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Abstract for TR-461

Toxicology and Carcinogenesis Studies of Nitromethane in F344/N Rats and B6C3F1 Mice (Inhalation Studies)

CASRN: 75-52-5
Chemical Formula: CH3NO2
Molecular Weight: 61.04
Synonyms/Common Names: Nitrocarbol
Report Date: February 1997

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Abstract

Nitromethane is used as a rocket and engine fuel; as a synthesis intermediate for agricultural fumigants, biocides, and other products; as a solvent; and as an explosive in mining, oil-well drilling, and seismic exploration. It has been detected in air, in surface and drinking water, and in cigarette smoke. Nitromethane was studied because of the potential for widespread human exposure and because it is structurally related to the carcinogens 2-nitropropane and tetranitromethane. Male and female F344/N rats and B6C3F1 mice received nitromethane (purity 98% or greater) by inhalation for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and peripheral blood erythrocytes of mice.

16-Day Study in Rats

Groups of five male and five female rats were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 16 days. All rats survived until the end of the study. The mean body weight gain of male rats in the 1,500 ppm group was slightly but significantly less than that of the controls; the final mean body weights and mean body weight gains of exposed females were similar to those of the controls. Clinical findings in all male and female rats in the 1,500 ppm groups included increased preening, rapid breathing, hyperactivity early in the study, and hypoactivity and loss of coordination in the hindlimbs near the end of the study. The relative liver weights of all exposed groups of male rats and the absolute and relative liver weights of females exposed to 375 ppm or greater were significantly greater than those of the controls.

Minimal to mild degeneration of the olfactory epithelium was observed in the nose of males and females exposed to 375 ppm or greater. Sciatic nerve degeneration was present in all male and female rats exposed to 375 ppm or greater; rats exposed to 750 or 1,500 ppm also had reduced myelin around sciatic axons.

16-Day Study in Mice

Groups of five male and five female mice were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 16 days. All mice survived to the end of the study. The final mean body weights and weight gains of exposed males and females were similar to those of the controls. Clinical findings included hypoactivity and tachypnea in male and female mice in the 1,500 ppm groups. Absolute and relative liver weights of male mice in the 750 and 1,500 ppm groups and female mice in all exposed groups and the relative liver weight of males in the 375 ppm group were significantly greater than those of the controls. Degeneration of the olfactory epithelium of the nose was observed microscopically in all males and females exposed to 375 ppm or greater; this lesion was of minimal severity in males and minimal to mild severity in females.

13-Week Study in Rats

Groups of 10 male and 10 female rats were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 13 weeks. All rats survived to the end of the study. The final mean body weight and weight gain of male rats in the 1,500 ppm group were significantly less than those of the controls. Clinical findings included hindlimb paralysis in rats in the 750 and 1,500 ppm groups.

Inhalation exposure of rats to nitromethane resulted in an exposure concentration-dependent, microcytic, responsive anemia; anemia was most pronounced in males and females exposed to 375 ppm or greater. The presence of schistocytes, Heinz bodies, and spherocytes and increased mean cell hemoglobin concentration and methemoglobin concentration were evidence that a hemolytic process was occurring; this hemolytic process could have accounted, in part, for the anemia. Thrombocytosis accompanied the anemia and would be consistent with a reactive bone marrow or could have been due to the erroneous inclusion of small erythrocyte fragments as part of the platelet count. On day 23, transient decreases in serum triiodothyronine, thyroxine, and free thyroxine were observed in male rats exposed to 375 ppm or greater and female rats exposed to 750 or 1,500 ppm. There was little or no pituitary response to the thyroid hormone decreases, as evidenced by the lack of significantly increased concentrations of thyroid-stimulating hormone in exposed rats.

No biologically significant differences in organ weights were observed. The forelimb and hindlimb grip strengths of males in the 1,500 ppm group were significantly less than those of the controls. The hindlimb grip strengths of females in the 750 and 1,500 ppm groups were also significantly less than the control value.

Minimal to mild hyperplasia of the bone marrow was observed microscopically in male rats in the 750 and 1,500 ppm groups and in females exposed to 188 ppm or greater. Nasal lesions in exposed males and females included olfactory epithelial degeneration in males and females exposed to 375 ppm or greater and in one female exposed to 188 ppm and respiratory epithelial hyaline droplets and goblet cell hyperplasia in males and females in the 750 and 1,500 ppm groups; the severity of nasal lesions in males and females was minimal to mild. Males and females exposed to 375 ppm or greater had minimal to mild degeneration of the sciatic nerve and the lumbar spinal cord.

