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Abstract for TR-504

Toxicology and Carcinogenesis Studies of o-Nitrotoluene in F344/N Rats and B6C3F1 Mice (Feed Studies)

CASRN: 88-72-2
Chemical Formula: C7H7NO2
Molecular Weight: 137.14
Synonyms/Common Names: 2-Methylnitrobenzene; o-methylnitrobenzene; 2-nitrotoluene; 4-nitrotoluol
Report Date: May 2002

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Abstract

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 the National Institute for Occupational Safety and Health and the NTP based on it's 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 2,000 ppm 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 bone marrow and spleen and of hyperplasia of the mandibular lymph node (females) 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 concentrations 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

o-Nitrotoluene was not mutagenic in any of several strains of S. typhimurium, with or without metabolic activation enzymes (S9). Sister chromatid exchanges were significantly increased in cultured Chinese hamster ovary cells following exposure to o-nitrotoluene in the presence of S9; an equivocal response was seen without S9. o-Nitrotoluene did not induce chromosomal aberrations in cultured Chinese hamster ovary cells,with or without S9. o-Nitrotoluene did not induce a significant increase in the frequency of micronuclei in bone marrow polychromatic erythrocytes of male rats or male mice when administered by intraperitoneal injection. Results of a peripheral blood micronucleus test were equivocal for male mice and negative for female mice administered o-nitrotoluene in feed for 13 weeks.

Conclusions

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.

Studies

Summary of the 2-Year Carcinogenesis Studies of o-Nitrotoluene
 

Male
F344/N Rats

Female
F344/N Rats

Male
B6C3F1 Mice

Female
B6C3F1 Mice

Concentrations in feed

0, 625, 1,250, or 2,000 ppm and 2,000 and 5,000 ppm stop-exposure

0, 625, 1,250, or 2,000 ppm

0, 1,250, 2,500, or 5,000 ppm

0, 1,250, 2,500, or 5,000 ppm

Body weights

1,250, 2,000 and 2,000 and 5,000 ppm stop-exposure groups less than the control group

2,000 ppm group less than the control group

Exposed groups less than the control group  

2,500 and 5,000 ppm groups less than the control group

Survival rates

39/60, 18/60, 3/60, 0/60, 11/60, 0/60

47/60, 47/60, 39/60, 33/60

52/60, 34/60, 0/60, 0/60

52/60, 46/60, 47/60, 5/60

Nonneoplastic effects

Liver: eosinophilic focus (7/60, 18/60, 29/60, 24/60, 15/60, 13/60); mixed cell focus (5/60, 7/60, 12/60, 6/60, 12/60, 8/60); clear cell focus (29/60, 29/60, 34/60, 31/60, 30/60, 34/60); mixed cell cellular infiltration (1/60, 5/60, 11/60, 20/60, 15/60, 33/60)

Bone marrow: hyperplasia (2/60, 25/60, 43/60, 45/60, 37/60, 33/60)

Spleen: hematopoietic cell proliferation (7/60, 33/60, 38/60, 47/60, 36/60, 35/60)

Lung: alveolar epithelial hyperplasia (2/60, 8/60, 3/60, 7/60, 15/60, 29/60)

Mammary gland: hyperplasia (14/60, 36/60, 30/60, 19/60)

Liver: eosinophilic focus (5/60, 12/59, 25/60, 32/60); mixed cell focus (6/60, 9/59, 11/60, 28/60); clear cell focus (16/60, 30/59, 28/60, 33/60); basophilic focus (51/60, 56/59, 60/60, 54/60)

Bone marrow: hyperplasia (2/60, 7/60, 15/60, 24/60)

Spleen: hematopoietic cell proliferation (22/60, 38/59, 48/60, 48/59)

Lung: alveolar epithelial hyperplasia (6/60, 14/60, 16/60, 9/60)

Lymph node (mandibular): lymphoid hyperplasia (3/60, 5/60, 6/59, 15/59)

Liver: eosinophilic focus (3/60, 14/59, 1/57, 1/60); basophilic focus (0/60, 6/59, 4/57, 0/60); necrosis (1/60, 15/59, 27/57, 30/60); focal hepatocyte syncytial alteration (16/60, 26/59, 43/57, 39/60)

