechanisms of carcinogenicity 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 intestinal microflora. Incubation of onitrotoluene in vitro with hepatocytes isolated from male rats failed to induce DNA 

repair. [These results suggest that DNA damage and subsequent repair is dependent on 

metabolism, both mammalian and bacterial.] 

[The activation scheme discussed in Section 5.2 and depicted in Figure 5-2 explains the 

observations on sex and isomer differences in the covalent binding of nitrotoluene-related 

material in hepatic DNA, and it also explains the sex, isomer, and intestinal flora 

dependency of the in vivo–in vitro DNA repair assay. The significantly increased 

incidences of tumors in other 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 studies 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 microarray analysis in L5178Y Tk+/- mouse lymphoma 

cells to evaluate gene expression profiles for o-nitrotoluene, which they considered a 

nongenotoxic carcinogen. Genes were considered differentially expressed when 
RoC Background Document for o-Nitrotoluene 

6/20/08 65

logarithmic gene expression ratios in four independent 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 and metastatic phenotype of cancer cells, and IL7, 

which has antitumor properties, were upregulated. Akap10, a protein kinase A anchoring 

protein, was downregulated. 

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Lee et al. (2007) also performed cDNA microarray analysis in L5178Y cells exposed to 

200 μg/mL of o-nitrotoluene for 24 h. Data was anlysed by hierarchical clustering, and a 

few pathways (see Table 5-7) were identified in which gene expression 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 genes 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 genes and are often 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 transcription 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 were 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 IIα (Dnase2a), and cytokine inducible SH2-
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containing protein (Cish), all of which were down-regulated and have a role in promoting 

apoptosis. 

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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 and transcriptional repressor and 

was down-regulated in B6C3F1 mouse liver following treatment 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 onitrotoluene 

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) ↓ expressiona

inhibin β-A gene (Inhba) ↓ expression 

B6C3F1 mice – in vivo

(liver) 

Jun-B gene (Junb) ↓ expression 

Iida et al. 2005 

a

In 5,000-ppm males only. 
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Gene expression and mutations in oncogenes, tumor suppressor genes, and other critical genes 

in rodents 

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Several studies have examined the potential 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 colon 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 onitrotoluene-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, subcutaneous tissue, and mesentery) from 

B6C3F1 mice exposed to o-nitrotoluene in the NTP two-year bioassay (see Section 4.2.2) 

and in 15 spontaneously occurring subcutaneous hemangiosarcomas. Protein production 

(indicating gene expression) was assessed 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 identified in 1 tumor. Production of βcatenin protein also was detected in 47% of the o-nitrotoluene–induced tumors (in 6 of 

the 7 tumors with a deletion and 1 tumor without 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 profile 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 B6C3F1 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 compared 
RoC 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 

5 
RoC Background Document for o-Nitrotoluene 

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Table 5-8. p53, β-catenin, and K-ras mutations in hemangiosarcomas (subcutaneous 

tissue, skeletal muscle, and mesentery) from B6C3F1 mice orally exposed to onitrotoluene 

Treatment group, 

mutations 

p53 (exon 6-8), 

number (%)b Catnba, 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 sites and 2 deletions in other sites, 1 base substitution in codon 28. b

Percent of total mutations identified, not percent of hemangiosarcomas with mutations. Fifteen mutations in p53 were 

identified in 11 hemangiosarcomas (4 hemangiosarcomas had double mutations), 8 mutations in Catnb 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 studies 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 

2 

3 

4 
RoC Background Document for o-Nitrotoluene 

Table 5-9. p53, β-catenin, and K-ras mutations in cecal carcinomas from B6C3F1

mice orally exposed to o-nitrotoluene 

Treatment group, 

Mutations 

p53 (exon 7), 

number (%)b Catnba, number (%)b

K-ras (codon 10-13), 

number (%)b

Controlsc 

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) 

1d

 (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) 

9e

 (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-pair 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 double 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 normal 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 6/20/08
RoC 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 upregulated. 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 growth 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. 

