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o-Nitrotoluene is used to synthesize agricultural and rubber chemicals, azo and
sulfur dyes, and
dyes for cotton, wool,
silk, leather, and paper.
o-Nitrotoluene was nominated for study by NIOSH and the NTP based on its considerable human exposure as well as the absence of long-term studies of carcinogenicity in rodents. Male and female F344/N rats and B6C3F1 mice were exposed to
o-nitrotoluene (greater than 99% pure) in feed for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-YEAR STUDY IN RATS: In the core study, groups of 60 male and 60 female rats were fed diets containing 625, 1,250, or 2,000 ppm
o-nitrotoluene (equivalent to average daily doses of approximately 25, 50, or 90 mg
o-nitrotoluene/kg
body weight to males and 30, 60, or 100 mg/kg to females) for 105 weeks. In a 3-month stop-exposure study, groups of 70 male rats were fed diets containing 2,000 or 5,000 ppm
o-nitrotoluene (equivalent to average daily doses of approximately 125 or 315 mg/kg) for 13 weeks followed by undosed feed for the remainder of the study. A group of 70 male rats receiving undosed feed served as a control group for both male rat studies; 60 female rats receiving undosed feed were the control group for the female core study. Ten control males and 10 males from each stop-exposure group were sacrificed at 3 months. Survival,
Body Weights, and Feed Consumption: All 2,000 ppm core study, all 5,000 ppm stop-exposure, and all but three core study 1,250 ppm male rats died before the end of the studies. Survival of 625 ppm core study and 2,000 ppm stop-exposure males and of 2,000 ppm females was significantly less than that of the controls. Mean
body weights of all exposed groups of males except the 625 ppm group were generally less than those of the controls throughout the study. Mean
body weights of 2,000 ppm females were less than those of the controls during year 2 of the study. Feed consumption by exposed groups of rats was similar to that by the controls.
Biomarkers of Exposure: Three urinary metabolites were followed during the study as
biomarkers of exposure. The ratios of o-nitrobenzoic
acid to
creatinine and of o-nitrobenzylmercapturic
acid to
creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. The ratio of o-aminobenzoic
acid to
creatinine was not related to exposure concentration. Pathology Findings: The incidences of
malignant mesothelioma in male rats occurred with positive trends in both the core and stop-exposure studies and were significantly greater in exposed groups than in the controls. Incidences of subcutaneous
skin neoplasms (
fibroma,
fibrosarcoma, and
lipoma) were increased in exposed groups of males, while the incidences of
fibroma or
fibrosarcoma (combined) were increased in exposed females. In all exposed groups of males and females except 2,000 ppm core study males, the incidences of mammary gland
fibroadenoma were significantly increased. The incidences of mammary gland
hyperplasia were significantly increased in 625 and 1,250 ppm females. Increased incidences of
mesothelioma,
skin neoplasms, and mammary gland
fibroadenoma in the stop-exposure males indicated that 3 months of dosing were sufficient to produce a carcinogenic effect. Liver weights of 5,000 ppm stop-exposure males were significantly greater than those of the controls at 3 months. The incidences of
hepatocellular adenoma in 2,000 ppm core study males and females and of
hepatocellular adenoma or
carcinoma (combined) in 2,000 ppm core study and 5,000 ppm stop-exposure males were significantly increased.
Cholangiocarcinoma occurred in three 5,000 ppm stop-exposure males, and a single hepatocholangiocarcinoma occurred in a 625 ppm male and in a 2,000 ppm core study male. Nonneoplastic lesions of the liver included eosinophilic, mixed cell, and clear cell foci in exposed groups of males and females and mixed cell infiltrate in exposed males and basophilic focus in exposed females. The incidences of alveolar/bronchiolar
adenoma and alveolar/bronchiolar
adenoma or
carcinoma (combined) were significantly increased in 5,000 ppm stop-exposure males, as were alveolar/bronchiolar
hyperplasia in most exposed groups of males and females. The incidences of hematopoietic cell proliferation of the spleen and of
hyperplasia of the mandibular lymph node (females) and bone marrow were increased in exposed groups of males at 3 months and/or 2 years and in exposed groups of females at 2 years. The incidences of mononuclear cell
leukemia were significantly decreased in all groups of males exposed to 1,250 ppm or greater and in all exposed groups of females; the incidence of testicular interstitial cell
adenoma was significantly decreased in 5,000 ppm stop-exposure males. 2-YEAR STUDY IN MICE: Groups of 60 male and 60 female mice were fed diets containing 0, 1,250, 2,500, or 5,000 ppm
o-nitrotoluene (equivalent to average daily doses of approximately 165, 360, or 700 mg/kg to males and 150, 320, or 710 mg/kg to females) for 105 weeks. Survival,
Body Weights, and Feed Consumption: All 2,500 and 5,000 ppm males died before the end of the study. Survival of 1,250 ppm males and 5,000 ppm females was significantly less than that of the controls. Mean
body weights of exposed males and 5,000 ppm females were generally less than those of the controls throughout the study, and those of 2,500 ppm females were less during the second year of the study. Feed consumption by 5,000 ppm males was less than that by the controls.
Biomarkers of Exposure: Three urinary metabolites were followed during the study as
biomarkers of exposure. The ratios of o-nitrobenzoic
acid to
creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. The concentrations of o-nitrobenzylmercapturic
acid and o-aminobenzoic
acid were below the limit of quantitation at most time points. Pathology Findings: The incidences of
hemangiosarcoma in all exposed groups of males and in 5,000 ppm females were significantly greater than those in the controls. Large intestine (cecum)
carcinomas were observed in all exposed groups except 5,000 ppm males. The incidences of hepatocellular
neoplasms were significantly increased in 2,500 and 5,000 ppm females. Nonneoplastic liver lesions including eosinophilic and basophilic foci and minimal to mild
necrosis were enhanced in exposed males and females. Also present were focal hepatocyte syncytial alteration in exposed males and hepatocyte
necrosis and focal hepatocyte cytoplasmic vacuolization in 5,000 ppm females. Renal tubule pigmentation occurred more frequently in exposed groups of males and in 5,000 ppm females than in the controls. Olfactory epithelial degeneration occurred in every male and female mouse exposed to 2,500 or 5,000 ppm, and the severity of this lesion increased with increasing exposure concentration.
GENETIC TOXICOLOGY: Under the conditions of these studies, there was clear evidence of carcinogenic activity* of
o-nitrotoluene in male rats based on increased incidences of
malignant mesothelioma, subcutaneous
skin neoplasms, mammary gland
fibroadenoma, and
liver neoplasms. The increased incidences of
lung neoplasms in male rats were also considered to be exposure related. There was clear evidence of carcinogenic activity of
o-nitrotoluene in female rats based on increased incidences of subcutaneous
skin neoplasms and mammary gland
fibroadenoma. The increased incidence of
hepatocellular adenoma in female rats was also considered to be exposure related. There was clear evidence of carcinogenic activity of -
o-nitrotoluene in male and female mice based on increased incidences of
hemangiosarcoma,
carcinoma of the large intestine (cecum), and hepatocellular
neoplasms (females only). Exposure to
o--nitrotoluene caused increased incidences of nonneoplastic lesions of the mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats and of the liver, kidney, and nose in male and female mice. Decreased incidences of mononuclear cell
leukemia occurred in exposed groups of rats; the incidence of testicular interstitial cell
adenoma was decreased in exposed male rats. [tables: see text]