Subacute and chronic oral toxicities of trinotroglycerin (TNG) were studied in beagle dogs, CD rats, and CD-1 mice. No adverse effects were seen in dogs given up to 1 mg/kd/day of TNG for 4 weeks, then 5 mg/kg/day for 9 more weeks. Dogs given 25 to 200 mg/kg/day for 5 days had transient and dose-related severe methemoglobinemia, while 200 mg/kg/day produced depression; dogs given 1, 5, or 25 mg/kg/day for 12 months had transient and dose-related mild methemoglobinemia. Rats fed 0.1% TNG for 5 weeks, then 0.5% (230-234 mg/kd/day) for 8 more weeks had decreases in feed consumption and weight gain after the increase in dosage; rats fed 2.5% TNG (1406 or 1416 mg/kg/day for males and females, respectively) for 13 weeks suffered adverse effects, including weight loss, compensated anemia, and testicular degeneration, but they resumed gaining weight as feeding continued. Rats fed 1% TNG (363 or 434 mg/kg/day for males and females, respectively) for 2 years had decreased weight gain, decreased grooming, methemoglobinemia and its sequelae, cholangiofibrosis , hepatocellular carcinoma, and interstitial cell tumors of the testis. A decrease in the naturally occurring pituitary chromophobe adenoma and mammary tumors increased the life span of the females. Some rats fed 0.1% TNG (31.5 or 38.1 mg/kg/day for males and females, respectively) had mild hepatic lesions similar to those seen in rats fed the larger doses. No adverse effects were seen in mice fed up to 0.1% TNG for 3 weeks, then 0.5% for 10 more weeks. Mice fed 1% TNG (1022 or 1058 mg/kg/day for males and females, respectively) for 2 years had decreased weight gain, decreased grooming, and methemoglobinemia and its sequelae, but no obvious cellular changes as found in the rats.