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Kim et al. (2006) investigated the genes involved in peritoneal mesotheliomas induced in 

male F344 rats by exposure to o-nitrotoluene or bromochloroacetic acid. Mesotheliomas 

were collected from four rats, RNA was isolated, and gene expression analysis was 

conducted. The analysis identified 169 differentially expressed cancer-related genes that 

were categorized according to cancer-related function. The major carcinogenic pathways 

involved in peritoneal mesothelioma formation 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 pathways 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 + 

B6C3F1 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 + 

B6C3F1 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 6/20/08
RoC Background Document for o-Nitrotoluene 

6/20/08 73

1 5.5 Carcinogenicity and genotoxicity of o-nitrotoluene analogues and metabolites 

An NTP (1992) study included comparative toxicity evaluations of the o-, m-, and pnitrotoluene isomers in rats and mice. The NTP conducted a two-year carcinogenicity 

study of rats and mice exposed to p-nitrotoluene; however, no studies describing chronic 

exposure to m-nitrotoluene were found. A great deal of research also has been done on 

the metabolism, mutagenicity, and carcinogenicity of the dinitrotoluenes, and the 

bioactivation of 2,4-dinitrotoluene 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. 

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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 organs and systems (e.g., clitoral gland, skin, 

hematopoietic system or spleen, testis, and lung) (NTP 2002a). However, most of the 

increased incidences either were not significantly 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 incidences 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 B6C3F1 mice, based on increased 

incidences of alveolar/bronchiolar neoplasia, and no evidence of carcinogenicity in 

female mice. 
RoC Background Document for o-Nitrotoluene 

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Table 5-11. Neoplastic lesions identified in rats and mice exposed to o-nitrotoluene 

and p-nitrotoluene 

o-Nitrotoluene p-Nitrotoluene Organ or 

system F344/N rats B6C3F1 mice F344/N rats B6C3F1 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 potential 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 pnitrotoluene, also binds 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 nitrobenzyl 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). 

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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 studies on the stability of o-, m-, and p-nitrotoluene DNA adducts are 
RoC 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. 

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5.5.2 Dinitrotoluenes 

In 1984 Rickert et al. reviewed the metabolism, mutagenicity, and carcinogenicity of 

dinitrotoluenes. At the time of their review, three independent chronic bioassays, all of 

which were feeding studies, had been completed: (1) a National Cancer Institute (NCI) 

(1978) study of 2,4-dinitrotoluene in F344 rats and B6C3F1 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 Institute) study of 2,4-

dinitroluene in Sprague-Dawley rats and Swiss mice. 

In the NCI study, 2,4-dinitrotoluene (95% pure, contaminants 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 B6C3F1 mice for 78 weeks followed by an additional 26 weeks of observation 

for rats and 13 weeks for mice. In male rats, a significantly increased incidence of 

fibroma of the skin and subcutaneous tissue occurred in both dose groups, and a 

statistically significant incidence 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 mixture 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 carcinomas or neoplastic nodules) were observed in high- and mid-dose 

males and females. Technical-grade dinitrotoluene 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-dinitrotoluene (mg/kg per day) 
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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. 

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Initiation-promotion studies in male F344 rats have demonstrated that technical-grade 

dinitrotoluene and 2,6-dinitrotoluene have initiating and promoting activity in the rat 

liver, whereas 2,4-dinitrotoluene had only promoting activity (reviewed by Rickert et al.

1984b). Leonard et al. (1987) noted that 2,4-dinitrotoluene did not induce hepatic tumors 

in F344 rats in the NCI study. However, technical-grade dinitrotoluene caused a 100% 

liver tumor incidence by 55 weeks in F344 rats in the CIIT study. Leonard et al. noted 

that rats in the CIIT study received a 5- to 10-times higher dose of 2,6-dinitrotoluene than 

the rats in the NCI study, which was based 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 dosing 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 three 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-dinitrotoluene and 18% 2,6-dinitrotoluene), and 

the two purified isomers (2,4- and 2,6-dinitrotoluene). Rats were fed 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 technical-grade dinitrotoluene for 52 weeks. At 

52 weeks, hepatocellular carcinomas 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 pulmonary tumors. Both compounds were inactive 

by oral or intraperitoneal administration 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 

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Mutagenicity studies of the dinitrotoluenes in bacterial and yeast systems showed activity 

for all the isomers (Rickert et al. 1984b). The increased activity of putative reduced 

metabolites of the dinitrotoluenes together with the inactivity in systems lacking 

nitroreductase suggests that nitroreduction is necessary for mutagenic activity. Studies in 

in vitro mammalian systems gave uniformly negative results for all isomers of 

dinitrotoluene, suggesting that extrahepatic metabolism was necessary 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. 