13-Week Study in Mice

Groups of 10 male and 10 female mice were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 13 weeks. All mice survived to the end of the study. The final mean body weights and weight gains of exposed mice were generally similar to those of the controls. There were no treatment-related clinical findings.

The absolute right kidney weights of all groups of exposed male mice except the 1,500 ppm group and of females exposed to 188 ppm or greater and the relative right kidney weights of all groups of exposed males and of females in the 750 and 1,500 ppm groups were significantly greater than those of the controls. The absolute liver weight of male mice in the 750 ppm group and the relative liver weights of males exposed to 375 ppm or greater were significantly greater than those of the controls.

Olfactory epithelial degeneration and respiratory epithelial hyaline droplets were observed microscopically in all male and female mice exposed to 375 ppm or greater. Degeneration also occurred in females in the 188 ppm group, and hyaline droplets occurred in females in the 94 and 188 ppm groups. The average severity of the nasal lesions ranged from minimal to mild in males. In females, the average severity of olfactory epithelial degeneration ranged from minimal to mild and the severity of respiratory epithelial hyaline droplets ranged from minimal to moderate. All males and nine females in the 1,500 ppm groups also had minimal extramedullary hematopoiesis of the spleen.

2-Year Study in Rats

Groups of 50 male and 50 female rats were exposed to 0, 94, 188, or 375 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 103 weeks.

Survival,Body Weights, and Clinical Findings

There were no significant differences in survival rates between exposed and control male or female rats. The mean body weight of females in the 375 ppm group was slightly greater than that of the control group; the mean body weights of exposed males were generally similar to the mean body weight of the controls throughout the study. Clinical findings were consistent with incidences of mammary gland neoplasms in females exposed to 188 or 375 ppm; no hindlimb paralysis, as occurred in rats in the 13-week study, was observed in male or female rats in the 2-year study.

Pathology Findings

The incidences of mammary gland fibroadenoma and fibroadenoma, adenoma, or carcinoma (combined) in female rats in the 188 and 375 ppm groups were significantly greater than those in the controls. Additionally, the incidences of mammary gland carcinoma in the 375 ppm group were significantly greater than those in the controls.

2-Year Study in Mice

Groups of 50 male and 50 female mice were exposed to 0, 188, 375, or 750 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 103 weeks.

Survival,Body Weights, and Clinical Findings

The survival rate of females in the 750 ppm group was marginally greater than that of the controls. The mean body weights of exposed females were generally slightly greater than the mean body weights of the controls during the study but were generally similar to the mean body weight of the controls at the end of the study. The mean body weights of exposed males were similar to those of the controls throughout the study. Clinical findings included swelling around the eyes and exophthalmos in exposed males and females; these findings were coincident with harderian gland neoplasms.

Pathology Findings

The incidences of harderian gland adenoma and adenoma or carcinoma (combined) in exposed mice increased with increasing exposure concentration and were significantly greater in males and females in the 375 and 750 ppm groups than those in the controls. The incidences of harderian gland carcinoma in males and females in the 375 and 750 ppm groups were also slightly greater than those in the controls.

Female mice in the 188 and 750 ppm groups had significantly greater incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) than the controls. The incidences of liver eosinophilic focus increased with increasing exposure concentration, and the incidences in the 375 and 750 ppm groups were significantly greater than the control incidence.

The incidences of alveolar/bronchiolar carcinoma in male mice in the 750 ppm group and female mice in the 375 ppm group were significantly greater than those in the controls. Females in the 750 ppm group also had a significantly greater incidence of alveolar/bronchiolar adenoma or carcinoma (combined) and a slightly greater incidence of alveolar/bronchiolar adenoma than the controls. Females in the 375 ppm group had a significantly greater incidence of cellular infiltration of histiocytes in the lung than the controls.

The incidences of degeneration and metaplasia of the olfactory epithelium and hyaline degeneration of the respiratory epithelium were significantly greater in exposed male and female mice than those in the controls. Additionally, males in the 375 and 750 ppm groups had significantly greater incidences of inflammation of the nasolacrimal duct than did the controls.

