NTP toxicology and carcinogenesiss studies of citral (microencapsulated) (CAS No. 5392-40-5) in F344/N rats and B6C3F1 mice (feed studies)

Citral is used primarily as lemon flavoring in foods, beverages, and candies. It is also used as a lemon fragrance in detergents, perfumes, and other toiletries. Citral was nominated by the National Cancer Institute for study because of its widespread use in foods, beverages, cosmetics, and other consumer products and its structure as a representative beta-substituted vinyl aldehyde. Male and female F344/N rats and B6C3F1 mice were exposed to microencapsulated citral (greater than 96% pure) in feed for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, mouse bone marrow cells, and mouse peripheral blood erythrocytes. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing starch microcapsules with a load of 31.3% citral. The concentration of citral in the diet was 3,900, 7,800, 15,600, or 31,300 ppm microencapsulated citral (equivalent to average daily doses of approximately 345, 820, 1,785, and 1,585 mg citral/kg body weight to males and 335, 675, 1,330, and 2,125 mg/kg to females) for 14 weeks. Additional groups of 10 male and 10 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). In the second week of the study, all rats in the 31,300 ppm groups were killed moribund. Mean body weights of exposed males and females that survived to the end of the study were generally significantly less than those of the vehicle controls. Feed consumption by 15,600 and 31,300 ppm males and females was less than that by the vehicle controls during the first week of the study. Males and females in the 31,300 ppm groups exhibited listlessness, hunched posture, absent or slow paw reflex, and dull eyes. Exposure of rats to citral may have been associated with forestomach epithelial hyperplasia and hyperkeratosis, bone marrow atrophy and hemorrhage, and nephrotoxicity. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 3,900, 7,800, 15,600, or 31,300 ppm microencapsulated citral (equivalent to average daily doses of approximately 745, 1,840, 3,915, and 8,110 mg/kg to males and 790, 1,820, 3,870, and 7,550 mg/kg to females) for 14 weeks. Additional groups of 10 male and 10 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). In the second week of the study, four males in the 31,300 ppm group were killed moribund. Mean body weights of all exposed groups of males and females were significantly less than those of the vehicle controls. Feed consumption by females exposed to 7,800 ppm or greater was less than that by the vehicle controls during the first week of the study. By the end of the study, feed consumption by all exposed groups was greater than that by the vehicle controls. Mice in the 15,600 and 31,300 ppm groups were generally thin and lethargic; a few males in the 7,800 ppm group were also thin. The incidences of ovarian atrophy were significantly increased in females exposed to 15,600 or 31,300 ppm. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were fed diets containing 1,000, 2,000, or 4,000 ppm microencapsulated citral for 2 years. Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1,000, 2,000, and 4,000 ppm delivered average daily doses of approximately 50, 100, and 210 mg/kg to males and females. Survival of all exposed groups of males was significantly greater than that of the vehicle control group. Mean body weights of rats exposed to 4,000 ppm were generally less than those of the vehicle controls from week 49 (males) or 25 (females) to the end of the study. Feed consumption by exposed groups was similar to that by the vehicle controls. No neoplasms or nonneoplastic lesions were attributed to exposure to citral. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were fed diets containing 500, 1,000, or 2,000 ppm microencapsulated citral for 2 years. Additional groups of 50 male and 50 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 500, 1,000, and 2,000 ppm delivered average daily doses of approximately 60, 120, and 260 mg/kg to males and females. Survival of exposed males and females was similar to that of the vehicle control groups. Mean body weights of mice exposed to 1,000 or 2,000 ppm were generally less than those of the vehicle controls throughout the study, and mean body weights of 500 ppm females were less from week 30 to the end of the study. Feed consumption by the exposed groups was similar to that by the vehicle controls. The incidences of malignant lymphoma occurred with a positive trend in female mice, and the incidence in 2,000 ppm females was significantly greater than that in the vehicle control group. Tissues most commonly affected by malignant lymphoma were the spleen, mesenteric lymph node, thymus, and, to a lesser extent, the ovary. GENETIC TOXICOLOGY: Citral was not mutagenic in S. typhimurium strain TA98, TA100, TA1535, or TA1537 with or without induced rat or hamster liver S9 enzymes. In cytogenetic tests with cultured Chinese hamster ovary cells, citral induced sister chromatid exchanges with and without S9, but chromosomal aberrations were not significantly increased after exposure to citral, with or without S9. Negative results were obtained in an in vivo bone marrow micronucleus test in male B6C3F1 mice treated by intraperitoneal injection with 250 to 750 mg/kg daily for 3 days. Likewise, no increases in the frequencies of micronucleated erythrocytes were observed in peripheral blood samples collected from male and female mice within 24 hours of the final exposure in the 14-week study. In conclusion, citral gave negative results in in vitro and in vivo tests for genotoxicity, with the exception of the in vitro mammalian cell test for sister chromatid exchange induction CONCLUSIONS: Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of citral in male or female F344/N rats exposed to 1,000, 2,000, or 4,000 ppm. There was no evidence of carcinogenic activity of citral in male B6C3F1 mice exposed to 500, 1,000, or 2,000 ppm. There was equivocal evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of malignant lymphoma.     

Toxicology and carcinogenesis studies of microencapsulated trans-cinnamaldehyde in rats and mice.

trans-Cinnamaldehyde is a widely used natural ingredient that is added to foods and cosmetics as a flavoring and fragrance agent. Male and female F344/N rats and B6C3F(1) mice were exposed to microencapsulated trans-cinnamaldehyde in the feed for three months or two years. All studies included untreated and vehicle control groups. In the three-month studies, rats and mice were given diets containing 4100, 8200, 16,500, or 33,000 ppm trans-cinnamaldehyde. In rats, feed consumption was reduced in all exposed groups. In mice, feed consumption was reduced in the highest dose groups. Body weights of all treated males were less than controls. Body weights were reduced in female rats exposed to 16,500 or 33,000 ppm and female mice exposed to 8200 ppm or greater. All rats survived to the end of the study but some male mice in the highest dose groups died due to inanition from unpalatability of the dosed feed. The incidence of squamous epithelial hyperplasia of the forestomach was significantly increased in rats exposed to 8200 ppm or greater and female mice exposed to 33,000 ppm. In mice, the incidence of olfactory epithelial degeneration of the nasal cavity was significantly increased in males and females exposed to 16,500 ppm and females exposed to 33,000 ppm. In the two-year studies, rats and mice were exposed to 1000, 2100, or 4100 ppm trans-cinnamaldehyde. Body weights were reduced in mice exposed to 2100 ppm and in rats and mice exposed to 4100 ppm. In rats, hippuric acid excretion was dose proportional indicating that absorption, metabolism, and excretion were not saturated. No neoplasms were attributed to trans-cinnamaldehyde in rats or mice. Squamous cell papillomas and carcinomas of the forestomach were observed in male and female mice but the incidences were within the NTP historical control range and were not considered to be related to trans-cinnamaldehyde exposure.     

Toxicity and carcinogenicity studies of nalidixic acid in rodents

Toxicity and carcinogenicity studies of nalidixic acid, an antimicrobial agent used to treat bacterial infections of the urinary tract, were conducted in F344/N rats and B6C3F1 mice of each sex for 13 weeks or 2 years. In the 13-week studies, nalidixic acid was administered at dietary concentrations ranging from 1,000 to 16,000 ppm. Body weights of both rats and mice were reduced in the groups receiving diet containing 8,000 and 16,000 ppm, and feed consumption of rats in the highest treatment groups was approximately two-thirds that of controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was observed in male rats that received 16,000 ppm; no other compound-related histopathologic effects were observed in either species. Two-year studies were conducted by feeding diets containing 0, 2,000, or 4,000 ppm nalidixic acid to groups of 50 rats and mice/sex/group. The average daily feed consumption was slightly reduced compared to control groups and resulted in approximate daily doses of 82 or 175 mg nalidixic acid/kg for low dose and high dose rats, and 220 or 475 mg/kg for low dose and high dose mice. Mean body weights of dosed rats and mice were lower than those of controls, except for groups of low dose female rats and male mice. The incidences of preputial gland neoplasms in dosed male rats and of clitoral gland neoplasms in dosed female rats were significantly increased compared to those in controls; responses in low dose groups were similar to those in high dose groups. There were decreased incidences of leukemia and mammary gland neoplasms in dosed female rats and of pituitary gland neoplasms in dosed male rats. Subcutaneous tissue fibrosarcomas were marginally increased in dosed male mice. There were no increased incidences of neoplasms in dosed female mice. Under the conditions of these studies, the dietary administration of nalidixic acid was carcinogenic for rats, causing preputial gland or clitoral gland neoplasms in males and females, respectively. The association of subcutaneous neoplasms with administration of nalidixic acid to male mice was equivocal.     

