title: NTP Toxicology and Carcinogenesis Studies of Tetranitromethane (CAS No. 509-14-8) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).
abstract: Tetranitromethane is a volatile contaminant formed during the manufacture of TNT and has been used as a rocket fuel and biochemical reagent. Toxicology and carcinogenesis studies were conducted in F344/N rats and B6C3F1 mice of each sex by whole body exposure to tetranitromethane vapor (greater than 99% pure), 6 hours per day, 5 days per week for 14 days, 13 weeks, or 2 years. Additional groups of male mice were exposed to tetranitromethane for evaluation at 1 year. Genetic toxicology studies were performed in Salmonella typhimurium and Chinese hamster ovary (CHO) cells. Fourteen-Day Studies: Exposure concentrations ranged from 2 to 25 ppm for rats and from 2 to 50 ppm for mice. All rats exposed to 25 ppm and all mice exposed at the top concentration of 50 ppm died by day 2; reduced survival was seen in mice exposed to 25 ppm and in rats exposed to 10 ppm. Pulmonary edema in rats and inflammation of the lung in mice were seen in those animals in the 25- and 50-ppm exposure groups examined microscopically. Thirteen-Week Studies: Exposure concentrations ranged from 0.2 to 10 ppm for rats and mice. No exposure-related deaths occurred in rats. The final mean body weight of rats exposed to 10 ppm was 16% lower than that of controls for males and 6% lower for females. Exposure-related histologic effects included squamous metaplasia of the respiratory epithelium of the nasal mucosa and chronic inflammation of the lung. No deaths of mice could be clearly related to exposure to tetranitromethane. The final mean body weights of mice exposed to 5 or 10 ppm were 5% or 12% lower than that of controls for males and 9% or 12% lower for females. Exposure-related histologic effects in mice included inflammation and squamous metaplasia of the respiratory epithelium of the nasal mucosa and hyperplasia of the bronchiolar epithelium. Based on the incidences and severity of lesions in the respiratory at the higher concentrations used in the 13-week studies, exposure concentrations chosen for the 2-year studies were 0, 2, and 5 ppm for groups of 50 rats of each sex and 0, 0.5, and 2 ppm for groups of 50 mice of each sex. Additional groups of 6 or 10 male mice were exposed at concentrations of 0, 0.5, or 2 ppm for 1 year. Body Weights and Survival in the Two-Year Studies: Mean body weights of male and female rats exposed to 5 ppm were approximately 5%-15% lower than those of controls after week 70. Survival of rats at 104 weeks was as follows: male: control, 18/50; 2 ppm, 17/50; 5 ppm, 4/50; female: 25/50; 34/50; 15/50; survival of rats at the top concentration was reduced due to neoplasia. Mean body weights of exposed mice were variable and ranged as much as 10% below those of controls during the second year of the studies. Survival of exposed male mice at 104 weeks was significantly lower than that of controls due to neoplasia (control, 37/50; 0.5 ppm, 26/50; 2 ppm, 15/50). Survival of female mice was not significantly affected by exposure to tetranitromethane (31/50; 28/50; 24/50). Neoplastic and Nonneoplastic Effects in the Two-Year Studies: Effects of exposure to tetranitromethane were limited to the respiratory tract. Hyperplasia of the alveolar and bronchiolar epithelium was observed at increased incidences in exposed rats. The incidence of alveolar/bronchiolar adenomas and carcinomas were markedly increased in exposed male and female rats, with carcinomas (many of which metastasized to other sites) occurring in nearly all rats exposed to the top concentration of 5 ppm (adenomas or carcinomas-- male: control, 1/50; 2 ppm, 33/50; 5 ppm, 46/50; female: 0/50; 22/50; 50/50). Many of the rats exposed to 5 ppm also had squamous cell carcinomas of the lung (male: 0/50; 1/50; 19/50; female: 0/50; 1/50; 12/50). Hyperplasia of the respiratory epithelium and chronic inflammation of the nasal mucosa were observed at increased incidences in exposed male and female rats. Squamous metaplasia of the respiratory epithelium was increased in exposed male rats. No neoplasms of the nasal passage were seen. In exposed mice, hyperplasia of the alveolar and br were seen. In exposed mice, hyperplasia of the alveolar and bronchiolar epithelium was observed at increased incidences. Alveolar/bronchiolar neoplasms, primarily carcinomas (many of which metastasized to other sites), were increased in exposed male and female mice (male: control, 12/50; 0.5 ppm, 27/50; 2 ppm, 47/50; female: 4/49; 24/50; 49/50). Chronic inflammation of the nasal mucosa and hyperplasia and squamous metaplasia of the respiratory epithelium of the nasal cavity occurred at increased incidences in female mice exposed to 2 ppm. No primary neoplasms of the nasal passage were observed in mice. Oncogene Analysis: DNA from 14/19 rat and 4/4 mouse lung neoplasms caused morphologic transformation after transfection into cultured NIH/3T3 fibroblasts. The transforming gene from both rat and mouse lung neoplasms was determined by Southern blot analysis to be an activated K-ras oncogene. Further studies showed a GC-->AT transition in the second base of the 12th codon of the K-ras oncogene. Genetic Toxicology: Tetranitromethane was mutagenic in S. typhimurium strains TA98, TA100, and TA1535 with and without exogenous metabolic activation (S9); no mutagenic activity was observed in TA1537 with or without S9. Chromosomal aberrations were observed in CHO cells treated in vitro with tetranitromethane in the presence of S9. Sister chromatid exchanges were induced in CHO cells in the absence of S9. Conclusions: Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of tetranitromethane for male and female F344/N rats and male and female B6C3F1 mice, based on increased incidences of alveolar/bronchiolar neoplasms in both species and squamous cell carcinomas of the lung in rats. Chronic inflammation of the nasal mucosa was related to exposure in rats and female mice, and hyperplasia and squamous metaplasia of the respiratory epithelium were increased in exposed male rats. Synonym: TNM