Report on Carcinogens, Twelfth Edition (2011)

National Toxicology Program, Department of Health and Human Services

o-Nitrotoluene

CAS No. 88-72-2

Reasonably anticipated to be a human carcinogen

First listed in the Twelfth Report on Carcinogens (2011)

Also known as 2-nitrotoluene

CH3

NO2

Carcinogenicity

o-Nitrotoluene is reasonably anticipated to be a human carcinogen 

based on sufficient evidence of carcinogenicity from studies in experimental animals and supporting data on mechanisms of carcinogenesis. 

Cancer Studies in Experimental Animals

Oral exposure to o-nitrotoluene caused tumors at several different tissue sites in rats and mice and early onset of cancer in male rats. Malignant mesothelioma and mesothelial-cell hyperplasia of the tunica 

vaginalis of the epididymis were observed in male rats administered 

o-nitrotoluene in their feed for 13 weeks (NTP 1992). Bile-duct cancer (cholangiocarcinoma) was observed after 26 weeks, both in rats 

exposed to o-nitrotoluene for 26 weeks and in rats exposed for 13 

weeks and then observed for 13 more weeks without exposure (NTP 

1996). o-Nitrotoluene caused cancer at several tissue sites in two-year 

chronic exposure studies of rats and mice of both sexes and in a study 

in which male rats were exposed to o-nitrotoluene for 13 weeks and 

evaluated at two years (NTP 2002). In rats, o-nitrotoluene caused 

(1) subcutaneous skin tumors and mammary-gland tumors (fibroadenoma) in both sexes, (2) malignant mesothelioma and benign or 

malignant tumors of the liver (hepatocellular adenoma or carcinoma 

or cholangiocarcinoma) and lung (alveolar/bronchiolar adenoma or 

carcinoma) in males, and (3) benign liver tumors (hepatocellular adenoma) in females. In mice, it caused malignant blood-vessel tumors 

(hemangiosarcoma) in both sexes, malignant tumors of the large intestine (cecal carcinoma) in males, and benign or malignant liver tumors (hepatocellular adenoma or carcinoma) in females (NTP 2002). 

Studies on Mechanisms of Carcinogenesis

Following oral administration to rats and mice, o-nitrotoluene is 

absorbed into the blood and rapidly cleared; the serum half-life 

is 1.5  hours in rats (NTP 2002). In the rat liver, o-nitrotoluene is 

metabolized to o-nitrobenzyl alcohol and can follow several metabolic pathways: (1) glucuronidation to o-nitrobenzyl glucuronide, 

(2) sulfation and subsequent reaction with glutathione and acetylcysteine to o-nitrobenzyl sulfate, S-(o-nitrobenzyl)glutathione, and S-(onitrobenzyl)-N-acetylcysteine, or (3) metabolism to o-aminobenzyl 

alcohol followed by oxidation to o-aminobenzoic acid. The metabolites are eliminated primarily in the urine. The major metabolites are 

o-nitrobenzyl glucuronide and o‐nitrobenzoic acid major metabolites 

in rats and mice and o‐aminobenzyl alcohol and S-(o-nitrobenzyl)-

N-acetylcysteine in rats. Female rats excrete less than half as much 

of the dose in the form of o‐aminobenzyl alcohol, o‐nitrobenzyl alcohol, or S-(o-nitrobenzyl)-N-acetylcysteine as male rats (NTP 2002). 