Toxicity and Carcinogenicity Studies of Nalidixic Acid in Rodents

ABSTRACT

Toxicity and carcinogenicity studies of nalidixic acid, an antimicrobial agent used to treat bacterial infections of the urinary tract, were conducted in F344/N rats and B6C3F mice of each sex for 13 weeks or 2 years. In the 13–week studies, nalidixlc acid was administered at dietary concentrations ranging from 1,000 to 16,000 pp. Body weights of both rats and mice were reduced in the groups receiving diet containing 8,000 and 16,000 ppm, and feed consumption of rats in the highest treatment groups was approximately tw-thirds that of controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was observed in male rats that received 16,000 ppm; no other compound-related histopathologic effects were observed in either species. TWo-year studies were conducted by feeding diets containing 0, 2,000, or 4,000 ppm nalidixic acid to groups of 50 rats and mice/sex/group. The average daily feed consumption was slightly reduced compared to control groups and resulted in approximate daily doses of 82 or 175 mg nalidixic acidfig for low dose and high dose rats, and 220 or 475 mg/kg for low dose and high dose mice. Mean body weights of dosed rats and mice were lower than those of controls, except for groups of low dose female rats and male mice. The incidences of preputial gland neoplasms in dosed male rats and of clitoral gland neoplasms in dosed female rats were significantly increased compared to those in controls; responses in low dose groups were similar to those in high dose groups. There were decreased incidences of leukemia and mammary gland neoplasms in dosed female rats and of pituitary gland neoplasms in dosed male rats. Subcutaneous tissue fibrosarcomas were marginally increased in dosed male mice. There were no increased incidences of neoplasms in dosed female mice. under the conditions of these studies, the dietary administration of nalidixic acid was carcinogenic for rats, causing preputial gland or clitoral gland neoplasms in males and females, respectively. The association of subcutaneous neoplasms with administration of nalidixic acid to male mice was equivocal.     

Toxicology and carcinogenesis studies of 1-bromopropane (CAS No. 106-94-5) in F344/N rats and B6C3F1 mice (inhalation studies)

In the early to mid 1990s, 1-bromopropane was used primarily as an intermediate in the production of pesticides, quaternary ammonium compounds, flavors and fragrances, pharmaceuticals, and other chemicals in well-controlled, closed processes. In the mid to late 1990s, it was introduced as a less toxic replacement for methylene chloride in emissive applications such as vapor and immersion degreasing operations and critical cleaning of electronics and metals. 1-Bromopropane was also introduced as a nonflammable, nontoxic, fast-drying, and inexpensive solvent for adhesive resins, and has been marketed as a replacement for ozone depleting refrigerants. 1-Bromopropane was nominated for study by the Occupational Safety and Health Administration based on the potential for widespread occupational and environmental exposure and a lack of toxicity and carcinogenicity data. Male and female F344/N rats and B6C3F1 mice were exposed to 1-bromopropane (99% or greater pure) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli and mouse peripheral blood. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were exposed to 1-bromopropane vapor at concentrations of 0, 125, 250, 500, 1,000, or 2,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats survived to the end of the study except one 500 ppm male. Mean body weights of 2,000 ppm rats were significantly less than those of the chamber controls. The absolute kidney weight of 1,000 ppm males, relative kidney weights of all exposed groups of males, and absolute and relative kidney weights of all exposed groups of females were significantly increased. The absolute and relative liver weights of 1,000 ppm males, relative liver weights of 500 and 2,000 ppm males, and absolute and relative liver weights of 500 ppm or greater females were significantly increased. Nasal lesions included suppurative inflammation in males exposed to 500 ppm or greater, respiratory epithelial necrosis in 1,000 and 2,000 ppm males, and respiratory epithelial regeneration in 1,000 and 2,000 ppm females. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were exposed to 1-bromopropane vapor at concentrations of 0, 125, 250, 500, 1,000, or 2,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days. All 2,000 ppm males, two 2,000 ppm females, four 500 ppm males, one 1,000 ppm male, and one 1,000 ppm female died early. The mean body weight gain of 1,000 ppm males was significantly less than that of the chamber controls. Abnormal breathing, lethargy, and eye discharge were observed primarily during week 1 in groups exposed to 500 ppm or greater. Liver weights of 1,000 ppm males and of females exposed to 500 ppm or greater were significantly increased. Kidney weights of 1,000 and 2,000 ppm females were significantly increased. Microscopic lesions related to 1-bromopropane exposure occurred in the lung, liver, and nose of males and females and were primarily seen in mice exposed to 500 ppm or greater. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to 1-bromopropane vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study. Mean body weights of 1,000 ppm males were significantly less than those of the chamber controls. The increases in sorbitol dehydrogenase activities in 500 ppm males and 1,000 ppm males and females were consistent with the histopathologic evidence of mild hepatotoxicity caused by 1-bromopropane. Liver weights of males exposed to 250 ppm or greater and of females exposed to 125 ppm or greater were significantly increased. Spleen and kidney weights of 1,000 ppm females were significantly increased. Exposure concentration-related decreases of 28% in sperm motility and 37% in sperm counts were seen in the 1,000 ppm group of male rats. Female rats in all three exposure groups evaluated exhibited altered estrous cycles, spending significantly more time in extended estrus and less time in extended diestrus. The incidences of cytoplasmic vacuolization of the liver were significantly increased in males exposed to 250 ppm or greater and in females exposed to 500 ppm or greater. Hepatocyte degeneration was also observed in 1,000 ppm females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed to 1-bromopropane vapor at concentrations of 0, 62.5, 125, 250, or 500 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks. One 250 ppm male and four males and five females in the 500 ppm groups died early. Mean body weights of exposed groups were similar to those of the chamber controls. Lethargy was observed in males and females exposed to 500 ppm, and abnormal breathing was observed in moribund mice. The kidney, liver, and lung weights of 500 ppm females were significantly greater than those of the chamber controls. The kidney weights of 500 ppm males were significantly decreased. Sperm counts in the 500 ppm group of male mice were 28% less than that in the chamber controls. Female mice exhibited altered estrous cycles, with females in the 500 ppm group spending significantly more time in extended diestrus and those in the 250 ppm group spending significantly more time in extended estrus compared to the chamber controls. Nonneoplastic lesions were observed in the nose, larynx, trachea, lung, and liver of 500 ppm males and females and in the adrenal cortex of 500 ppm females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to 1-bromopropane vapor at concentrations of 0, 125, 250, or 500 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 105 weeks. Survival of 500 ppm males was significantly less than that of the chamber control group. Mean body weights of exposed groups were similar to those of the chamber controls. Increased incidences of macroscopic, soft, pale-yellow to green, variably sized nodules were seen predominantly in the nose and skin of exposed rats. The number of animals with multiple masses was increased in the 500 ppm groups. In most cases, these lesions were microscopically shown to be suppurative inflammation, many with Splendore-Hoeppli material. The incidence of adenoma of the large intestine (colon or rectum) was significantly greater in 500 ppm females than in the chamber control group. The incidence of adenoma of the large intestine in 250 ppm males exceeded the historical control ranges for inhalation studies and all routes. The incidences of keratoacanthoma, basal cell adenoma, basal cell carcinoma, or squamous cell carcinoma (combined) were significantly greater in all exposed groups of males than in the chamber control group and exceeded the historical control range for inhalation studies. The incidences of keratoacanthoma and of keratoacanthoma or squamous cell carcinoma (combined) in 250 and 500 ppm males were also significantly increased and exceeded the historical control ranges for inhalation studies. In 500 ppm females, the incidence of squamous cell papilloma, keratoacanthoma, basal cell adenoma, or basal cell carcinoma (combined) exceeded the historical control range for inhalation studies. The incidence of malignant mesothelioma was significantly greater in 500 ppm males than in the chamber control group. The incidences of pancreatic islet adenoma in all exposed groups of males and of pancreatic islet adenoma or carcinoma (combined) in 125 and 250 ppm males were significantly increased. Treatment-related nonneoplastic lesions were observed in the respiratory system of exposed male and female rats. In the nose, the incidences of suppurative chronic inflammation, chronic active inflammation, glandular hyperplasia, respiratory epithelial hyperplasia (females), and respiratory metaplasia of the olfactory epithelium (females) were increased in all exposed groups. In the larynx, the incidences of chronic active inflammation and squamous metaplasia (except 125 ppm females) were increased in all exposed groups, and the incidences of suppurative chronic inflammation were increased in the 500 ppm groups. Also, chronic inflammation of the lung was observed in the 500 ppm females. In the trachea, there were increased incidences of chronic active inflammation in all exposed groups of females and 500 ppm males, and the incidence of epithelial hyperplasia was increased in 500 ppm females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed to 1-bromopropane vapor at concentrations of 0, 62.5, 125, or 250 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 105 weeks. Survival of exposed groups was similar to that of the chamber controls. Mean body weights of all exposed groups were similar to those of the chamber controls throughout the study. In the females, there were increased incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined); the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in all exposed groups of females. There were significantly increased incidences of cytoplasmic vacuolization of the bronchiolar epithelium in all exposed male groups and regeneration of the bronchiolar epithelium in all exposed groups of males and females. In the nose, there were significantly increased incidences of cytoplasmic vacuolization of the respiratory epithelium in all exposed groups of males and in 125 and 250 ppm females. There were significantly increased incidences of respiratory epithelial hyperplasia in all exposed female groups and in 62.5 and 250 ppm males. (ABSTRACT TRUNCATED)     

NTP toxicology and carcinogenesis studies of trans-cinnamaldehyde (CAS No. 14371-10-9) in F344/N rats and B6C3F1 mice (feed studies)

