Toxicology and carcinogenesis studies of p-nitrotoluene (CAS no. 99-99-0) in F344/N rats and B6C3F(1) mice (feed studies).
p-Nitrotoluene is used to synthesize agricultural and rubber chemicals, azo and sulfur dyes, and dyes for cotton, wool, silk, leather, and paper. p-Nitrotoluene was nominated by the National Institute for Occupational Safety and Health and the NTP for study based on its considerable human exposure as well as the absence of long-term studies of its carcinogenicity in rodents. Male and female F344/N rats and B6C3F1 mice were exposed to p-nitrotoluene (greater than 99% pure) in feed for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, and rat and mouse bone marrow cells. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were fed diets containing 0, 1,250, 2,500, or 5,000 ppm p-nitrotoluene (equivalent to average daily doses of approximately 55, 110, or 240 mg p-nitrotoluene/kg body weight to males and 60, 125, or 265 mg/kg to females) for 105 or 106 weeks. Survival, Body Weights, and Feed Consumption: Survival of all exposed groups of rats was similar to that of the control groups. Mean body weights of 5,000 ppm male and 2,500 and 5,000 ppm female rats were less than those of the controls during most of the study; mean body weights of 1,250 ppm females were less during the second year of the study. Feed consumption by 5,000 ppm females was less than that by the controls during year 2 of the study. Biomarkers of Exposure: Two urinary metabolites were followed during the study as biomarkers of exposure. The ratios of p-nitrobenzoic acid to creatinine and of p-acetamidobenzoic acid to creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. Pathology Findings: The incidence of clitoral gland adenoma or carcinoma (combined) was significantly greater in 2,500 ppm females than that in the controls and exceeded the historical control ranges. The incidence of clitoral gland neoplasms was not increased in 5,000 ppm females, possibly because of the lower body weights in this group. The incidences of subcutaneous fibroma and of subcutaneous fibroma or fibrosarcoma (combined) in 2,500 ppm male rats were significantly increased and exceeded the historical control ranges. The incidences of several nonneoplastic kidney lesions were significantly increased in exposed groups of rats, and the severities of these lesions generally increased with increasing exposure concentration. In the spleen, incidences of hematopoietic cell proliferation and pigmentation were significantly increased in the 2,500 and 5,000 ppm groups. Significantly increased incidences of various types of altered cell foci in the liver of males and females were associated with exposure. Incidences of germinal epithelial atrophy of the testis in 5,000 ppm males and endometrial cystic hyperplasia of the uterus in 2,500 and 5,000 ppm females were significantly increased. The incidences of mononuclear cell leukemia were significantly decreased in all exposed groups except 1,250 ppm females. The incidence of interstitial cell adenoma of the testis in 5,000 ppm males was significantly decreased. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were fed diets containing 0, 1,250, 2,500, or 5,000 ppm p-nitrotoluene (equivalent to average daily doses of approximately 170, 345, or 690 mg/kg to males and 155, 315, or 660 mg/kg to females) for 105 or 106 weeks. Survival, Body Weights, and Feed Consumption: Survival of all exposed groups of male and female mice was similar to that of the control groups. Mean body weights of 5,000 ppm males and females were less than those of the control groups during most of the study. Mean body weights of 2,500 ppm males were less than those of the controls after week 92. Feed consumption by all exposed groups of mice was similar to that by the control groups. Pathology Findings: The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was significantly greater in 5,000 ppm male mice than in the controls, as was the incidence of alveolar epithelial hyperplasia in this group. The incidences of alveolar epithelial bronchiolization were significantly increased in all exposed groups of males and females.


RESULTS
p-Nitrotoluene was not mutagenic in any of several strains of S. typhimurium, with or without metabolic activation enzymes (S9). A positive response to p-nitrotoluene was observed in the L5178Y mouse lymphoma cell assay in trials with S9. Significantly increased sister chromatid exchange frequencies were observed in cultured Chinese hamster ovary cells treated with p-nitrotoluene with and without S9. Chromosomal aberrations were also induced in Chinese hamster ovary cells treated with p-nitrotoluene in the presence of S9; no increased aberrations were seen without S9. p-Nitrotoluene did not induce a significant reproducible increase in the frequency of micronuclei in bone marrow polychromatic erythrocytes of male rats or male mice when administered by intraperitoneal injection.


CONCLUSIONS
Under the conditions of these 2-year feed studies there was equivocal evidence of carcinogenic activity* of p-nitrotoluene in male F344/N rats based on increased incidences of subcutaneous skin neoplasms. There was some evidence of carcinogenic activity of p-nitrotoluene in female F344/N rats based on increased incidences of clitoral gland neoplasms. There was equivocal evidence those of the control groups during most of the study. Mean body weights of 2,500 ppm males were less than those of the controls after week 92. Feed consumption by all exposed groups of mice was similar to that by the control groups. Pathology Findings: The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was significantly greater in 5,000 ppm male mice than in the controls, as was the incidence of alveolar epithelial hyperplasia in this group. The incidences of alveolar epithelial bronchiolization were significantly increased in all exposed groups of males and females.


RESULTS
p-Nitrotoluene was not mutagenic in any of several strains of S. typhimurium, with or without metabolic activation enzymes (S9). A positive response to p-nitrotoluene was observed in the L5178Y mouse lymphoma cell assay in trials with S9. Significantly increased sister chromatid exchange frequencies were observed in cultured Chinese hamster ovary cells treated with p-nitrotoluene with and without S9. Chromosomal aberrations were also induced in Chinese hamster ovary cells treated with p-nitrotoluene in the presence of S9; no increased aberrations were seen without S9. p-Nitrotoluene did not induce a significant reproducible increase in the frequency of micronuclei in bone marrow polychromatic erythrocytes of male rats or male mice when administered by intraperitoneal injection.


CONCLUSIONS
Under the conditions of these 2-year feed studies there was equivocal evidence of carcinogenic activity of p-nitrotoluene in male F344/N rats based on increased incidences of subcutaneous skin neoplasms. There was some evidence of carcinogenic activity of p-nitrotoluene in female F344/N rats based on increased incidences of clitoral gland neoplasms. There was equivocal evidence in male and female rats, testis in male rats, and lung in male and female mice. Decreased incidences of mononuclear cell leukemia in male and female rats and testicular interstitial cell adenoma in male rats were attributed to exposure to p-nitrotoluene.
D000284:Administration, Oral; D000818:Animals; D016466:CHO Cells; D015197:Carcinogenicity Tests; D002273:Carcinogens / Q000008:administration & dosage / Q000378:metabolism / Q000633:toxicity*; D006224:Cricetinae; D004032:Diet; D004305:Dose-Response Relationship, Drug; D005260:Female; D066298:In Vitro Techniques; D007274:Injections, Intraperitoneal; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009152:Mutagenicity Tests; D009153:Mutagens / Q000008:administration & dosage / Q000378:metabolism / Q000633:toxicity*; D009374:Neoplasms, Experimental / Q000139:chemically induced*; D051381:Rats; D011916:Rats, Inbred F344; D014050:Toluene / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity*
D002273:Carcinogens; D009153:Mutagens; D014050:Toluene; C017572:4-nitrotoluene