Methacrylonitrile is an unsaturated aliphatic nitrile. It is widely used in the preparation of homopolymers and copolymers, elastomers, and plastics and as a chemical intermediate in the preparation of acids, amides, amines, esters, and other nitriles. Methacrylonitrile is also used as a replacement for acrylonitrile in the manufacture of an acrylonitrile/butadiene/styrene-like polymer that provides improved barrier properties to gases such as carbon dioxide in carbonated beverage containers. Methacrylonitrile was nominated for study by the National Cancer Institute because of the potential for human exposure, structural similarity to the known carcinogen acrylonitrile, demonstrated toxic effects in several animal species, and a lack of toxicity and carcinogenicity data. Male and female F344/N rats and B6C3F1 mice received methacrylonitrile (greater than 99% pure) in deionized water by gavage for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Drosophila melanogaster, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 3, 10, or 30 mg methacrylonitrile/kg body weight in deionized water by gavage, 5 days per week for 104 to 105 weeks. This dose selection was based on the results of an NTP 13-week gavage study where rats were administered 0, 7.5, 15, 30, 60, or 120 mg methacrylonitrile/kg body weight. In the 13-week study, clinical signs of toxicity were observed early in the study. In addition, lower mean body weights and survival were observed in male and female rats administered 60 or 120 mg/kg. In the 2-year study, survival of all dosed groups of rats was similar to that of the vehicle control groups. Mean body weights of the 30 mg/kg groups were less than those of the vehicle controls after weeks 21 and 37 for males and females, respectively. No changes in the incidences of neoplasms were attributed to exposure to methacrylonitrile. The incidences of olfactory epithelial atrophy and metaplasia of the nose were significantly greater for 30 mg/kg males and females than for the vehicle controls. Increased incidences of cytoplasmic vacuolization occurred in the liver of males and females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered methacrylonitrile in deionized water by gavage at doses of 0, 1.5, 3, or 6 mg/kg, 5 days per week for 104 to 105 weeks. This dose selection was based on the results of an NTP 13-week gavage study; where methacrylonitrile was administered at doses of 0, 0.75, 1.5, 3, 6, or 12 mg/kg for 32 days or 13 weeks. Mice administered 6 or 12 mg/kg exhibited clinical signs of toxicity. In addition, slight decreases in the mean body weight gains of 12 mg/kg male and female mice were observed at 13 weeks. Additionally, one 12 mg/kg male from the 32-day interim evaluation and one 12 mg/kg female from the 13-week study died during the first week of treatment; other deaths were accidental. In the 2-year study, methacrylonitrile had no effect on survival. The mean body weights of all dosed groups were generally similar to those of the vehicle controls throughout the study. No neoplasms or nonneoplastic lesions were attributed to the methacrylonitrile administration. RESULTS Methacrylonitrile did not induce mutations in any of five strains of Salmonella typhimurium, with or without S9 activation, and did not induce sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster fed methacrylonitrile during the larval stage. Results of in vivo bone marrow micronucleus tests with methacrylonitrile in male rats and mice were also negative. Finally, no increase in the frequency of micronucleated erythrocytes was seen in peripheral blood of male or female mice treated with methacrylonitrile for 13 weeks by gavage. CONCLUSIONS Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of methacrylonitrile in male or female F344/N rats administered 3, 10, or 30 mg/kg. There was no evidence of carcinogenic activity of methacrylonitrile in male or female B6C3F1 mice administered 1.5, 3, or 6 mg/kg. In male and female rats, methacrylonitrile administration caused significant increases in the incidences of nonneoplastic lesions of the nose and liver.