Cinnamaldehyde is used in foods, beverages, medical products, perfumes, cosmetics, soaps, detergents, creams, and lotions. Cinnamaldehyde has been used as a filtering agent and a rubber reinforcing agent and is used as a brightener in electroplating processes, as an animal repellent, as an insect attractant, and as an antifungal agent. trans-cinnamaldehyde was nominated for study by the Food and Drug Administration based on its widespread use as a flavor and fragrance ingredient and its structural similarity to cinnamyl anthranilate and 3,4,5-trimethoxy cinnamaldehyde, two known rodent carcinogens. Male and female F344/N rats and B6C3F1 mice were exposed to trans-cinnamaldehyde (at least 95% pure) in feed for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing 4,100, 8,200, 16,500, or 33,000 ppm microencapsulated trans-cinnamaldehyde (equivalent to average daily doses of approximately 275, 625, 1,300, or 4,000 mg trans-cinnamaldehyde/kg body weight to males and 300, 570, 1,090, or 3,100 mg/kg to females) for 3 months. Additional groups of 10 male and 10 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). All rats survived to the end of the study. Mean body weights of all exposed groups of males and 16,500 and 33,000 ppm females were significantly less than those of the vehicle controls, and 33,000 ppm males lost weight during the study. Feed consumption by exposed groups of males and females was less than that by the vehicle controls throughout the study. Clinical chemistry results of these studies indicated that trans-cinnamaldehyde administration, at the doses selected, induced an increase in serum bile acid concentration that suggests a hepatic effect in both male and female rats. Gross lesions observed at necropsy included multifocal to diffuse white nodules of the forestomach mucosa in 8,200 ppm or greater males and females. Increased incidences of nonneoplastic lesions of the forestomach included squamous epithelial hyperplasia in 8,200 ppm or greater males and females and chronic active inflammation in 33,000 ppm males and 16,500 and 33,000 ppm females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 4,100, 8,200, 16,500, or 33,000 ppm microencapsulated trans-cinnamaldehyde (equivalent to average daily doses of approximately 650, 1,320, 2,550, and 5,475 mg/kg to males and 625, 1,380, 2,680, and 5,200 mg/kg to females) for 3 months. Additional groups of 10 male and 10 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). One vehicle control male, one 4,100 ppm male, and one 33,000 ppm male died during the first week of the study due to inanition that resulted from difficulty with the feeder. Five 16,500 ppm and eight 33,000 ppm male mice died during weeks 2 and 3 due to unpalatability of the dosed feed. Mean body weights of all exposed groups of males and of females exposed to 8,200 ppm or greater were significantly less than those of the vehicle controls. Feed consumption by 16,500 and 33,000 ppm mice was less than that by the vehicle controls during weeks 1 and 2. The incidence of squamous epithelial hyperplasia of the forestomach mucosa in 33,000 ppm females was significantly increased, and olfactory epithelial degeneration of the nasal cavity occurred in 16,500 and 33,000 ppm males and females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were fed diets containing 1,000, 2,100, or 4,100 ppm microencapsulated trans-cinnamaldehyde for 2 years. Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1,000, 2,100, or 4,100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg to males and females. Survival of 4,100 ppm males was greater than that of the vehicle controls. Mean body weights of 4,100 ppm males and females were generally less than those of the vehicle controls throughout the study. Feed consumption by 2,100 and 4,100 ppm males and 4,100 ppm females was less than that by the vehicle controls at the beginning and end of the study. There were no neoplasms or nonneoplastic lesions that were attributed to exposure to trans-cinnamaldehyde. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were fed diets containing 1,000, 2,100, or 4,100 ppm microencapsulated trans-cinnamaldehyde for 2 years. Additional groups of 50 male and 50 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1,000, 2,100, or 4,100 ppm delivered average daily doses of approximately 125, 270, or 550 mg/kg to males and females. Survival of males in the 2,100 ppm group was less than that of the vehicle control group. Mean body weights of 2,100 and 4,100 ppm males and females were generally less than those of the vehicle controls throughout the study, and mean body weights of 1,000 ppm males were less after week 74. Feed consumption by exposed mice was similar to that by the vehicle controls. The incidences of olfactory epithelial pigmentation in 4,100 ppm males and in 2,100 and 4,100 females were significantly greater than those in vehicle controls. There were no neoplasms that were attributed to exposure to trans-cinnamaldehyde. GENETIC TOXICOLOGY: trans-cinnamaldehyde was mutagenic in S. typhimurium strain TA100 in the presence of induced mouse liver S9 activation enzymes only. All other strain and activation combinations, including the standard rat and hamster derived liver S9 fractions yielded negative results. trans-cinnamaldehyde induced sister chromatid exchanges in Chinese hamster ovary cells with and without induced rat liver S9 activation. No significant increase in the frequency of chromosomal aberrations occurred in Chinese hamster ovary cells cultured with trans-cinnamaldehyde, with or without induced rat liver S9. In tests for induction of germ cell genetic damage in male Drosophila melanogaster, trans-cinnamaldehyde induced a significant increase in the frequency of sex-linked recessive lethal mutations when administered by abdominal injection; however, no induction of reciprocal translocations occurred in germ cells of treated males. No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood of male or female mice administered trans-cinnamaldehyde in dosed feed for 3 months. CONCLUSIONS: Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of transcinnamaldehyde in male or female F344/N rats exposed to 1,000, 2,100, or 4,100 ppm. There was no evidence of carcinogenic activity of trans-cinnamaldehyde in male or female B6C3F1 mice exposed to 1,000, 2,100, or 4,100 ppm. Exposure to trans-cinnamaldehyde resulted in olfactory epithelial pigmentation in male and female mice.     

Toxicology and carcinogenesis study of styrene-acrylonitrile trimer in F344/N rats (perinatal and postnatal feed studies)