The glucuronidated form can also be excreted in the bile; when the 

glucuronidated form in the bile is excreted into the small intestine, 

intestinal bacteria can deconjugate it and reduce the nitro group to 

an amino group, forming aminobenzyl alcohol. Aminobenzyl alcohol can be reabsorbed from the intestine and further metabolized 

by the liver to reactive compounds (carbonium and nitrenium ions) 

that can covalently bind to DNA or to proteins (Chism and Rickert 

1985, NTP 2002, 2008). Thus, microbial metabolism in the intestine 

is an important step in the carcinogenicity of o‐nitrotoluene. However, neither o‐aminobenzyl alcohol nor its metabolites have been detected in mouse urine after exposure to o-nitrotoluene (NTP 2002); 

therefore, other unidentified biochemical pathways leading to tumor 

formation most likely are involved.

o-Nitrotoluene did not cause mutations in bacteria. In studies 

of its ability to cause genetic damage in cultured mammalian cells, 

the results were mixed. o-Nitrotoluene caused (1) sister chromatid 

exchange in Chinese hamster ovary (CHO) cells, (2) chromosomal 

aberrations in Chinese hamster lung (CHL) cells and human peripheral lymphocytes but not in CHO cells, (3)  micronucleus formation in CHL cells but not in CHO-K1 cells, and (4) DNA damage 

in L5178Y mouse lymphoma cells (NTP 2008). It did not induce DNA 

repair in rat or human hepatocytes (NTP 2008). In rats and mice exposed in vivo, o-nitrotoluene caused a slight increase in micronucleus formation in peripheral normochromatic erythrocytes in male 

mice at a high dose level; this finding was not considered conclusive. 

o-Nitrotoluene did not induce micronucleus formation in peripheral 

normochromatic erythrocytes in female mice or in polychromatic 

erythrocytes in the bone marrow of male rats or mice (NTP 2002). 

Following in vivo exposure of rats to o-nitrotoluene, DNA repair was 

increased in liver cells isolated from males, but not from females or 

germ-free males. These results, together with o-nitrotoluene’s inability to induce DNA repair in hepatocytes in vitro, suggest that activation of o-nitrotoluene to become genotoxic is sex-specific and 

depends on both mammalian metabolism and metabolism by intestinal bacteria (Doolittle et al. 1983). However, o-nitrotoluene also 

caused tumors in other tissues in rats and mice of both sexes, suggesting that other activation mechanisms exist. 

In rats exposed to o-nitrotoluene in vivo, DNA adducts were detected in the liver of males but not females (NTP 2008). Formation 

of DNA adducts was consistent with the reaction of intermediate 

compounds derived from o-aminobenzyl alcohol with guanine or 

adenine bases (Jones et al. 2003). The pattern of mutations in oncogenes from o-nitrotoluene–induced tumors was also consistent with 

guanine adduct formation: the majority of p53 mutations in hemangiosarcomas were G:C to A:T transitions, and almost all the K-ras mutations in cecal carcinomas were G:C to T:A transversions (Hong et 

al. 2003, Sills et al. 2004). Mutations in the p53, β‐catenin, and K-ras

genes also were found in hemangiosarcomas from mice exposed to 

o-nitrotoluene, but not in spontaneously occurring hemangiosarcomas from unexposed mice (Hong et al. 2003). 

In factory workers exposed to o-nitrotoluene, o-nitrotoluene–hemoglobin adducts were detected in the blood (Jones et al. 2005a), and 

o-nitrobenzoic acid and o-nitrobenzyl alcohol were detected in the 

urine (Jones et al. 2005b), providing evidence that human exposure 

to o-nitrotoluene results in production of a reactive metabolite(s). 

In addition, adducts between hemoglobin and 2-methylaniline (a 

metabolite of o-nitrotoluene) were identified in both exposed workers and exposed rats, and the level of 2-methylaniline–hemoglobin 

adducts in the blood of rats was proportional to the level of 2-methylaniline–DNA adducts in the livers of rats (Jones and Sabbioni 2003, 

Jones et al. 2003). 