Genetic Toxicology

Nitromethane was not mutagenic in any tests performed by the NTP. It did not induce mutations in Salmonella typhimurium, with or without S9 metabolic activation, and no induction of sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells exposed to nitromethane was noted with or without S9. No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood samples of male and female mice at the end of the 13-week inhalation study of nitromethane.

Conclusions

Under the conditions of these 2-year inhalation studies, there was no evidence of carcinogenic activity of nitromethane in male F344/N rats exposed to 94, 188, or 375 ppm. There was clear evidence of carcinogenic activity of nitromethane in female F344/N rats based on increased incidences of mammary gland fibroadenomas and carcinomas. There was clear evidence of carcinogenic activity of nitromethane in male B6C3F1 mice based on increased incidences of harderian gland adenomas and carcinomas. There was clear evidence of carcinogenic activity in female B6C3F1 mice, based on increased incidences of liver neoplasms (primarily adenomas) and harderian gland adenomas and carcinomas. Increased incidences of alveolar/bronchiolar adenomas and carcinomas in male and female mice exposed to nitromethane were also considered to be related to chemical administration.

Exposure to nitromethane by inhalation for 2 years resulted in increased incidences of nasal lesions including degeneration and metaplasia of the olfactory epithelium and degeneration of the respiratory epithelium in male and female mice.

Studies

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Nitromethane
  Male F344/N Rats Female F344/N Rats Male B6C3F1 Mice Female B6C3F1 Mice
Exposure concentrations 0, 94, 188, or 375 ppm 0, 94, 188, or 375 ppm <0, 188, 375, or 750 ppm 0, 188, 375, or 750 ppm
Body weights Exposed groups similar to controls 375 ppm group slightly greater than controls Exposed groups similar to controls Exposed groups similar to controls
2-Year survival rates 13/50, 16/50, 14/50, 8/50 28/50, 19/50, 30/50, 23/50 31/50, 36/50, 30/50, 29/50 25/50, 28/50, 26/50, 36/50
Nonneoplastic effects None None Nose: olfactory epithelium, degeneration (0/50, 10/49, 50/50, 50/50); olfactory epithelium, metaplasia (0/50, 1/49, 41/50, 49/50); respiratory epithelium, hyaline degeneration (5/50, 5/49, 50/50, 50/50) Nose: olfactory epithelium, degeneration (0/50, 22/49, 50/50, 50/50); olfactory epithelium, metaplasia (0/50, 2/49, 46/50, 48/50); respiratory epithelium, hyaline degeneration (16/50, 39/49, 50/50, 50/50)
Neoplastic effects None Mammary gland: fibroadenoma (19/50, 21/50, 33/50, 36/50); carcinoma (2/50, 7/50, 1/50, 11/50); fibroadenoma, adenoma, or carcinoma (21/50, 25/50, 34/50, 41/50) Harderian gland: adenoma (9/50, 10/50, 19/50, 32/50); carcinoma (1/50, 1/50, 6/50, 5/50); adenoma or carcinoma (10/50, 11/50, 25/50, 37/50)
Lung: alveolar/ bronchiolar carcinoma (2/50, 3/50, 3/50, 11/50); alveolar/bronchiolar adenoma or carcinoma (13/50, 13/50, 12/50, 20/50)
Harderian gland: adenoma (5/50, 7/50, 16/50, 19/50); adenoma or carcinoma (6/50, 9/50, 20/50, 21/50)
Liver: hepatocellular adenoma (14/50, 25/49, 17/49, 35/50); hepatocellular adenoma or carcinoma (19/50, 34/49, 22/49, 40/50)
Lung: alveolar/ bronchiolar adenoma (3/50, 3/50, 2/49, 9/50); alveolar/bronchiolar carcinoma (0/50, 3/50, 5/49, 3/50); alveolar/bronchiolar adenoma or carcinoma (3/50, 6/50, 6/49, 12/50)
Level of evidence
of carcinogenic activity
No evidence Clear evidence Clear evidence Clear evidence
Genetic Toxicology of Nitromethane
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: Negative with and without S9
Chromosomal aberrations Cultured Chinese hamster ovary cells in vitro: Negative with and without S9
Micronucleated erythrocytes Mouse peripheral blood in vivo: Negative