Styrene-acrylonitrile trimer (SAN Trimer) is a mixture of isomers formed by the condensation of two moles of acrylonitrile and one mole of styrene and has a molecular weight of 210. The mixture is composed of two structural forms: 4-cyano-1,2,3,4-tetrahydro-a-methyl-1-naphthaleneacetonitrile (THNA, CAS No. 57964-39-3) and 4-cyano-1,2,3,4-tetrahydro-1-naphthalenepropionitrile (THNP, CAS No. 57964-40-6). The THNA form consists of four stereoisomers. [Structure:see text]. The THNP form consists of two stereoisomers. [Structure:see text]. SAN Trimer is a by-product of the production of acrylonitrile styrene plastics and is created in specific manufacturing processes for polymers of acrylonitrile and styrene. In June 1998, due to community concerns about the toxicity of SAN Trimer, it was nominated to the NTP for carcinogenicity testing by a member of Congress. Male and female F344/N rats were exposed to SAN Trimer in feed in perinatal and postnatal studies for 7 weeks, 18 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, and in rat reticulocytes, leukocytes, liver cells, and brain cells. In vivo comet and micronucleus assays were performed in the juvenile rats. 7-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 250, 500, 1,000, 2,000, or 4,000 ppm SAN Trimer (equivalent to average daily doses of approximately 50, 90, 175, 270, or 410 mg SAN Trimer/kg body weight to males and 45, 90, 185, 295, or 430 mg/kg to females) for 2 weeks postweaning; the dams of these rats were fed the same concentrations of SAN Trimer from gestation day 7 until the pups were weaned. One 4,000 ppm male rat died 3 days after weaning; all other rats that started the postweaning phase survived to the end of the study. Mean body weights of 1,000, 2,000, and 4,000 ppm males and 2,000 and 4,000 ppm females were significantly less than those of the controls; weaning mean body weights were reduced in 4,000 ppm males and females and in 2,000 ppm females. Feed consumption by 2,000 and 4,000 ppm males and females was less than that by the control groups. Thinness in 4,000 ppm male rats was the only clinical finding related to SAN Trimer exposure. Nonneoplastic lesions were observed in the brain, thymus, spleen, liver, kidney, and reproductive organs of males and females and were considered due to overt toxicity. 18-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets of 0, 100, 200, 400, 800, or 1,600 ppm SAN Trimer (equivalent to average daily doses of 10, 20, 40, 80, or 150 mg/kg to males and females) for 3 months postweaning; the dams of these rats were fed the same concentrations from gestation day 7 until the pups were weaned. All rats survived to the end of the study. Mean body weights of 1,600 ppm males and females exposed to 200 ppm or greater were significantly less than those of the controls. At termination, brown staining of the urogenital fur was observed in females exposed to 200 ppm or greater. The liver weights of all exposed groups of males and the spleen weights of 800 and 1,600 ppm males and 1,600 ppm females were significantly greater than those of the controls. There were no significant differences in sperm parameters of male rats or the estrous cyclicity of female rats administered 400, 800, or 1,600 ppm in the diet when compared to the control groups. No exposure-related histopathologic lesions were observed. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female core study rats were fed diets of 0, 400, 800, or 1,600 ppm SAN Trimer (equivalent to average daily doses of approximately 20, 40, or 75 mg/kg to males and 20, 40, or 85 mg/kg to females) for 2 years. Special study groups of 20 males and 20 females were fed the same exposure concentrations and were evaluated at 27, 52, and 78 weeks for hematology and clinical chemistry or at 26, 51, and 77 weeks for urinalysis. The dams of core and special study rats were fed the same concentrations from gestation day 7 until the pups were weaned. Mean body weights of 1,600 ppm males were less than 90% of the controls after week 1; mean body weights of 800 and 1,600 ppm females were less than 90% of the controls after weeks 41 and 13, respectively. Feed consumption by exposed groups of males and females was generally similar to that by the control groups. Brown staining of the urogenital fur was observed in all exposed groups, and the number of animals affected increased with increasing exposure concentration. Rare neoplasms were present in the central nervous system of male and female rats. In the original evaluation, the 800 and 1,600 ppm groups of male rats each had one astrocytoma and one granular cell tumor in the brain. Also in the brain, one 400 ppm female had a granular cell tumor and one control, one 400 ppm, and one 800 ppm female had a mixed cell glioma. In the spinal cord, one astrocytoma was noted in a 1,600 ppm male in the original evaluation. In the expanded review of the spinal cord, one granular cell tumor was found in a 400 ppm male and one meningioma was found in an 800 ppm female. There were statistically significant increases in the incidence of spinal nerve root degeneration in 1,600 ppm males and the incidences of sciatic nerve degeneration in 800 and 1,600 ppm females. More importantly, there were increases in the severities of both nerve lesions in males and in the severity of spinal nerve root degeneration in females. The incidences of bone marrow hyperplasia were significantly increased in 1,600 ppm males and females and 800 ppm females. Incidences of bone marrow granulomatous inflammation were increased in 1,600 ppm males and 800 and 1,600 ppm females, and the increase in the 800 ppm females was significant. Because this lesion is very rare and did not occur in control animals, it should be considered biologically significant. In the liver, the incidence of eosinophilic focus was significantly increased in 1,600 ppm males and the incidences of mixed cell focus were significantly increased in 400 and 1,600 ppm males. Incidences of mixed cell focus were increased in the liver of all exposed groups of females, and the increase was significant in the 1,600 ppm group. The incidence of transitional epithelial hyperplasia of the urinary bladder in 1,600 ppm females was significantly greater than that in the controls. There were significant decreases in the incidences of pituitary gland pars distalis adenoma in 1,600 ppm males and females, and the incidences in both sexes occurred with negative trends. The incidences of mammary gland fibroadenoma occurred in females with a negative trend, and the incidences in 800 and 1,600 ppm females were significantly less than that in the control group. The incidences of mononuclear cell leukemia in all exposed groups of males and females were significantly less than those in the controls. GENETIC TOXICOLOGY: SAN Trimer (Batch 3) was not mutagenic in Salmonella typhimurium strains TA98 or TA100 or in Escherichia coli strain WP2 uvrA/pKM101 in tests conducted with and without exogenous metabolic activation. In vivo, however, results of a comet assay indicated significantly increased levels of DNA damage in brain cells of male and female juvenile rats following administration of SAN Trimer (Batch 3) by oral gavage. Dose-related increases in DNA damage in liver cells of these rats were also observed, but the increases were smaller than those observed in brain cells and were judged to be equivocal in both males and females. Indications of DNA damage following exposure to SAN Trimer were also seen in leukocytes of male and female rats. Increases in male rats were significant, but in females, observed levels of DNA damage did not correlate with dose. Therefore, the results were judged to be positive in males and equivocal in females. In addition to the positive comet assay results, significant increases in the frequencies of micronucleated reticulocytes were observed in peripheral blood of male and female juvenile rats dosed with SAN Trimer. CONCLUSIONS: Under the conditions of this 2-year feed study preceded by perinatal exposure, there was no evidence of carcinogenic activity of SAN Trimer in male and female F344/N rats given feed containing 400, 800, or 1,600 ppm SAN Trimer. Exposure to SAN Trimer resulted in increased incidences and/or severities of peripheral nerve degeneration in male and female F344/N rats, increased incidences of nonneoplastic lesions of the bone marrow and liver in male and female F344/N rats, and of nonneoplastic urinary bladder lesions in female F344/N rats. The incidences of pituitary gland adenoma and mononuclear cell leukemia in male and female F344/N rats and mammary gland fibroadenoma in female F344/N rats were decreased.     

Toxicology and carcinogenesis studies of o-nitrotoluene sulfone (CAS no. 88-72-2) in F344/N rats and B6C3F(1) mice (feed studies)

[structure: see text] o-Nitrotoluene is used to synthesize agricultural and rubber chemicals, azo and sulfur dyes, and dyes for cotton, wool, silk, leather, and paper. o-Nitrotoluene was nominated for study by NIOSH and the NTP based on its considerable human exposure as well as the absence of long-term studies of carcinogenicity in rodents. Male and female F344/N rats and B6C3F1 mice were exposed to o-nitrotoluene (greater than 99% pure) in feed for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-YEAR STUDY IN RATS: In the core study, groups of 60 male and 60 female rats were fed diets containing 625, 1,250, or 2,000 ppm o-nitrotoluene (equivalent to average daily doses of approximately 25, 50, or 90 mg o-nitrotoluene/kg body weight to males and 30, 60, or 100 mg/kg to females) for 105 weeks. In a 3-month stop-exposure study, groups of 70 male rats were fed diets containing 2,000 or 5,000 ppm o-nitrotoluene (equivalent to average daily doses of approximately 125 or 315 mg/kg) for 13 weeks followed by undosed feed for the remainder of the study. A group of 70 male rats receiving undosed feed served as a control group for both male rat studies; 60 female rats receiving undosed feed were the control group for the female core study. Ten control males and 10 males from each stop-exposure group were sacrificed at 3 months. Survival, Body Weights, and Feed Consumption: All 2,000 ppm core study, all 5,000 ppm stop-exposure, and all but three core study 1,250 ppm male rats died before the end of the studies. Survival of 625 ppm core study and 2,000 ppm stop-exposure males and of 2,000 ppm females was significantly less than that of the controls. Mean body weights of all exposed groups of males except the 625 ppm group were generally less than those of the controls throughout the study. Mean body weights of 2,000 ppm females were less than those of the controls during year 2 of the study. Feed consumption by exposed groups of rats was similar to that by the controls. Biomarkers of Exposure: Three urinary metabolites were followed during the study as biomarkers of exposure. The ratios of o-nitrobenzoic acid to creatinine and of o-nitrobenzylmercapturic acid to creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. The ratio of o-aminobenzoic acid to creatinine was not related to exposure concentration. Pathology Findings: The incidences of malignant mesothelioma in male rats occurred with positive trends in both the core and stop-exposure studies and were significantly greater in exposed groups than in the controls. Incidences of subcutaneous skin neoplasms (fibroma, fibrosarcoma, and lipoma) were increased in exposed groups of males, while the incidences of fibroma or fibrosarcoma (combined) were increased in exposed females. In all exposed groups of males and females except 2,000 ppm core study males, the incidences of mammary gland fibroadenoma were significantly increased. The incidences of mammary gland hyperplasia were significantly increased in 625 and 1,250 ppm females. Increased incidences of mesothelioma, skin neoplasms, and mammary gland fibroadenoma in the stop-exposure males indicated that 3 months of dosing were sufficient to produce a carcinogenic effect. Liver weights of 5,000 ppm stop-exposure males were significantly greater than those of the controls at 3 months. The incidences of hepatocellular adenoma in 2,000 ppm core study males and females and of hepatocellular adenoma or carcinoma (combined) in 2,000 ppm core study and 5,000 ppm stop-exposure males were significantly increased. Cholangiocarcinoma occurred in three 5,000 ppm stop-exposure males, and a single hepatocholangiocarcinoma occurred in a 625 ppm male and in a 2,000 ppm core study male. Nonneoplastic lesions of the liver included eosinophilic, mixed cell, and clear cell foci in exposed groups of males and females and mixed cell infiltrate in exposed males and basophilic focus in exposed females. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 5,000 ppm stop-exposure males, as were alveolar/bronchiolar hyperplasia in most exposed groups of males and females. The incidences of hematopoietic cell proliferation of the spleen and of hyperplasia of the mandibular lymph node (females) and bone marrow were increased in exposed groups of males at 3 months and/or 2 years and in exposed groups of females at 2 years. The incidences of mononuclear cell leukemia were significantly decreased in all groups of males exposed to 1,250 ppm or greater and in all exposed groups of females; the incidence of testicular interstitial cell adenoma was significantly decreased in 5,000 ppm stop-exposure males. 2-YEAR STUDY IN MICE: Groups of 60 male and 60 female mice were fed diets containing 0, 1,250, 2,500, or 5,000 ppm o-nitrotoluene (equivalent to average daily doses of approximately 165, 360, or 700 mg/kg to males and 150, 320, or 710 mg/kg to females) for 105 weeks. Survival, Body Weights, and Feed Consumption: All 2,500 and 5,000 ppm males died before the end of the study. Survival of 1,250 ppm males and 5,000 ppm females was significantly less than that of the controls. Mean body weights of exposed males and 5,000 ppm females were generally less than those of the controls throughout the study, and those of 2,500 ppm females were less during the second year of the study. Feed consumption by 5,000 ppm males was less than that by the controls. Biomarkers of Exposure: Three urinary metabolites were followed during the study as biomarkers of exposure. The ratios of o-nitrobenzoic acid to creatinine determined at 2 weeks and at 3, 12, and 18 months were linearly related to exposure concentration in males and females. The concentrations of o-nitrobenzylmercapturic acid and o-aminobenzoic acid were below the limit of quantitation at most time points. Pathology Findings: The incidences of hemangiosarcoma in all exposed groups of males and in 5,000 ppm females were significantly greater than those in the controls. Large intestine (cecum) carcinomas were observed in all exposed groups except 5,000 ppm males. The incidences of hepatocellular neoplasms were significantly increased in 2,500 and 5,000 ppm females. Nonneoplastic liver lesions including eosinophilic and basophilic foci and minimal to mild necrosis were enhanced in exposed males and females. Also present were focal hepatocyte syncytial alteration in exposed males and hepatocyte necrosis and focal hepatocyte cytoplasmic vacuolization in 5,000 ppm females. Renal tubule pigmentation occurred more frequently in exposed groups of males and in 5,000 ppm females than in the controls. Olfactory epithelial degeneration occurred in every male and female mouse exposed to 2,500 or 5,000 ppm, and the severity of this lesion increased with increasing exposure concentration. GENETIC TOXICOLOGY: o-Nitrotoluene was not mutagenic in any of several strains of S. typhimurium, with or without metabolic activation enzymes (S9). Sister chromatid exchanges were significantly increased in cultured Chinese hamster ovary cells following exposure to o-nitrotoluene in the presence of S9; an equivocal response was seen without S9. o-Nitrotoluene did not induce chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9. o-Nitrotoluene did not induce a significant increase in the frequency of micronuclei in bone marrow polychromatic erythrocytes of male rats or male mice when administered by intraperitoneal injection. Results of a peripheral blood micronucleus test were equivocal for male mice and negative for female mice administered o-nitrotoluene in feed for 13 weeks. CONCLUSIONS: Under the conditions of these studies, there was clear evidence of carcinogenic activity* of o-nitrotoluene in male rats based on increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma, and liver neoplasms. The increased incidences of lung neoplasms in male rats were also considered to be exposure related. There was clear evidence of carcinogenic activity of o-nitrotoluene in female rats based on increased incidences of subcutaneous skin neoplasms and mammary gland fibroadenoma. The increased incidence of hepatocellular adenoma in female rats was also considered to be exposure related. There was clear evidence of carcinogenic activity of -o-nitrotoluene in male and female mice based on increased incidences of hemangiosarcoma, carcinoma of the large intestine (cecum), and hepatocellular neoplasms (females only). Exposure to o--nitrotoluene caused increased incidences of nonneoplastic lesions of the mammary gland (females only), liver, bone marrow, spleen, lung, and mandibular lymph node (females only) in male and female rats and of the liver, kidney, and nose in male and female mice. Decreased incidences of mononuclear cell leukemia occurred in exposed groups of rats; the incidence of testicular interstitial cell adenoma was decreased in exposed male rats. [tables: see text]     

