title: NTP Toxicity Studies of beta-Bromo-beta-Nitrostyrene (CAS No. 7166-19-0) Administered by Gavage to F344/N Rats and B6C3F1 Mice.
abstract: beta-Bromo-beta-nitrostyrene is a wide-spectrum biocide most frequently used as a fungicide to combat the formation of slime in paper and pulp mill operations. Toxicity studies were conducted by administering beta-bromo-beta-nitrostyrene (99% pure, trans isomer) to groups of 10 male and 10 female F344/N rats and B6C3F1 mice by gavage, 5 days per week for 4 weeks. Doses of 0, 37, 75, 150, 300, or 600 mg/kg were administered in a corn oil vehicle. The parameters evaluated included hematology, clinical chemistry (rats only), and histopathology. The genetic toxicity of b-bromo-b- nitrostyrene was evaluated in Salmonella typhimurium and in peripheral blood erythrocytes of mice. In addition, the absorption, distribution, metabolism, and excretion of b-bromo-b- nitrostyrene were studied in male F344 rats following intravenous, dermal, or oral administration. In the 4-week study in rats, two males in the 150 mg/kg group, one male and one female in the 300 mg/kg groups, and all rats in the 600 mg/kg groups died or were killed moribund before the end of the study. The mean body weight gains and absolute and relative thymus weights of male and female rats in the 300 mg/kg groups were lower than those of the controls. Hematology evaluations in rats indicated the development of a mild anemia and monocytosis consistent with and likely related to inflammatory and ulcerative lesions that occurred in the gastrointestinal tract. Clinical chemistry evaluations indicated lower alkaline phosphatase activities and serum total protein and albumin concentrations in treated rats than in the controls. Treatment-related lesions in rats were observed in the forestomach, glandular stomach, cecum, nasal passages, and testis. Males were generally affected at lower doses than females. The most prominent lesions were in the forestomach and were characterized by inflammation, hemorrhage, and necrosis in rats dying early. In rats surviving to the end of the study, forestomach lesions included necrosis, ulceration, and regenerative epithelial hyperplasia and hyperkeratosis. Inflammation of the glandular stomach and cecum also occurred in rats dying early. Inflammation and degeneration of the nasal passage in treated rats were attributed to reflux of the irritant chemical in the gavage fluid. Testicular degeneration was seen in rats dying early and was characterized by necrotic germ cells and a decreased number of spermatozoa in the epididymal tubules and by multinucleated syncytial cells in the seminiferous tubules. In the 4-week study in mice, one male in the 300 mg/kg group and all mice in the 600 mg/kg groups died or were killed moribund before the end of the study. No significant changes in final mean body weights or mean body weight gains were observed in males or females. Hematologic changes consistent with inflammatory lesions occurred in male and female mice in the 300 mg/kg groups. Treatment-related lesions in mice occurred in the forestomach, gallbladder, and testis. Forestomach lesions were similar to those described in rats and were only present in male and female mice given doses of 300 mg/kg or greater. At these dose levels, inflammation and degeneration/necrosis of the gallbladder mucosa also occurred in male and female mice, but these lesions were absent in the bile ducts or liver. Testicular degeneration occurred in mice dying early and was similar to that observed in rats. In comparative disposition and metabolism studies in male F344 rats, clear differences were found between the fate of b-bromo- b-nitrostyrene following oral administration and the fate of radiolabeled beta-bromo-beta-nitrostyrene following intravenous or dermal administration. Oral exposure resulted in significant absorption of nonhydrolyzed beta-bromo-beta-nitrostyrene and the formation of parent compound metabolites, primarily 1-phenyl-2-nitroethyl-1-sulfonic acid (PNSA), a product of a sulfation reaction at the alpha carbon. Following dermal exposure, a limited amount of beta-bromo-beta-nitrostyrene entered the systemic circulation (approximately 10% per 24 hours from a 10 mg/cm2 dose) although lower doses were more completely absorbed. Once beta-bromo-beta-nitrostyrene entered the circulation, significant amounts of the dose were hydrolyzed or bound to macromolecules. PNSA was not a major metabolite in dermal or intravenous studies. Regardless of the route of administration, only low levels of radioactive label from beta-bromo-beta-nitrostyrene were retained in tissues following exposure, and most beta-bromo-beta-nitrostyrene metabolites were excreted in the urine and feces within 24 to 48 hours. beta-bromo-beta-nitrostyrene was mutagenic in S. typhimurium strains TA98 and TA100 in the absence of exogenous metabolic activation (S9). No mutagenic activity was observed with S9 in either of these strains, and no mutagenic activity was observed in strains TA97 or TA1535, with or without S9. The frequency of micronucleated normochromatic erythrocytes was significantly increased in the peripheral blood of male mice, but not female mice, following 4 weeks of exposure to beta-bromo-beta-nitrostyrene by corn oil gavage. In summary, under the conditions of these 4-week gavage studies, rats were more sensitive to the toxic and irritant effects of beta-bromo-beta-nitrostyrene than mice, and males were more affected by beta-bromo-beta-nitrostyrene than females. Although the specific cause of the early deaths could not be determined, significant inflammation and necrosis developed in the forestomach of rats and mice, in the glandular stomach and cecum of rats, and in the gallbladder of mice. Similar lesions in the nasal passages of rats were attributed to reflux of gavage materials. The no-observed- adverse-effect level (NOAEL) for histopathologic lesions was 37 mg/kg per day for rats and 150 mg/kg per day for mice.