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These studies showed that sulfotransferase inhibition in vivo decreased the covalent 

binding of the hepatocarcinogen 2,6-dinitrotoluene 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 mechanisms of the dinitrotoluenes (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 related to sex differences in tumor formation. 

The metabolism of 2,6-dinitrotoluene and related 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-dinitrobenzyl glucuronide 

than do females. Once in the intestine, the glucuronic acid moiety is cleaved, and one of 

the nitro groups is reduced before reabsorption. 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 

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Several studies have investigated the potential for dinitrotoluenes to cause cancer in 

human subjects (see Section 3.2.1). These data suggest the possibility of carcinogenicity 

of dinitrotoluenes in humans. 

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5.5.3 o-Toluidine hydrochloride 

o-Toluidine (o-aminotoluene) hydrochloride administered in feed to F344 rats and 

B6C3F1 mice for 101 to 104 weeks (NCI 1979) increased the incidences of mesothelioma 

of the abdominal cavity or scrotum 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 and 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 equimolar 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, kidney, and urinary bladder differed. 

Epidemiological data indicate an increased risk of bladder cancer in workers exposed to 

o-toluidine and aniline (see Section 3.2.2). o-Toluidine and its hydrochloride 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).

27 5.6 Toxicity 

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, or 
RoC Background Document for o-Nitrotoluene 

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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 the studies but males showed more treatmentrelated toxicity than females. Toxic effects were graded on a scale of 1 to 4 where 1 

represented minimal effects and 4 represented severe 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), kidney 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 hepatotoxic effects included oval-cell hyperplasia, 

PGST+ foci, and increased serum bile acids, 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) were 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 ppm, and no liver lesions were observed in 

females at any concentration tested. 

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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 α2uglobulin levels in male rats (the m-isomer was not tested) (Dunnick et al. 1994, NTP 

1992, 1996). 

NTP (1992) conducted 13-week studies with B6C3F1 mice exposed to o-nitrotoluene (< 

96% purity), to compare clinical toxicity, histopathology, 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 controls. The only treatment-related 

histopathological lesions were olfactory epithelium degeneration/metaplasia in both male 
RoC Background Document for o-Nitrotoluene 

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and female mice (see Table 5-12). The observed changes included moderate thinning of 

the olfactory nuclear layer, decreased diameter of the associated nerve fiber bundles in 

the lamina propria, replacement of the olfactory epithelium by cuboidal respiratory 

epithelium, and dilation of the submucosa underlying Bowman’s glands (Dunnick et al.

1994). 

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3 

4 

5 

Table 5-12. Treatment-related lesions in B6C3F1 mice following dietary exposure to 

o-nitrotoluene for 13 weeksa

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. 

6 5.7 Summary 

7 5.7.1 Absorption, distribution, metabolism, and excretion 

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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 indicate that absorption 

occurs from skin contact and 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 first 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)-Nacetylcysteine 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-aminobenzyl 
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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 accounted for 4.9% of the dose in males and 

0.4% in females. Metabolites excreted through the bile can be reabsorbed and further 

metabolized. 

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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, renal, or splenic histopathology, 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 glucuronic 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-related 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 exchange in Chinese hamster ovary (CHO) cells; 

(2) chromosomal aberrations in Chinese hamster lung cells (CHL) and human peripheral 

lymphocytes (but not in CHO cells); (3) micronuclei in CHL 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 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-dependent increase in the percentage 
RoC Background Document for o-Nitrotoluene 

82 6/20/08

of hepatocytes undergoing DNA repair and the extent 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 metabolic activation. Differences 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 B6C3F1 mice except for a slight increase in the 

frequency of micronucleated NCEs in male mice exposed to 10,000 ppm in one study. 

1 

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3 

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19 

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21 

22 

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24 

25 

26 

27 

28 

29 

Gene expression studies with mouse L5178Y Tk+/- lymphoma cells in vitro identified 

four genes and a few pathways whose expression was consistently affected by onitrotoluene. A study of early changes in gene expression in mouse liver after exposure to 

o-nitrotoluene in vivo for 2 weeks identified changes in tumor suppressor 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 adequate 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 increased incidences of tumors in other 

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.] 