Carcinogenicity of <Emphasis Type="Italic">ortho</Emphasis>-phenylenediamine dihydrochloride in rats and mice by two-year drinking water treatment

The carcinogenicity of ortho-phenylenediamine (o-PD) was examined by administrating o-phenylenediamine dihydrochloride (o-PD2HCl) in dinking water to groups of 50 F344/DuCrj rats and 50 Crj:BDF₁ mice of both sexes for 2 years. The drinking water concentration of o-PD2HCl was 0, 500, 1,000 or 2,000 ppm (wt/wt) for male rats; 0, 250, 500 or 1,000 ppm for female rats; 0, 500, 1,000 or 2,000 ppm for male mice; and 0, 1,000, 2,000 or 4,000 ppm for female mice. Two-year administration of o-PD2HCl produced a dose-dependent increase in the incidences of hepatocellular adenomas and carcinomas in rats of both sexes and in female mice, and hepatocellular adenomas in male mice. In mice, the incidences of hepatocellular adenomas were increased at the lowest dose used in both males and females. Metastasis from hepatocellular carcinomas of rats occurred predominantly in the lung. Incidences of transitional cell papillomas and carcinomas in the urinary bladder were noted in male rats administered 2,000 ppm, together with an increased incidence of papillary and/or nodular hyperplasia of transitional epithelium. In mice, the incidence of papillary adenomas in the gall bladder, which is rare in mice, was increased in both males and females administered 2,000 ppm. Thus, o-PD is carcinogenic in two species, i.e., rats and mice of both sexes.     

Toxicology and carcinogenesis studies of dibromoacetic acid (Cas No. 631-64-1) in F344/N rats and B6C3F1 mice (drinking water studies)

Dibromoacetic acid is a water disinfection by-product. Dibromoacetic acid was nominated to the National Toxicology Program by the United States Environmental Protection Agency for toxicity and carcinogenicity studies in rats and mice because of widespread human exposure and because a related dihaloacetate, dichloroacetate, was found to be carcinogenic to the liver of rats and mice. Drinking water was selected as the route of exposure to mimic human exposure to this chemical. Male and female F344/N rats and B6C3F1 mice were exposed to dibromoacetic acid (greater than 99% pure) in drinking water for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and peripheral blood erythrocytes of exposed mice. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were exposed to 0, 125, 250, 500, 1,000, or 2,000 mg/L dibromoacetic acid in drinking water for 2 weeks, equivalent to average daily doses of approximately 17, 32, 67, 134, 270 (males), or 257 (females) mg dibromoacetic acid/kg body weight. All rats survived to the end of the study. Mean body weight gains of 1,000 mg/L males and of 500 mg/L females were significantly greater than those of the controls. Water consumption by exposed and control groups was similar. Liver weights of exposed males and females were significantly increased. Right testis weights of males exposed to 500 mg/L or greater were significantly decreased. The incidences of hepatocytic cytoplasmic alteration were significantly increased in males exposed to 500 mg/L or greater and in 2,000 mg/L females. Testicular lesions, characterized by a delay in spermiation and retained spermatids, were noted in males exposed to 500 mg/L or higher concentrations. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were exposed to 0, 125, 250, 500, 1,000, or 2,000 mg/L dibromoacetic acid (equivalent to average daily doses of approximately 24, 47, 95, 178, or 370 mg/kg to males and 22, 53, 88, 166, or 309 mg/kg to females) in drinking water for 2 weeks. All mice survived to the end of the study. Mean body weight gains of 250 and 500 mg/L males were significantly greater than those of the controls. Water consumption by exposed and control groups was similar. Liver weights of males and females in the 1,000 and 2,000 mg/L groups were significantly increased. Thymus weights of males and females in the 1,000 and 2,000 mg/L groups were significantly less than those of controls. The incidences of thymus atrophy were significantly increased in 1,000 and 2,000 mg/L males and 2,000 mg/L females. The incidences of morphological changes to the germinal epithelium of the testes were increased in males exposed to 1,000 or 2,000 mg/L. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to 0, 125, 250, 500, 1,000, or 2,000 mg/L dibromoacetic acid (equivalent to average daily doses of approximately 10, 20, 40, 90, and 166 mg/kg to males and 12, 23, 48, 93, and 181 mg/kg to females) in drinking water for 3 months. All rats survived to the end of the study. Mean body weights of male and female rats in the 2,000 mg/L group were significantly less than those of controls. Water consumption by the 2,000 mg/L males at weeks 1 and 13 and by females at week 13 was less than that by controls. Small decreases in the erythron and platelet counts occurred in rats exposed to 2,000 mg/L; minimally impaired erythropoiesis was also seen in 1,000 mg/L rats. Liver weights of all exposed groups of males and females were significantly increased. Male rats in the 2,000 mg/L group had significantly decreased testis weights. Testicular atrophy was noted in the 2,000 mg/L group, and retained spermatids were observed in the 500 and 1,000 mg/L groups. In the pituitary gland of male rats exposed to 2,000 mg/L, the incidence of cellular hypertrophy was significantly increased. The incidences of hepatocellular vacuolization were significantly increased in males exposed to 500 mg/L or greater and in females exposed to 2,000 mg/L. Hematopoietic cell proliferation was noted in females in the 2,000 mg/L group. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed to 0, 125, 250, 500, 1,000, or 2,000 mg/L dibromoacetic acid (equivalent to average daily doses of approximately 16, 30, 56, 115, and 230 mg/kg to males and 17, 34, 67, 132, and 260 mg/kg to females) in drinking water for 3 months. All mice survived to the end of the study. Mean body weights and body weight gains of female mice in the 2,000 mg/L group and the mean body weight gain of 2,000 mg/L males were significantly less than those of controls. Water consumption by males in the 2,000 mg/L group was decreased at weeks 1 and 13 relative to controls. Small decreases in mean cell hemoglobin and platelet counts occurred in 2,000 mg/L male mice. Liver weights of males and females exposed to 500 mg/L or greater were significantly increased. Hepatocellular cytoplasmic vacuolization was present in most mice and the severity was increased in 1,000 and 2,000 mg/L males and females. The incidences of abnormal testicular morphology were significantly increased in 1,000 and 2,000 mg/L males. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to drinking water containing 0, 50, 500, and 1,000 mg/L dibromoacetic acid for 2 years (equivalent to average daily doses of approximately 2, 20, and 40 mg/kg to males and 2, 25, and 45 mg/kg to females). Survival of exposed rats was similar to that of the control groups. Mean body weights of 1,000 mg/L males and females were less than those of the controls after weeks 29 and 53, respectively, and those of 500 mg/L males and females were less after weeks 57 and 85, respectively. Water consumption by males and females exposed to 1,000 mg/L was less than that by controls during year 2 of the study. The incidence of malignant mesothelioma was significantly increased in 1,000 mg/L male rats. A positive trend in the incidence of mononuclear cell leukemia occurred in female rats, and the incidence in 1,000 mg/L females was significantly increased. The incidences of mononuclear cell leukemia were increased in 50 and 500 mg/L males. The incidences of cystic degeneration of the liver were significantly increased in all exposed groups of male rats. The incidences of alveolar epithelial hyperplasia were significantly increased in 500 and 1,000 mg/L females, and the incidences of nephropathy were significantly increased in all exposed groups of females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed to drinking water containing 0, 50, 500, and 1,000 mg/L dibromoacetic acid for 2 years (equivalent to average daily doses of approximately 4, 45, and 87 mg/kg to males and 4, 35, and 65 mg/kg to females). Survival of exposed mice was similar to that of the controls. Mean body weights of 50 and 500 mg/L male mice were greater than those of the controls after week 85. Water consumption by exposed mice was generally similar to that by controls throughout the study. The incidences of liver neoplasms occurred with positive trends in male and female mice. The incidences of multiple hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) were significantly increased in all exposed groups of males and in 500 and 1,000 mg/L females. The incidences of hepatoblastoma were significantly increased in 500 and 1,000 mg/L males, and the incidences of hepatocellular carcinoma were significantly increased in 1,000 mg/L males and 500 mg/L females. The incidences of alveolar/bronchiolar adenoma occurred with positive trends in males and females, and the incidence in 500 mg/L male mice was significantly greater than that in controls. GENETIC TOXICOLOGY: Dibromoacetic acid was mutagenic in Salmonella typhimurium strain TA100 with and without rat or hamster liver metabolic activation enzymes (S9); no activity was detected in strain TA98, with or without S9. Increased frequencies of micronucleated normochromatic erythrocytes were observed in peripheral blood samples from male, but not female, mice administered dibromoacetic acid in drinking water for 3 months. CONCLUSIONS: Under the conditions of these studies, there was some evidence of carcinogenic activity of dibromoacetic acid in male rats based on an increased incidence of malignant mesothelioma. The increased incidences of mononuclear cell leukemia in male rats may have been related to dibromoacetic acid exposure. There was some evidence of carcinogenic activity of dibromoacetic acid in female rats based on an increased incidence and positive trend of mononuclear cell leukemia. There was clear evidence of carcinogenic activity of dibromoacetic acid in male and female mice based on increased incidences of hepatocellular neoplasms and hepatoblastoma (males only). Increased incidences of lung neoplasms in male mice were also considered to be exposure related. The slight increased incidence of lung neoplasms in female mice may have been related to dibromoacetic acid exposure. Exposure to dibromoacetic acid for 2 years caused increased incidences of cystic degeneration of the liver in male rats, increased incidences of alveolar epithelial hyperplasia and nephropathy in female rats, and increased incidences of splenic hematopoiesis in male mice.     

