### 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 CH 3 NO 2 

### Carcinogenicity o -Nitrotoluene is reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in ex
perimental animals and supporting data on mechanisms of carcino
genesis. Cancer Studies in Experimental Animals Oral exposure to o - nitrotoluene caused tumors at several different tis
sue sites in rats and mice and early onset of cancer in male rats. Ma
lignant 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 can
cer (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 (fibro
adenoma) 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 ad
enoma) in females. In mice, it caused malignant blood-vessel tumors (hemangiosarcoma) in both sexes, malignant tumors of the large in
testine (cecal carcinoma) in males, and benign or malignant liver tu
mors (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 met
abolic pathways: (1) glucuronidation to o - nitrobenzyl glucuronide, (2) sulfation and subsequent reaction with glutathione and acetylcys
teine to o - nitrobenzyl sulfate, S
- ( o -nitrobenzyl)glutathione, and S
- ( o nitrobenzyl)-N -acetylcysteine, or (3) metabolism to o - aminobenzyl alcohol followed by oxidation to o - aminobenzoic acid. The metabo
lites 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 al
cohol, 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 alco
hol 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. How
ever, neither o‐ aminobenzyl alcohol nor its metabolites have been de
tected 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 pe
ripheral lymphocytes but not in CHO cells, (3)  micronucleus for
mation 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 ex
posed in vivo , o - nitrotoluene caused a slight increase in micronu
cleus formation in peripheral normochromatic erythrocytes in male mice at a high dose level; this finding was not considered conclusive. o - Nitro toluene 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 - nitro toluene’s in
ability to induce DNA repair in hepatocytes in vitro, suggest that ac
tivation of o - nitrotoluene to become genotoxic is sex-specific and depends on both mammalian metabolism and metabolism by intes
tinal bacteria (Doolittle et al . 1983). However, o - nitrotoluene also caused tumors in other tissues in rats and mice of both sexes, sug
gesting that other activation mechanisms exist. In rats exposed to o - nitrotoluene in vivo , DNA adducts were de
tected 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 onco
genes from o - nitrotoluene–induced tumors was also consistent with guanine adduct formation: the majority of p53 mutations in heman
giosarcomas were G:C to A:T transitions, and almost all the K
- rasmu tations 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 - nitro toluene, but not in spontaneously occurring hemangiosarco
mas from unexposed mice (Hong et al. 2003). In factory workers exposed to o - nitrotoluene, o - nitrotoluene–he
moglobin 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 work
ers and exposed rats, and the level of 2
- methylaniline–hemoglobin adducts in the blood of rats was proportional to the level of 2
- meth
ylaniline–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 spe
cifically to o - nitrotoluene. One cohort study of workers involved in 

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

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### 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 ex
cess 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 iso
mers 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 tempera
ture 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 - nitro toluene 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 K ow 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-chloro
toluene, 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 ag
ricultural 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. Re
ports filed between 1986 and 2002 under EPA’s Toxic Substances Con
trol 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 - nitro toluene or chemical mixtures containing o - nitro toluene, (2) emissions directly into the environment, or (3) breakdown prod
ucts of dinitro toluenes (DNT) and trinitrotoluenes (TNT). o - Nitro
toluene has been detected in U.S. air and water. The National Response Center database contains reports of two spills reported as o - nitrotol
uene in 1990 and one spill reported as nitrotoluene ( o -, p -, and mix
tures) in 2000 (NRC 2008). Two ambient-air samples collected in Boise, Idaho, in the winter of 1986–87 contained o - nitrotoluene va
por at concentrations of 0.03 and 0.29 ng/m 3 (Nishioka and Lewtas 1992). o‐ Nitrotoluene was also detected in a paper-mill waste-treat
ment 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 mil
itary explosives, and o - nitrotoluene has been found in groundwater, private well water, surface water, and soil at or near munitions produc
tion 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 Ten
nessee 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 pro
duction site in Wisconsin, o - nitrotoluene was detected in off-site pri
vate 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 con
centration of 1.4 ppm (Radtke et al. 2002). No information was found on the number of U.S. workers poten
tially exposed to o - nitrotoluene in the production of chemical inter
mediates. o - Nitrotoluene was detected at a concentration of 47 ng/m 3 in ambient air at a chemical manufacturing plant in New Jersey (IARC 1996) and at air concentrations of up to 2.0 mg/m 3 in the nitrotolu
ene 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 methemoglo
bin in blood be used as a biological exposure index for o -nitrotolu
ene (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/m 3 ). 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/m 3 ). Potential for dermal absorption. 

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