Pyridine is used as a denaturant in alcohol and anti freeze mixtures, as a solvent for paint, rubber, and polycarbonate resins, and as an intermediate in the manufacture of insecticides, herbicides, and fungicides. It is used in the production of piperidine, an intermediate in the manufacture of rubber and mepiquat chloride, and as an intermediate and solvent in the preparation of vitamins and drugs, dyes, textile water repellants, and flavoring agents in food. Pyridine was nominated for study because of its large production volume and its use in a variety of food, medical, and industrial products. Male and female F344/N rats, male Wistar rats, and male and female B6C3F1 mice were exposed to pyridine (approximately 99% pure) in drinking water for 13 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse bone marrow cells.
Thirteen-week study in F344/N rats
Groups of 10 male and 10 female F344/N rats were exposed to pyridine in drinking water at concentrations of 0, 50, 100, 250, 500, or 1,000 ppm (equivalent to average daily doses of 5, 10, 25, 55, or 90 mg pyridine/kg body weight). Two females exposed to 1,000 ppm died during week 1. Final mean body weights of 1,000 ppm males and females and 500 ppm females were significantly less than controls. Water consumption by female rats exposed to 1,000 ppm was less than that by controls. At study termination, evidence of anemia persisted in the 500 and 1,000 ppm males and all exposed groups of females. There was evidence of hepatocellular injury and/or altered hepatic function demonstrated by increased serum alanine aminotransferase and sorbitol dehydrogenase activities and bile acid concentrations in 500 and 1,000 ppm rats. The estrous cycle length of 1,000 ppm females was significantly longer than that of the controls. Liver weights of males and females exposed to 250 ppm or greater were significantly greater than controls. In the liver, the incidences of centrilobular degeneration, hypertrophy, chronic inflammation, and pigmentation were generally increased in 500 and 1,000 ppm males and females relative to controls. In the kidney, the incidences of granular casts and hyaline degeneration (hyaline droplets) were significantly increased in 1,000 ppm males and slightly increased in 500 ppm males; these lesions are consistent with 2u-globulin nephropathy. Additionally, there were increased incidences and/or severities of protein casts, chronic inflammation, mineralization, and regeneration primarily in 500 and 1,000 ppm males.
Thirteen-week study male Wistar rats
Groups of 10 male Wistar rats were exposed to pyridine in drinking water at concentrations of 0, 50, 100, 250, 500, or 1,000 ppm (equivalent to average daily doses of 5, 10, 30, 60, or 100 mg/kg). One male rat exposed to 500 ppm died during week 1. Final mean body weights of rats exposed to 250, 500, or 1,000 ppm were significantly less than those of the controls. Water consumption by rats exposed to 1,000 ppm was lower than that by controls. There was evidence of hepatocellular injury and/or altered hepatic function in the 500 and 1,000 ppm groups, similar to that observed in the 13-week study in F344/N rats. Incidences of centrilobular degeneration, hypertrophy, chronic inflammation, and pigmentation in the liver of rats exposed to 500 or 1,000 ppm were significantly increased relative to controls.
Thirteen-week study in mice
Groups of 10 male and 10 female B6C3F1 mice were exposed to pyridine in drinking water at concentrations of 0, 50, 100, 250, 500, or 1,000 ppm (equivalent to average daily doses of 10, 20, 50, 85, or 160 mg/kg for males and 10, 20, 60, 100, or 190 mg/kg for females). One female mouse exposed to 250 ppm died during week 2. Final mean body weights of female mice exposed to 1,000 ppm were significantly less than those of controls. Water consumption by exposed female mice was lower than that by controls at week 1 but generally slightly higher than controls at week 13. Sperm motility in exposed male mice was significantly decreased relative to controls. Liver weights were significantly increased relative to controls in males exposed to 100 ppm or greater and in 250 and 500 ppm females. No chemical-related lesions were observed in male or female mice.
Two-year study in F344/N rats
Groups of 50 male and 50 female F344/N rats were exposed to pyridine in drinking water at concentrations of 0, 100, 200, or 400 ppm (equivalent to average daily doses of 7, 14, or 33 mg/kg) for 104 (males) or 105 (females) weeks.
Survival, body weights, and water consumption
Survival of exposed males and females was similar to that of controls. Mean body weights of 400 ppm males and females were generally less than those of the controls throughout the study, and those of 200 ppm males and females were less during the second year of the study. Water consumption by males and females exposed to 200 or 400 ppm was generally greater than that by controls.
Pathology findings
Incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) in male rats exposed to 400 ppm were significantly increased compared to controls and exceeded the historical control ranges. The findings from an extended evaluation (step section) of the kidneys did not reveal additional carcinomas, but additional adenomas were observed in each group of males. In the standard evaluation, an increased incidence of renal tubule hyperplasia was observed in 400 ppm males compared to controls. Incidences of mononuclear cell leukemia in female rats were significantly increased in the 200 and 400 ppm groups, and the incidence in the 400 ppm group exceeded the historical control range.