Toxicology and carcinogenesis studies of alpha-methylstyrene (Cas No. 98-83-9) in F344/N rats and B6C3F1 mice (inhalation studies)

alpha-Methylstyrene is used in the production of acrylonitrile-butadiene-styrene resins and copolymers, which improve the impact and heat-resistant properties of polymers, specialty grades of plastics, rubber, and protective coatings. alpha-Methylstyrene also moderates polymerization rates and improves product clarity in coatings and resins. Low molecular weight liquid polymers are used as plasticizers in paints, waxes, adhesives, and plastics. alpha-Methylstyrene was nominated by the U.S. Environmental Protection Agency for toxicologic evaluation and genotoxicity studies based on its high production volume and limited information available on its toxicity. Male and female F344/N rats and B6C3F1 mice were exposed to alpha-methylstyrene (99.5% pure) by inhalation for 3 months or 2 years. Inhalation studies were conducted because the primary route of human exposure is via inhalation. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were exposed by whole-body inhalation to alpha-methylstyrene at concentrations of 0, 75, 150, 300, 600, or 1,000 ppm for 6 hours per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Kidney weights were significantly increased in 1,000 ppm males and 600 and 1,000 ppm females. Statistically significant increases in liver weights occurred in 150 ppm or greater males and 600 and 1,000 ppm females. The incidences of renal hyaline droplet accumulation were similar between exposed groups and chamber control groups, but the severity of hyaline droplet accumulation in 600 and 1,000 ppm males was greater than in chamber controls. Consistent with the hyaline droplet accumulation, an exposure-related increase in alpha2μ-globulin was detected in the kidneys of males exposed to alpha-methylstyrene. Morphologic changes were not detected in the liver. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed by whole-body inhalation to alpha-methylstyrene at concentrations of 0, 75, 150, 300, 600, or 1,000 ppm for 6 hours per day, 5 days per week for 14 weeks. Two female mice in the 1,000 ppm group died before exposure on day 3. Final mean body weights of 600 and 1,000 ppm males and 75, 300, and 1,000 ppm females were significantly less than those of the chamber controls; final mean body weight gains of mice exposed to 300 ppm or greater were also significantly less. Moderate to severe sedation (males only) and ataxia were observed in 1,000 ppm mice. The absolute liver weights of 600 and 1,000 ppm females and the relative liver weights of 300, 600, and 1,000 ppm males and females were significantly increased. The estrous cycle lengths of 600 and 1,000 ppm female mice were significantly longer than that of the chamber controls. Minimal to mild centrilobular hypertrophy was present in the livers of male and female mice exposed to 600 or 1,000 ppm alpha-methylstyrene. The incidences of exposure-related nasal lesions, including atrophy and hyperplasia of Bowman's glands and atrophy and metaplasia of the olfactory epithelium, were significantly increased in all exposed groups of males and females. The incidences of hyaline degeneration, characterized by the accumulation of eosinophilic globules in the cytoplasm of the respiratory epithelium, were significantly increased in females exposed to 150 ppm or greater. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed by whole body inhalation to alpha-methylstyrene at concentrations of 0, 100, 300, or 1,000 ppm for 6 hours per day, 5 days per week, except holidays, for 105 weeks. Survival rates of exposed male and female rats were similar to those of the chamber controls. The mean body weights of 1,000 ppm males and females were less than those of the chamber control groups during year 2 of the study. Two 1,000 ppm males and one 300 ppm male had renal tubule carcinomas, and one 300 ppm male had a renal tubule adenoma. Because of the neoplasms observed in 300 and 1,000 ppm males at the end of the 2-year study and the finding of alpha2μ-globulin accumulation in the kidneys at 3 months, which is often associated with kidney neoplasms, additional step sections of kidney were prepared; additional males with focal hyperplasia or adenoma were identified. The incidences of renal tubule adenoma and carcinoma (combined) in the 1,000 ppm males were significantly greater than those in the chamber controls when the single and step sections were combined. The incidence of mineralization of the renal papilla was significantly increased in 1,000 ppm males. The incidence of mononuclear cell leukemia in 1,000 ppm males was significantly increased compared to the chamber controls. In the nose, the incidences of basal cell hyperplasia were significantly increased in all exposed groups of males and females, and the incidences of degeneration of the olfactory epithelium were increased in 1,000 ppm males and females and 300 ppm females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed by whole body inhalation to alpha-methylstyrene at concentrations of 0, 100, 300, or 600 ppm for 6 hours per day, 5 days per week, except holidays, for 105 weeks. Survival of all exposed male and female mice was similar to that of the chamber control groups. Mean body weights of 600 ppm males were less than those of the chamber control group throughout the study, and those of 600 ppm females were less after week 13. The mean body weights of 300 ppm males and females were less than those of the chamber controls during much of the study, but these groups recovered by the end of the study. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in the 100 and 600 ppm males and in all exposed groups of females. The incidences of hepatocellular adenoma were significantly increased in all exposed groups of females, and the incidences in all exposed groups of males and females exceeded the historical range for chamber controls. The incidences of hepatocellular carcinoma and eosinophilic foci of the liver were significantly increased in 600 ppm females. The incidences of olfactory epithelial metaplasia and hyperplasia of the glands overlying the olfactory epithelium were significantly increased in all exposed groups of males and females. In addition, atrophy of the olfactory epithelium was significantly increased in 300 and 600 ppm males. The incidence and severity of nephropathy were increased in 600 ppm females compared to chamber controls. Epithelial hyperplasia of the forestomach also was present in male mice. GENETIC TOXICOLOGY: alpha-Methylstyrene was not mutagenic in four strains of Salmonella typhimurium, with or without rat or hamster liver metabolic activation enzymes (S9). alpha-Methylstyrene did not induce chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9 activation, but did significantly increase the frequency of sister chromatid exchanges in cultures exposed in the presence of S9. In vivo, no significant increases in the frequencies of micronucleated erythrocytes were seen in blood samples of male mice obtained at the conclusion of the 3-month study. However, in female mice from the 3-month study, a significant increase in micronucleated erythrocytes was observed in the 1,000 ppm group. CONCLUSIONS: Under the conditions of this 2-year inhalation study, there was some evidence of carcinogenic activity of alpha-methylstyrene in male F344/N rats based on increased incidences of renal tubule adenomas and carcinomas (combined). The increased incidence of mononuclear cell leukemia in 1,000 ppm male F344/N rats may have been related to alpha-methylstyrene exposure. There was no evidence of carcinogenic activity of alpha-methylstyrene in female F344/N rats exposed to 100, 300, or 1,000 ppm. There was equivocal evidence of carcinogenic activity of alpha-methylstyrene in male B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma or carcinoma (combined). There was clear evidence of carcinogenic activity of alpha-methylstyrene in female B6C3F1 mice based on increased incidences of hepatocellular adenomas and carcinomas. Exposure of rats to alpha-methylstyrene resulted in kidney toxicity, which in males exhibited some features of alpha2μ-globulin nephropathy. Exposure to alpha-methylstyrene resulted in nonneoplastic lesions of the nose in male and female rats and mice and of the liver and kidney in female mice.     

