title: NTP Toxicology and Carcinogenesis Studies of Tribromomethane (Bromoform) (CAS No. 75-25-2) in F344/N Rats and B6C3F1 Mice (Gavage Studies).
abstract: Tribromomethane, a chemical intermediate and solvent, has been identified as a drinking water contaminant resulting from water chlorination. Toxicology and carcinogenesis studies were conducted by administering tribromomethane (95%-97% pure) in corn oil by gavage to groups of F344/N rats and B6C3F1 mice of each sex once or for 14 days, 13 weeks, or 2 years. Single-Administration, Fourteen-Day, and Thirteen-Week Studies: All rats that received 2,000 mg/kg and 3/5 males and 3/5 females that received 1,000 mg/kg tribromomethane died before the end of the single-administration studies. All mice that received 2,000 mg/kg, 4/5 males and 2/5 females that received 1,000 mg/kg, and 1/5 males that received 500 mg/kg died before the end of the studies. Shallow breathing was observed for rats and male mice that received 1,000 or 2,000 mg/kg tribromomethane. In the 14-day studies, all rats that received 600 or 800 mg/kg and 1/5 males that received 400 mg/kg tribromomethane died before the end of the studies. The final mean body weight of male rats that received 400 mg/kg was 14% lower than that of vehicle controls. One of five male mice that received 600 mg/kg and 1/5 female mice that received 800 mg/kg died before the end of the studies. Final mean body weights of dosed and vehicle control mice were comparable. None of the rats died before the end of the 13-week studies (doses ranged from 12 to 200 mg/kg). Final mean body weights were comparable for dosed and vehicle control rats. All male rats that received 100 or 200 mg/kg tribromomethane and all female rats that received 200 mg/kg were lethargic. The incidences of cytoplasmic vacuolization of hepatocytes in dosed male rats were slightly increased compared with that in vehicle controls. The severity of this lesion was increased in the 200 mg/kg group. One of 10 female mice that received 100 mg/kg tribromomethane died before the end of the 13-week studies. The final mean body weight of mice that received 400 mg/kg was 8% lower than that of vehicle controls for males and was comparable to that of vehicle controls for females. Cytoplasmic vacuolization of hepatocytes was observed in the liver of 5/10 male mice that received 200 mg/kg and in 8/10 male mice that received 400 mg/kg tribromomethane. Based on these results, 2-year studies of tribromomethane were conducted by administering 0, 100, or 200 mg/kg tribromomethane in corn oil by gavage, 5 days per week for 103 weeks, to groups of 50 F344/N rats of each sex and 50 female B6C3F1 mice. Male B6C3F1 mice were administered 0, 50, or 100 mg/kg tribromomethane on the same schedule. Body Weights and Survival in the Two-Year Studies: Mean body weights of high dose male and female rats were 10%-28% lower than those of vehicle controls throughout the second year of the studies. Survival of the high dose group of male rats was significantly lower than that of the vehicle controls after week 91; no significant differences in survival were observed between any groups of female rats (male: vehicle control, 34/50; low dose, 30/50; high dose, 11/50; female: 34/50; 28/50; 28/50). Reduced survival for male rats given 200 mg/kg tribromomethane lowered the sensitivity of this group to detect a carcinogenic response. Mean body weights of dosed and vehicle control male mice were comparable throughout the study. Mean body weights of dosed female mice were 5%-16% lower than those of vehicle controls from week 28 to the end of the study. No significant differences in survival were observed between any groups of male mice; the survival of both dosed groups of female mice was significantly lower than that of the vehicle controls after week 77 (male: 41/50; 37/50; 36/50; female: 25/49; 15/50; 20/50). Reduced survival in all groups of female mice was partly due to a utero-ovarian infection; nonetheless, survival of all groups of female mice was at least 50% by week 92. Nonneoplastic and Neoplastic Effects in the Two-Year Studies: Uncommon adenomatous polyps or adenocarcinomas (combined) of the large intestine (colon or rectum) were induced in three male ratarge intestine (colon or rectum) were induced in three male rats (vehicle control, 0/50; low dose, 0/50; high dose, 3/50) and in nine female rats (0/50; 1/50; 8/50); the historical incidence of neoplasms of the large intestine is less than 0.2&percnt; in approximately 2,000 corn oil vehicle control male F344/N rats, and none has been observed in approximately 2,000 corn oil vehicle control female F344/N rats. Three of the neoplasms of the large intestine (one in the high dose male rats and two in the high dose female rats) were adenocarcinomas. Focal or diffuse fatty change of the liver