Cancer Studies in Humans 

The data available from epidemiological studies are inadequate to 

evaluate the relationship between human cancer and exposure specifically to o-nitrotoluene. One cohort study of workers involved in 

Table of Contents: http://ntp.niehs.nih.gov/go/roc12

Page 331
Report on Carcinogens, Twelfth Edition (2011)

National Toxicology Program, Department of Health and Human Services

the manufacture of magenta dye mentioned exposure of workers 

to o-nitrotoluene as part of the manufacturing process. A large excess of bladder cancer was reported; however, the workers were also 

exposed to other chemicals  — o-toluidine (2-methylaniline) and 

4,4′-methylenebis(2-methylaniline)—that are suspected of causing 

bladder cancer (Rubino et al. 1982). Two other studies of magenta 

manufacturing workers also reported an excess of bladder cancer, but 

did not report whether the workers were exposed to o-nitrotoluene 

(Case and Pearson 1954, Vineis and Magnani 1985).

Properties

o-Nitrotoluene is a nitroaromatic compound. 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). 

At room temperature, o-nitrotoluene is a yellow liquid 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 of o-nitrotoluene may explode when heated 

(HSDB 2010). Physical and chemical properties of o-nitrotoluene are 

listed in the following table.

Property Information

Molecular weight 137.1

Specific gravity 1.162 at 19°C/15°C

Melting point –9.5°C (needles); –2.9°C (crystals)

Boiling point 222°C at 760 mm Hg

Log Kow 2.30

Water solubility 650 mg/L at 30°C

Vapor pressure (mm Hg) 0.188 at 25°C

Vapor density relative to air 4.73

Source: HSDB 2010.

Use

o-Nitrotoluene is used primarily in the production of o‐toluidine (also 

known as o‐aminotoluene or 2-methylaniline), 2-amino-4-chlorotoluene, 2-amino-6-chlorotoluene, o‐toluidine-4-sulfonic acid, and 

other chemicals that are intermediates in the production of various 

azo dyes (IARC 1996). It is also used in the manufacture of (or the 

manufacture of intermediates for) other dyes, such as magenta and 

various sulfur dyes for cotton, wool, silk, leather, and paper (IARC 

1996, HSDB 2010). In addition, it is used as an intermediate in the 

synthesis of (or the synthesis of intermediates for) explosives and a 

variety of organic chemicals, including compounds used in the agricultural chemical, pesticide, petrochemical, pharmaceutical, and 

rubber industries (HSDB 2010).

Production

o-Nitrotoluene is produced principally by the nitration of toluene 

with a mixture of nitric acid and either sulfuric, aromatic sulfonic, or 

phosphoric acid (IARC 1996). U.S. production of o-nitrotoluene was 

calculated as 13 billion grams (29 million pounds) for 1981 (HSDB 

2010) and was estimated at 16,120 metric tons (35.5 million pounds) 

for 1993 (Dunlap 1998). The U.S. Environmental Protection Agency 

lists o-nitrotoluene as a high-production-volume chemical. In 2010, 

o-nitrotoluene was produced by one U.S. manufacturer (SRI 2010) 

and was available from 14 U.S. suppliers (ChemSources 2010). No 

data on U.S. imports or exports of o-nitrotoluene were found. Reports filed between 1986 and 2002 under EPA’s Toxic Substances Control Act Inventory Update Rule indicated that U.S. production plus 

imports of o-nitrotoluene totaled 10 million to 50 million pounds 

(EPA 2004). 

Exposure

Exposure to o-nitrotoluene in the United States is expected to result 

primarily from dermal and inhalation exposure during its production 

and use. The general population may be exposed to o-nitrotoluene as a 

result of its occurrence in the environment from (1) inadvertent spills 

of o-nitrotoluene or chemical mixtures containing o-nitrotoluene, 

(2) emissions directly into the environment, or (3) breakdown products of dinitrotoluenes (DNT) and trinitrotoluenes (TNT). o-Nitrotoluene has been detected in U.S. air and water. The National Response 

Center database contains reports of two spills reported as o-nitrotoluene in 1990 and one spill reported as nitrotoluene (o-, p-, and mixtures) in 2000 (NRC 2008). Two ambient-air samples collected in 

Boise, Idaho, in the winter of 1986–87 contained o-nitrotoluene vapor at concentrations of 0.03 and 0.29 ng/m3

 (Nishioka and Lewtas 

1992). o‐Nitrotoluene was also detected in a paper-mill waste-treatment lagoon (concentration and location not reported) (HSDB 2010) 

and at concentrations ranging from 320 to 16,000 μg/L in the effluent 

of a U.S. plant producing TNT (IARC 1996, HSDB 2010).