Chronic Toxicity/Oncogenicity of Dimethylformamide in Rats and Mice Following Inhalation Exposure

The potential chronic toxicity and oncogenicity of dimethylformamide(DMF) was evaluated by exposing male and female rats and miceto 0, 25, 100, or 400 ppm DMF for 6 hr/day, 5 days/week for18 months (mice) or 2 years (rats). Clinical pathology was evaluatedat 3, 6, 12, 18, and 24 (rats only) months. An interim euthanasiafor rats occurred at 12 months and hepatic cell proliferationin rats and mice was examined at 2 weeks, 3 months, and 12 months.No compound-related effects on clinical observations or survivalwere observed. Body weights of rats exposed to 100 (males only)and 400 ppm were reduced. Conversely, body weights were increasedin 400 ppm mice. No hematologic changes were observed in eitherspecies. Serum sorbitol dehydrogenase activity was increasedin rats exposed to 100 or 400 ppm. There were no compound-relatedeffects on the estrous cycle of rats or mice at any concentration.Compound-related morphological changes were observed only inthe liver. In rats, exposure to 100 and 400 ppm produced increasedrelative liver weights, centrilobular hepatocellular hypertrophy,lipofuscin/hemosiderin accumulation in Kupifer cells, and centrilobularsingle cell necrosis (400 ppm only). In mice, increased liverweights (100 ppm males, 400 ppm both sexes), centrilobular hepatocellularhypertrophy, accumulation of lipofuscin/hemosiderin in Kupffercells, and centrilobular single cell necrosis were observedin all exposure groups. These observations occurred in a dose-responsefashion and were minimal at 25 ppm. No increase in hepatic cellproliferation was seen in mice or female rats. Slightly higherproliferation was seen in male rats exposed to 400 ppm at 2weeks and 3 months but not at 12 months. Dimethylformamide wasnot oncogenic under these experimental conditions in eitherthe rat or mouse.     

Toxicology and carcinogenesis studies of p,p'-dichlorophenyl sulfone (CAS No. 80-07-9) in F344/N rats and B6C3F1 mice (feed studies)

p,pN-Dichlorodiphenyl sulfone is used as a starting material in the production of polysulfones and polyethersulfones and as a component in reactive dyes in the textile industry; it is also a by-product of pesticide production. p,pN-Dichlorodiphenyl sulfone was nominated for study by the National Cancer Institute because of its history of high production and use, the prospect of increased production and use, and the absence of adequate toxicity testing. Male and female F344/N rats and B6C3F1 mice were exposed top,pN-dichlorodiphenyl sulfone (greater than 99% pure)in feed for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium,cultured Chinese hamster ovary cells, and mouse bone marrow. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 30, 100, 300, 1,000, or 3,000 ppm p,pN-dichlorodiphenyl sulfone (equivalent to average daily doses of approximately 2, 6, 19, 65, or 200 mgp,pN-dichlorodiphenyl sulfone/kg body weight) for 14 weeks. All rats survived until the end of the study. Mean body weights of groups exposed to 300 ppm or greater were significantly less than those of the controls. Liver weights of groups exposed to 100 ppm or greater and kidney weights of 1,000 and 3,000 ppm male rats were significantly greater than those of the controls. Centrilobular hepatocyte hypertrophy of the liver was observed in most male rats exposed to 100 ppm or greater and in all female rats exposed to 300 ppm or greater, and the severities were increased in 300 ppm males and 1,000 and 3,000 ppm males and females. The incidences of nephropathy in 1,000 and 3,000 ppm female rats were significantly increased. Dose-related increases in severity of nephropathy were observed in male rats. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 0, 30, 100, 300, 1,000, or 3,000 ppm p,pN-dichlorodiphenyl sulfone (equivalent to average daily doses of approximately 3.5, 15, 50, 165,or 480 mg/kg) for 14 weeks. All mice survived until the end of the study. Mean body weights of groups exposed to 300 ppm or greater were significantly less than those of the controls. Liver weights of groups exposed to 300 ppm or greater were significantly increased. Centrilobular hypertrophy of the liver was observed in most males exposed to 100 ppm or greater and in all females exposed to 1,000 or 3,000 ppm, and the severities generally increased with increasing exposure concentration. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were fed diets containing 0, 10 (males), 30, 100, or 300 (females) ppm p,pN-dichlorodiphenyl sulfone for 105 weeks. Dietary concentrations of 10, 30, and 100 ppm resulted in average daily doses of approximately 0.5, 1.5, and 5.0 mg/kg to males. Dietary concentrations of 30, 100,and 300 ppm resulted in average daily doses of approximately 1.6, 5.4, and 17 mg/kg to females. Additional groups of 10 male and 10 female rats were fed the same p,pN-dichlorodiphenyl sulfone-containing diets for 18 months and bled for plasma determinations of p,pN-dichlorodiphenyl sulfone at approximately 2 weeks and 3, 12, and 18 months. Survival of all exposed groups of male and female rats was similar to that of the control groups. Mean body weights of 30 and 100 ppm males were generally less than those of the controls during the latter part of the study, and mean body weights of 100 and 300 ppm female rats were less from weeks 30 and 18,respectively. Feed consumption by the exposed groups was similar to that by the controls throughout the study. The incidences of centrilobular hepatocyte hypertrophy in 100 ppm male and 100 and 300 ppm female rats were significantly greater than those in the controls. The incidences of bile duct hyperplasia and centrilobular degeneration were also significantly increased in 100 and 300 ppm females. No neoplasms were related to chemical exposure. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were fed diets containing 0, 30, 100, or 300 ppm p,pN-dichlorodiphenyl sulfone for 105 to 106 weeks. Dietary concentrations of 30, 100, and 300 ppm delivered average daily doses of approximately 4, 13, and 40 mg/kg to males and approximately 3, 10, and 33 mg/kg to females. Additional groups of 10 male and 10 female mice were fed the same p,pN-dichlorodiphenyl sulfone-containing diets for up to 12 months;three mice in each group were bled for plasma determinations of p,pN-dichloro-diphenyl sulfone at approximately 2 weeks or 3 or 12 months. Survival of all exposed groups of male and female mice was similar to that of the control groups. Mean body weights of 300 ppm mice were less than those of the controls throughout most of the study. Feed consumption by the exposed groups was similar to that by the controls throughout the study. The incidences of centrilobular hepatocyte hypertrophy in all exposed groups of male mice and in 100 and 300 ppm females were significantly greater than those in the controls. The incidence of eosinophilic foci in 300 ppm females was significantly increased. No neoplasms were related to chemical exposure. PHARMACOKINETICS OF p,pN-DICHLORODIPHENYL SULFONE: p,pN-Dichlorodiphenyl sulfone is rapidly absorbed from the gut and metabolized by a saturable process. Although some p,pN-dichlorodiphenyl sulfone is eliminated unchanged in feces and urine, most of the elimination is via metabolism. Mathematical modeling of the toxicokinetics supports the view that p,pN-dichlorodiphenyl sulfone induces enzymes involved in its metabolism. GENETIC TOXICOLOGY: p,pN-Dichlorodiphenyl sulfone was not mutagenic in any of several strains of Salmonella typhimurium, with or without metabolic activation enzymes (S9). Results of the sister chromatid exchange test in cultured Chinese hamster ovary cells were judged to be negative in the presence of S9 and equivocal in the absence of S9, but no induction of chromosomal aberrations was noted, with or without S9. In contrast to the in vitro results, positive results were obtained in an acute in vivo mouse bone marrow micronucleus assay with p,pN-dichlorodiphenyl sulfone administered by intraperitoneal injection three times over a dose range of 200 to 800 mg/kg. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity* of p,pN-dichlorodiphenyl sulfone in male F344/N rats exposed to 10, 30, or 100 ppm or in female F344/N rats exposed to 30, 100, or 300 ppm. There was no evidence of carcinogenic activity of p,pN-dichlorodiphenyl sulfone in male or female B6C3F1 mice exposed to 30,100, or 300 ppm. Exposure to p,pN-dichlorodiphenyl sulfone for 2 years caused increased incidences of nonneoplastic lesions of the liver in male and female rats and mice.     