In gene expression studies on mouse liver, a dose-dependent loss of expression of the 

fragile histidine triad gene (Fhit) and the WW domain-containing oxidoreductase (Wwox) 

gene was observed with exposure to o-nitrotoluene. The authors noted that these genes 

are human tumor suppressor genes and are often 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 transcription 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 

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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 cyclin 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 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). Major carcinogenic 

pathways involved in peritoneal mesothelioma formation in mice exposed to onitrotoluene also were similar to pathways activated in human mesotheliomas, including 

IGF-1, p38 MAPK, Wnt/B-catenin, and integrin signaling pathways. 

5.7.5 Carcinogenicity and genotoxicity of o-nitrotoluene analogues and metabolites 

The NTP also conducted cancer studies in experimental animals on another nitrotoluene 

isomer, p-nitrotoluene, and found equivocal evidence of carcinogenic activity 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 macromolecules, or (3) greater 

metabolism of o-nitrotoluene to the nitrobenzyl glucuronide, which gives rise to oaminobenzyl sulfate, the proposed proximal reactive metabolite. 
RoC Background Document for o-Nitrotoluene 

84 6/20/08

1 5.7.6 Toxicity 

2 

3 

4 

5 

6 

7 

8 

9 

10 

o-Nitrotoluene caused more treatment-related toxicity in male rats than in female rats, 

and most toxic effects occurred at or greater than 2,500 ppm in a 13-week study. oNitrotoluene exposure resulted in toxic effects in the kidney (hyaline droplet nephropathy 

and increased renal α2u-globulin levels) and liver (hepatocyte vacuolization, oval-cell 

hyperplasia, PGST+ foci, and increased serum 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 concentrations less than 2,500 ppm. 
RoC Background Document for o-Nitrotoluene 

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Glossary of Terms 

Adenoma: An ordinarily benign neoplasm of epithelial 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 substance at atmospheric pressure 

(101.3 kPa) unless a different pressure is stated. If the substance decomposes below or at 

the boiling point, this is noted (dec). The temperature 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 columnar 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 which 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/cm3

) and is generally assumed to refer to temperatures near room temperature unless 

otherwise stated. Values for gases are generally 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 atmospheric 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 characterized by rapidly proliferating cells 

derived from the blood vessels and lining 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 larger the Henry’s law 

constant the less soluble it is (greater 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 Update Rule is to assist 

EPA in keeping an inventory of chemical substances 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 the rule enacted in 2003 raised the threshold 

limit to 25,000 pounds and increased the reporting period to every 5 years. 

Koc: Soil organic adsorption coefficient, which is calculated as the ratio of the 

concentration of a chemical adsorbed to the organic matter component 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 Kow): The ratio of concentrations of a 

substance in octanol and in water, when dissolved in a mixture of octanol and water. For 

convenience, the logarithm of Kow is used. The octanol/water partition coefficient of a 

substance is useful as a means to predict soil adsorption, biological 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 between the melting point and the freezing point, a 

range is given. In case of hydrated substances (i.e., those with crystal water), the apparent 

melting point is given. If the substance decomposes 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 cells of the pleura and peritoneum 

that is composed of either epithelial-like cells, spindle cells, or both.

Methemoglobin: A compound formed from hemoglobin by oxidation of the iron atom 

from the ferrous (Fe2+) to the ferric (Fe3+) state with essentially ionic bonds, rendering it 

incapable of functioning reversibly as an oxygen carrier. Methemoglobin is present in 

small amounts in blood normally, but injury or toxic agents can increase the conversion.

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Molecular weight: The molecular weight of a substance 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 (pKa): 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 dissociation 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 layer 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 described variously as amorphous, powders, 

pellets, flakes, lumps, or crystalline; and the shape of the crystals is specified if available. 

Solids also may be described as hygroscopic or deliquescent depending upon their 

affinity for water. 

Pleomorphic: Occurring in various distinct forms.

Poly-3 test: Poly-3 test: A survival-adjusted statistical 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 number of animal years at risk. For analysis of 

a given tumor site, each animal is assigned either (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 time that it survived, raised to the 3rd power, if the animal 

died prior to terminal sacrifice and did not have 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 material to the density of a standard 

material, such as water at a specified temperature; 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 functions to mark the protein for proteolytic 

destruction. 

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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 liquid or solid, the value 

applies only to the vapor formed 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).