DNT and TNT are used in the production of commercial and military explosives, and o-nitrotoluene has been found in groundwater, 

private well water, surface water, and soil at or near munitions production facilities and military training grounds. o-Nitrotoluene was found 

at average concentrations of 42.6 mg/L (42,600 μg/L) (Best et al. 2001) 

and 2.9 mg/L (2,900 μg/L) (Spain et al. 1999) in groundwater at a Tennessee munitions arsenal. At a facility that has produced munitions 

since World War II, o-nitrotoluene has been detected sporadically 

during routine groundwater monitoring of both the Ogallala aquifer, 

at concentrations of 0.12 to 2.9 μg/L (both measured in 2004), and a 

perched aquifer above the Ogallala, at concentrations of 0.14 μg/L (in 

2003) to 5 μg/L (in 2004) (Pantex 2008). At a former munitions production site in Wisconsin, o-nitrotoluene was detected in off-site private well water at concentrations of up to 0.095 μg/L (WDHFS 2002). 

At a historical testing ground in Idaho, soil contaminated with TNT 

at a concentration of 39,100 ppm contained o-nitrotoluene at a concentration of 1.4 ppm (Radtke et al. 2002). 

No information was found on the number of U.S. workers potentially exposed to o-nitrotoluene in the production of chemical intermediates. o-Nitrotoluene was detected at a concentration of 47 ng/m3

in ambient air at a chemical manufacturing plant in New Jersey (IARC 

1996) and at air concentrations of up to 2.0 mg/m3 in the nitrotoluene production area of a Swedish plant producing pharmaceuticals 

and explosives (Ahlborg et al. 1985). The American Conference of 

Governmental Industrial Hygienists considers o-nitrotoluene to be 

an inducer of methemoglobin and recommends that methemoglobin in blood be used as a biological exposure index for o-nitrotoluene (and the other nitrotoluene isomers) (ACGIH 2009).

Regulations

Coast Guard, Department of Homeland Security 

Minimum requirements have been established for the safe transport of o‐nitrotoluene on barges.

Department of Transportation (DOT)

o‐Nitrotoluene is considered a hazardous material, and special requirements have been set for marking, 

labeling, and transporting this material.

Safety measures after spills or leaks are prescribed in accordance with o‐nitrotoluene being a 

combustible toxic hazardous material.

Environmental Protection Agency (EPA)

Comprehensive Environmental Response, Compensation, and Liability Act

Reportable quantity (RQ) = 1,000 lb.

(EPA has not carried out an Integrated Risk Information System assessment for o‐nitrotoluene.)

Occupational Safety and Health Administration (OSHA)

While this section accurately identifies OSHA’s legally enforceable PELs for this substance in 2010, 

specific PELs may not reflect the more current studies and may not adequately protect workers.
Report on Carcinogens, Twelfth Edition (2011)

National Toxicology Program, Department of Health and Human Services

Permissible exposure limit (PEL) = 5 ppm (30 mg/m3).

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.

Guidelines

American Conference of Governmental Industrial Hygienists (ACGIH)

Threshold limit value – time-weighted average (TLV-TWA) = 2 ppm.

Potential for dermal absorption.

Biological Exposure Index (BEI): Methemoglobin in blood not to exceed 1.5% of hemoglobin measured 

during or at the end of a shift.

National Institute for Occupational Safety and Health (NIOSH)

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

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

Potential for dermal absorption.

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