2-Methylimidazole is used in the manufacture of pharmaceuticals, photographic chemicals, dyes and pigments, agricultural chemicals, and rubber. It has been identified as a by-product in foods and has been detected in mainstream and sidestream tobacco smoke. 2-Methylimidazole was nominated by the National Cancer Institute for a long-term study because of the high potential for human exposure and a lack of carcinogenicity studies in rodents. Male and female F344/N rats and B6C3F1 mice were exposed to 2-methylimidazole (99.5% pure) in feed for 2 years. Fifteen-day and 14-week toxicity studies of 2-methylimidazole in F344/N rats and B6C3F1 mice are reported in NTP Toxicity Report No. 67 (NTP, 2004). Genetic toxicity studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood.
Two-year study in rats
Groups of 60 male and 60 female rats were fed diets containing 0, 300, 1,000, or 3,000 ppm 2-methylimidazole (males) or 0, 1,000, 2,500, or 5,000 ppm 2-methylimidazole (females) (equivalent to average daily doses of approximately 13, 40, or 130 mg 2-methylimidazole/kg body weight to males and 50, 120, or 230 mg/kg to females) for 106 weeks. Ten male and 10 female rats were necropsied at 6 months. Additional groups of 20 male and 20 female special study rats were exposed to the same concentrations for 8 days or 14 weeks and were evaluated for clinical chemistry, liver enzyme activity, and organ weights. Survival of 2,500 ppm females was significantly less than that of the controls. The mean body weights of 3,000 ppm males and 2,500 and 5,000 ppm females were generally less than those of the controls during most of the study. Feed consumption by 5,000 ppm females was less than that by the control group.
The hematology results at 6 months indicated that exposure of rats to 2-methylimidazole induced a decreased erythron that was characterized as microcytic, normochromic, and nonresponsive. The thyroid hormone data indicated that rats administered 2-methylimidazole developed alterations in thyroid hormone concentrations; serum thyroxine and triiodothyronine concentrations were decreased, and thyroid stimulating hormone levels were increased. In general, the thyroid hormone effects were most pronounced early in the study and ameliorated with time. The results for the tissue enzyme content analyses of these 2-year feed studies indicated that exposure of rats to 2-methylimidazole induced an increase in total hepatic UDP-glucuronosyltransferase at all time points evaluated through 6 months. The thyroid gland weights of 3,000 ppm males and 2,500 and 5,000 ppm females were significantly increased at 6 months.
At 6 months, two 5,000 ppm female rats had a thyroid gland follicular cell adenoma. The incidences of follicular cell adenoma, follicular cell carcinoma, and adenoma or carcinoma (combined) in the thyroid gland of 5,000 ppm females were significantly greater that those in the controls at 2 years. The incidence of follicular cell adenoma or carcinoma (combined) in the thyroid gland occurred with a positive trend in males. The incidences of follicular cell hyperplasia in all exposed groups of rats were significantly increased at 6 months and 2 years. The incidences of follicle mineralization of the thyroid gland in all exposed groups, except 300 ppm males at 6 months and in 1,000 and 5,000 ppm females at 2 years, were significantly greater than those of the controls.
In the liver, the incidences of hepatocellular adenoma or carcinoma (combined) in the two highest exposure groups of males and females exceeded the historical ranges for controls, and the incidences of hepatocellular adenoma in females occurred with a positive trend. The incidences of bile duct hyperplasia and granulomatous inflammation were increased in females, as were those of mixed cell focus in males and females. The incidence of granulomatous inflammation of the spleen in 5,000 ppm females was significantly increased. Lower body weights of female rats exposed to 5,000 ppm likely contributed to the decreased incidences of mammary gland fibroadenoma, pituitary gland adenoma, and clitoral gland adenoma in this group.
Two-year study in mice
Groups of 60 male and 60 female mice were fed diets containing 0, 625, 1,250, or 2,500 ppm 2-methylimidazole (equivalent to average daily doses of approximately 75, 150, or 315 mg/kg to males and 80, 150, or 325 mg/kg to females) for 105 weeks. Ten male and 10 female mice were necropsied at 6 months. Additional groups of 20 male and 20 female special study mice were exposed to the same concentrations for 8 days or 14 weeks and were evaluated for clinical chemistry, liver enzyme activity, and organ weights.