was observed at increased incidences in dosed rats (male: 23/50; 49/50; 50/50; female: 19/50; 39/49; 46/50). Active chronic inflammation was observed at increased incidences in dosed male and high dose female rats (male: 0/50; 29/50; 23/50; female: 9/50; 8/49; 27/50). The incidence of necrosis of the liver was increased in high dose male rats (7/50; 3/50; 20/50) and decreased in dosed females (11/50; 3/49; 2/50). Mixed cell focus was observed at increased incidences in dosed female rats (8/50; 25/49; 28/50). Other nonneoplastic lesions observed at increased incidences in dosed rats included chronic active inflammation and squamous metaplasia of the ducts of the salivary gland (squamous metaplasia-- male: 0/50; 15/50; 31/48; female: 0/49; 10/49; 16/50; chronic active inflammation--male: 0/50; 16/50; 25/48; female: 0/49; 9/49; 18/50), squamous metaplasia of the prostate gland (2/49; 6/46; 12/50), ulcers of the forestomach (male: 1/49; 5/50; 10/50), and chronic active inflammation of the lung (male: 1/50; 7/50; 15/50). Pigmentation of the spleen was also observed at an increased incidence in high dose female rats. The salivary gland and lung lesions were characteristic of infection by rat coronavirus, a virus to which a positive serologic reaction was observed early in the studies. The incidence of follicular cell hyperplasia of the thyroid gland was increased in high dose female mice (5/49; 4/49; 19/47), and fatty change of the liver was increased in both dosed groups of female mice (1/49; 9/50; 24/50). No chemically related adverse effects were observed in male mice. Neoplastic lesions that occurred at lower incidences in dosed animals compared with those in vehicle controls included preputial gland neoplasms in male rats (10/41; 5/38; 1/34), uterine stromal polyps in female rats (10/49; 9/50; 2/50), anterior pituitary gland adenomas in male and female rats (male: 12/50; 12/48; 2/45; female: 29/48; 12/46; 16/48), mammary gland fibroadenomas in female rats (22/50; 17/50; 6/50), and alveolar/bronchiolar neoplasms in male mice (11/50; 7/50; 2/49). Other than concomitant decreases in body weights, no other reasons are obvious to correlate these decreases with chemical administration. Genetic Toxicology: Tribromomethane exhibited equivocal mutagenicity in Salmonella typhimurium strain TA100 in the absence of exogenous metabolic activation and in strains TA97 and TA98 when exposure occurred in the presence of hamster S9; tribromomethane produced no increases in revertant colonies in TA1535 or TA1537 with or without exogenous metabolic activation. Tribromomethane induced trifluorothymidine (Tft) resistance in mouse L5178Y cells with and without metabolic activation. When tested incultured Chinese hamster ovary (CHO) cells for cytogenetic effects, tribromomethane produced an increase in both sister chromatid exchanges (SCEs) and chromosomal aberrations in the absence, but not in the presence, of exogenous metabolic activation. Tribromomethane caused sex-linked recessive lethal mutations in Drosophila when administered to adult males by feeding; no induction of mutations was observed when tribromomethane was administered by abdominal injection. Results of tests for reciprocal translocations in adult male Drosophila exposed to tribromomethane by feeding were negative. In vivo tests for cytogenetic effects in bone marrow cells of male B6C3F1 mice demonstrated that intraperitoneal injection of tribromomethane induced an increase in SCEs but no increase in chromosomal aberrations. Intraperitoneal injection of tribromomethane also induced an increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of B6C3F1 mice. Audit: The data, documents, and pathology materials from the 2-year studies of tribromomethane have been audited. The audit findings show that the conduct of the studies is documented adequately and support the data and results given in this Technical Report. Conclusions: Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of tribromomethane for male F344/N rats and clear evidence of carcinogenic activity for female F344/N rats, based on increased incidences of uncommon neoplasms of the large intestine. Reduced survival for male rats given 200 mg/kg tribromomethane lowered the sensitivity of this group to detect a carcinogenic response. Chemically related nonneoplastic lesions included fatty change and active chronic inflammation of the liver in male and female rats, minimal necrosis of the liver in male rats, and mixed cell foci of the liver in female rats. There was no evidence of carcinogenic activity for male B6C3F1 mice given 50 or 100 mg/kg tribromomethane or for female B6C3F1 mice given 100 or 200 mg/kg; male mice might have been able to tolerate a higher dose. Survival of female mice was reduced, partly due to a utero-ovarian infection. Synonym: bromoform