Kidney: renal tubule pigmentation (1/58, 6/59, 32/58, 35/60)

Nose: olfactory epithelial degeneration (0/60, 36/60, 60/60, 60/60)

Liver: eosinophilic focus (2/60, 3/59, 6/59, 28/60); basophilic focus (1/60, 6/59, 2/59, 6/60) necrosis (3/60, 0/59, 2/59, 13/60); focal hepatocyte necrosis (0/60, 0/59, 0/59, 6/60); focal hepatocyte cytoplasmic vacuolization (1/60, 2/59, 2/59, 9/60)

Kidney: renal tubule pigmentation (0/59, 1/56, 3/58, 35/59)

Nose: olfactory epithelial degeneration (0/60, 28/60, 59/59, 57/57)

Neoplastic effects

Mesothelium: malignant mesothelioma (2/60, 20/60, 29/60, 44/60, 44/60, 54/60)

Skin (subcutaneous): lipoma (0/60, 4/60, 13/60, 13/60, 10/60, 12/60); fibroma (5/60, 46/60, 52/60, 59/60, 45/60, 52/60); fibrosarcoma (0/60, 7/60, 17/60, 20/60, 8/60, 12/60); fibroma or fibrosarcoma (5/60, 47/60, 55/60, 59/60, 47/60, 53/60)

Mammary gland: fibroadenoma (0/60, 7/60, 10/60, 2/60, 13/60, 20/60)

Liver: hepatocellular adenoma (2/60, 3/60, 3/60, 7/60, 3/60, 4/60); hepatocellular adenoma or carcinoma (3/60, 3/60, 3/60, 8/60, 3/60, 6/60); cholangiocarcinoma (0/60, 0/60, 0/60, 0/60, 0/60, 3/60); hepato- cholangiocarcinoma (0/60, 1/60, 0/60, 1/60, 0/60, 0/60)

Lung: alveolar/ bronchiolar adenoma (1/60, 5/60, 1/60, 2/60, 3/60, 8/60); alveolar/ bronchiolar adenoma or carcinoma: (2/60, 5/60, 1/60, 2/60, 3/60, 11/60)

Skin (subcutaneous): fibroma (3/60, 3/60, 18/60, 20/60); fibroma or fibrosarcoma (3/60, 3/60, 21/60, 22/60)

Mammary gland: fibroadenoma (23/60, 47/60, 52/60, 56/60)

Liver: hepatocellular adenoma (1/60, 0/59, 1/60, 6/60)

Circulatory system: hemangiosarcoma (4/60, 17/60, 55/60, 60/60)

Large intestine (cecum): carcinoma (0/60, 12/60, 9/60, 0/60)

Circulatory system: hemangiosarcoma (0/60, 2/60, 3/60, 50/60)

Large intestine (cecum): carcinoma (0/60, 1/60, 4/60, 3/60)

Liver: hepatocullular adenoma (7/60, 5/59, 19/59, 29/60); hepatocellular carcinoma (2/60, 4/59, 6/59, 16/60); hepatocellular adenoma or carcinoma (9/60, 9/59, 24/59, 39/60)

Decreased incidences

Mononuclear cell leukemia: (30/60, 21/60, 3/60, 3/60, 13/60, 1/60)

Testis: Interstitial cell adenoma (55/60, 53/60, 51/60, 46/60, 50/60, 27/50)

Mononuclear cell leukemia: (21/60, 6/60, 4/60, 5/60)

None

None

Level of evidence of carcinogenic activity

Clear evidence

Clear evidence

Clear evidence

Clear evidence

Genetic Toxicology of o-Nitrotoluene
Assay Test System Results
Bacterial Mutagenicity Salmonella typhimurium gene mutations: Negative in strains TA98, TA100, TA1535, and TA1537 with and without S9
Sister chromatid exchanges Cultured Chinese hamster ovary cells in vitro: Positive with S9; equivocal without S9
Chromosomal aberrations Cultured Chinese hamster ovary cells in vitro: Negative with and without S9
Micronucleated erythrocytes Rat bone marrow in vivo: Negative in two protocols
Micronucleated erythrocytes Mouse bone marrow in vivo: Negative
Micronucleated erythrocytes Mouse peripheral blood in vivo: Equivocal in male mice; negative in female mice