Exposure concentration-related nonneoplastic liver lesions were observed in males and females, and the incidences were generally increased in groups exposed to 400 ppm. These included centrilobular cytomegaly, cytoplasmic vacuolization, periportal fibrosis, fibrosis, centrilobular degeneration and necrosis, and pigmentation. Bile duct hyperplasia occurred more often in exposed females than in controls.
Two-year study in male Wistar rats
Groups of 50 male Wistar rats were exposed to pyridine in drinking water at concentrations of 0, 100, 200, or 400 ppm (equivalent to average daily doses of 8, 17, or 36 mg/kg) for 104 weeks.
Survival, body weights, and water consumption
Survival of rats exposed to 200 or 400 ppm was significantly less than that of the controls. Mean body weights of rats exposed to 100, 200, or 400 ppm were significantly less than controls. Water consumption was similar by control and exposed rats.
Pathology findings
The incidence of testicular interstitial cell adenoma in rats exposed to 400 ppm was significantly increased compared to controls. Incidences of interstitial cell hyperplasia were observed in control and exposed groups and were slightly, but not significantly, increased in rats exposed to 200 or 400 ppm.
Severity of nephropathy was marked in all groups, and additional evidence of kidney disease, including mineralization in the glandular stomach, parathyroid gland hyperplasia, and fibrous osteodystrophy, was observed in 100 and 200 ppm rats. The incidences of hepatic centrilobular degeneration and necrosis, fibrosis, periportal fibrosis, and/or pigmentation were increased in one or more exposed groups.
Two-year study in mice
Groups of 50 male B6C3F1 mice were exposed to pyridine in drinking water at concentrations of 0, 250, 500, or 1,000 ppm (equivalent to average daily doses of 35, 65, or 110 mg/kg) for 104 weeks, and groups of 50 female B6C3F1 mice were exposed to pyridine in drinking water at concentrations of 0, 125, 250, or 500 ppm (equivalent to average daily doses of 15, 35, or 70 mg/kg) for 105 weeks.
Survival, body weights, and water consumption
Survival of exposed males and females was similar to that of the controls. Mean body weights of 250 and 500 ppm females were less than controls. Water consumption by males exposed to 250 or 500 ppm was generally greater than that by controls during the last year of the study; male mice exposed to 1,000 ppm consumed less water than controls throughout the study. Water consumption by exposed females was generally lower than that by controls during the first year of the study, but greater than controls during the second year.
Pathology findings
Hepatocellular neoplasms, including hepatoblastomas, in exposed male and female mice were clearly related to pyridine exposure. Additionally, many mice had multiple hepatocellular neoplasms. The incidences of hepatocellular neoplasms in exposed males and females generally exceeded the historical control ranges for drinking water studies. Neoplasms from control mice, 1,000 ppm males, and 500 ppm females were negative when stained for p53 protein.
Genetic toxicology
Pyridine was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 or in L5178Y mouse lymphoma cells, with or without S9 metabolic activation, and it did not induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9. Pyridine was tested for induction of sex-linked recessive lethal mutations in adult male Drosophila melanogaster, and mixed results were obtained. In one experiment, administration by injection gave negative results, but feeding produced an equivocal response. A second experiment generated negative results by injection and feeding. A third experiment showed significant increases in sex-linked recessive lethal mutations in flies treated with pyridine by injection but not by feeding. Overall, results of the sex-linked recessive lethal mutations test in Drosophila melanogaster were considered negative by feeding and equivocal by injection. Results of a single reciprocal translocation test in male Drosophila melanogaster were negative. No induction of chromosomal aberrations or micronuclei was noted in bone marrow cells of male mice administered pyridine via intraperitoneal injection.
Conclusions
Under the conditions of these 2-year drinking water studies, there was some evidence of carcinogenic activity of pyridine in male F344/N rats based on increased incidences of renal tubule neoplasms. There was equivocal evidence of carcinogenic activity of pyridine in female F344/N rats based on increased incidences of mononuclear cell leukemia. There was equivocal evidence of carcinogenic activity in male Wistar rats based on an increased incidence of interstitial cell adenoma of the testis. There was clear evidence of carcinogenic activity of pyridine in male and female B6C3F1 mice based on increased incidences of malignant hepatocellular neoplasms.
In F344/N rats, exposure to pyridine resulted in increased incidences of centrilobular cytomegaly and degeneration, cytoplasmic vacuolization, and pigmentation in the liver of males and females; periportal fibrosis, fibrosis, and centrilobular necrosis in the liver of males; and bile duct hyperplasia in females. In male Wistar rats, pyridine exposure resulted in increased incidences of centrilobular degeneration and necrosis, fibrosis, periportal fibrosis, and pigmentation in the liver, and, secondary to kidney disease, mineralization in the glandular stomach and parathyroid gland hyperplasia.