Survival of all exposed groups of mice was similar to that of the control groups. The mean body weights of 1,250 and 2,500 ppm males and 2,500 ppm females were less than those of the controls during most of the study. Feed consumption by all exposed groups of mice was similar to that by the control groups.
The hematology results at 6 months indicated that exposure of mice to 2-methylimidazole induced a decreased erythron that was characterized as macrocytic, normochromic to hypochromic, and responsive. The thyroid gland weights of 2,500 ppm male and female mice and 1,250 ppm females were increased at 6 months.
The incidence of follicular cell adenoma in the thyroid gland of 2,500 ppm males was significantly greater than that in the control group at 2 years. Follicular cell hypertrophy of the thyroid gland occurred in most exposed mice at 6 months, and the incidences of this lesion were significantly increased in the 1,250 and 2,500 ppm groups at 2 years; the incidences of follicular cell hyperplasia were significantly increased in 2,500 ppm males and females at 2 years.
The liver weights of 2,500 ppm female mice were significantly increased at 6 months. The incidences of hepatocellular adenoma occurred with positive trends in males and females and the incidences were significantly increased in the 2,500 ppm groups. The incidence of hepatocellular carcinoma was significantly increased in 1,250 ppm males and exceeded the historical control range in 2,500 ppm males. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in all exposed groups of males. The incidences of hepatocellular karyomegaly in 2,500 ppm males at 6 months and in 1,250 and 2,500 ppm males at 2 years, of hepatocellular cytoplasmic alteration in 1,250 and 2,500 ppm males at 2 years, and Kupffer cell pigmentation in 2,500 ppm males at 2 years were significantly increased.
In the spleen, the incidences of hematopoietic cell proliferation in all exposed groups of males and in 2,500 ppm females were significantly increased at 6 months and 2 years. Pigmentation was present in most 1,250 and 2,500 ppm mice at 6 months, and the incidences of this lesion were significantly increased in all exposed groups of males and in 1,250 and 2,500 ppm females at 2 years. The incidences of bone marrow hyperplasia were significantly increased in 1,250 and 2,500 ppm male mice at 2 years. Renal proximal tubule pigmentation was present in most 2,500 ppm male mice at 6 months and 2 years. The responses in the spleen, bone marrow, and kidney were considered to be related to the responsive anemia. In males, the incidences of chronic active inflammation of the epididymis at 1,250 and 2,500 ppm, sperm granuloma at 2,500 ppm, and of germinal epithelial atrophy of the testis at 1,250 and 2,500 ppm were significantly increased at 2 years.
Genetic toxicology
2-Methylimidazole was negative in the S. typhimurium mutation assay when tested in strains TA97, TA98, TA100, and TA1535, with and without S9 activation enzymes. Testing of 2-methylimidazole in vivo for induction of chromosomal damage, as measured by micronucleated erythrocyte frequency, produced mixed results. When administered by intraperitoneal injection three times at 24-hour intervals, 2-methylimidazole produced negative results in bone marrow micronucleus tests in rats and mice. However, in the 14-week study of 2-methylimidazole, a significant exposure-related increase in the frequency of micronucleated normochromatic erythrocytes was noted in peripheral blood of male and female mice. Exposure concentration-related increases in the percentage of micronucleated polychromatic erythrocytes in peripheral blood was also seen in male and female mice in the 14-week study.
Conclusions
Under the conditions of this 2-year feed study, there was some evidence of carcinogenic activity of 2-methylimidazole in male F344/N rats based on increased incidences of thyroid gland follicular cell neoplasms. The increased incidences of hepatocellular neoplasms in males may have been related to exposure. There was clear evidence of carcinogenic activity of 2-methylimidazole in female F344/N rats based on increased incidences of thyroid gland follicular cell neoplasms. The increased incidences of hepatocellular adenoma in females may have been related to exposure. There was some evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of thyroid gland follicular cell adenoma and hepatocellular neoplasms. There was some evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of hepatocellular adenoma.
Exposure to 2-methylimidazole resulted in nonneoplastic lesions in the thyroid gland and liver of male rats; the thyroid gland, liver, and spleen of female rats; the thyroid gland, liver, spleen, bone marrow, kidney, epididymis and testes of male mice; and the thyroid gland and spleen of female mice.