Inhalation toxicity studies of the alpha,beta-unsaturated ketones: 2-cyclohexene-1-one

2-Cyclohexene-1-one (CHX) is a cyclic alpha,beta-unsaturated ketone with broad human exposure. CHX is an environmental pollutant and is present in tobacco smoke and in soft drinks sweetened with cyclamate. Interest in the toxicity of this class of compounds is due to their structural similarity to the cytotoxin acrolein. In a pilot study, rats and mice were exposed to 0, 20, 40, or 80 ppm CHX for 6 h/day. The study was terminated after 4 days due to acute toxicity in the high-dose groups. In a subsequent 14-day study, mice and rats were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day. All animals survived exposure until terminal sacrifice. Body weights were not significantly different from controls after 14 days of exposure. Liver/body weights were increased in male and female mice exposed to 5 and 10 ppm, and in male and female rats exposed to 10 ppm CHX. Ninety-day toxicity studies were conducted to provide data required to design chronic toxicity and carcinogenicity studies of CHX if it is determined such studies are necessary. Groups of 10 male and female F-344 rats and B6C3F1 mice were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day for 13 wk. All animals survived until sacrifice. Body weights were not significantly different from controls after 13 wk of exposure. Liver weights were increased in male and female mice exposed to 5 and 10 ppm and in male and female rats exposed to 10 ppm CHX. No adverse effects on bone-marrow micronuclei, sperm motility, or vaginal cytology were observed. Microscopic lesions included hyperplasia, and squamous metaplasia in the nasal cavity in rats and mice of both sexes at all doses. Nasal-cavity erosion and suppurative inflammation also occurred in high-dose mice. Larynx and lung were not affected in either sex or species. Dose-related hepatic centrilobular cytoplasmic vacuolation was seen in male rats only. These data suggest that CHX acts as an alkylating agent primarily producing toxicity at the exposure site.     

Toxicology and carcinogenesis studies of p,p'-dichlorodiphenyl sulfone in rats and mice.

p,p'-Dichlorodiphenyl sulfone (DDS) is used as a starting material in the production of polysulfones and polyethersufones, a family of thermoplastics. DDS was studied because of its high production volume and use. In toxicology studies, 10 Fischer 344 rats and 10 B6C3F1 mice/sex/group were fed diets containing 0, 30, 100, 300, 1,000 or 3,000 ppm DDS for 14 weeks. All animals survived until the end of the studies. Mean body weights of groups exposed to 300 ppm or greater were significantly decreased. Liver and kidney in rats and liver in mice were the major target organs of DDS toxicity. Dose-related increases in liver weights and incidences of centrilobular hepatocyte hypertrophy were observed in DDS-exposed groups. Nephropathy was seen in male and female rats only at and above 300 ppm. Neurotoxicity evaluations were negative in DDS-treated animals. Clinical chemistry and hematology parameters were minimally affected. In the 2-year toxicity and carcinogenicity studies, 50 rats and 50 mice/sex/group were fed diets containing 0, 10 (male rats), 30, 100, or 300 ppm DDS for 104 to 105 weeks. Survival of exposed groups was not affected. There were no clinical signs of toxicity related to DDS exposure. Final mean body weights were 2-17% lower in DDS-treated groups. Liver was the only target organ of DDS-induced toxicity. The incidence of centrilobular hepatocyte hypertrophy in mice and rats, and the incidence of bile duct hyperplasia and centrilobular degeneration in female rats was significantly greater than in controls. A no-observed-adverse-effect level (NOAEL) of 30 ppm DDS in the diet (1.5 mg/kg body weight) was established for rats. DDS was not carcinogenic in these studies.     

Toxicology and carcinogenesis studies of ginseng (CAS No. 50647-08-0) in F344/N rats and B6C3F1 mice (gavage studies)

CHEMICAL AND PHYSICAL PROPERTIES: Ginseng is a perennial aromatic herb widely used in herbal remedies, dietary supplements, cosmetics, and as a food additive. Ginseng was nominated for study by the National Cancer Institute based on significant human exposure through the uses described above and the lack of information on its toxicity. Male and female F344/N rats and B6C3F1 mice were administered extracts of ginseng root by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were administered ginseng in 0.5% aqueous methylcellulose by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week for 16 days. All rats survived to the end of the study. Mean body weight gain of 2,000 mg/kg males was significantly greater than that of the vehicle controls. There were no chemical-related gross or microscopic findings attributed to the administration of ginseng. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were administered ginseng in 0.5% aqueous methylcellulose by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week for 17 days. All mice survived to the end of the study. The final mean body weight of 1,000 mg/kg males was significantly less than that of the vehicle controls. There were no significant chemical-related gross or histopathologic changes in dosed mice. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered ginseng in sterile water by gavage at doses of 0, 1,000, 2,000, 3,000, 4,000, or 5,000 mg/kg, 5 days per week for 14 weeks. All rats survived to the end of the study. Mean body weights of all dosed groups were similar to those of the vehicle control groups. No lesions that were observed by gross or histopathologic examination were attributed to the administration of ginseng. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered ginseng in sterile water by gavage at doses of 0, 1,000, 2,000, 3,000, 4,000, or 5,000 mg/kg, 5 days per week for 14 weeks. All mice survived to the end of the study. Mean body weights of all dosed groups were similar to those of the vehicle control groups. Although sporadic incidences of lesions were observed in the vehicle control and 5,000 mg/kg groups, there were no chemical-related gross or microscopic findings in dosed mice. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered ginseng in sterile water by gavage at doses of 0, 1,250, 2,500, or 5,000 mg/kg, 5 days per week for 104 to 105 weeks. Survival of 5,000 mg/kg females was significantly less than that of the vehicle controls; however, the deaths were not attributed to the administration of ginseng because no histopathologic findings attributable to ginseng were found. Mean body weights of 5,000 mg/kg females were less than those of the vehicle controls after week 61 of the study, and mean body weights of other dosed groups of rats were similar to those of the vehicle controls throughout the study. No increases in the incidences of neoplasms or nonneoplastic lesions were attributed to the administration of ginseng. The incidence of mammary gland fibroadenoma was significantly decreased in 5,000 mg/kg females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered ginseng in sterile water by gavage at doses of 0, 1,250, 2,500, or 5,000 mg/kg, 5 days per week for 105 weeks. Survival of dosed groups was similar to that of the vehicle control groups. Mean body weights of dosed mice were similar to those of the vehicle controls. No neoplasms or nonneoplastic lesions were attributed to the administration of ginseng. GENETIC TOXICOLOGY: Ginseng was not mutagenic in either of two independent bacterial mutagenicity assays, each conducted with or without exogenous metabolic activation enzymes. Bacterial strains tested included S. typhimurium strains TA97, TA98, TA100, TA102, TA104, and TA1535, as well as E. coli strain WP2 uvrA/pKM101. No significant increases were seen in the frequencies of micronucleated erythrocytes in the peripheral blood of male or female B6C3F1 mice exposed for 3 months to 1,000 to 5,000 mg/kg ginseng via gavage. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of ginseng in male or female F344/N rats or B6C3F1 mice administered 1,250, 2,500, or 5,000 mg/kg. The incidence of mammary gland fibroadenoma was significantly decreased in 5,000 mg/kg female rats.     

Toxicology and carcinogenesis studies of milk thistle extract (CAS No. 84604-20-6) in F344/N rats and B6C3F1 mice (Feed Studies)

Milk thistle extracts have been used as medicinal herbs in the treatment of liver cirrhosis, chronic hepatitis (liver inflammation), and gallbladder disorders. Treatment claims also include lowering cholesterol levels; reducing insulin resistance; reducing the growth of cancer cells in breast, cervical, and prostate gland cancers; and antiviral activity. Other reported uses of milk thistle in folk medicine include as a treatment for malarial fever, bronchitis, gallstones, jaundice, peritonitis, uterine congestion, varicose veins, and as a milk production stimulant for nursing mothers. The roots soaked in water overnight are used in food, and the despined leaves are added to salads. Roasted milk thistle fruit has been used as a coffee substitute. Milk thistle extract was nominated for study by the National Institute of Environmental Health Sciences because it is one of the most widely used herbs in the United States. Male and female F344/N rats and B6C3F1 mice were exposed to an ethanol/water extract of milk thistle fruit (milk thistle extract) containing approximately 65% silymarin in feed for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 260, 525, 1,050, 2,180, or 4,500 mg milk thistle extract/kilogram body weight to males and 260, 510, 1,050, 2,150, or 4,550 mg/kg to females) for 14 weeks. All rats survived to the end of the study. Mean body weights of exposed groups were within 10% of those of the controls. Feed consumption by exposed and control groups was similar. The sperm motility in 12,500, 25,000, and 50,000 ppm males was decreased by 5%, 11%, and 9%, respectively, relative to that of the controls; the total number of spermatid heads per testis decreased by 11%, 21%, and 9% in 12,500, 25,000, and 50,000 ppm males. No significant differences in estrous cyclicity were observed between exposed and control groups of female rats. No exposure-related histopathologic lesions were observed. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were fed diets containing 0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 640, 1,340, 2,500, 5,280, or 11,620 mg/kg to males and 580, 1,180, 2,335, 4,800, or 9,680 mg/kg to females) for 14 weeks. All mice survived to the end of the study. Mean body weights and feed consumption of all exposed groups were similar to those of the controls. Absolute and relative thymus weights were significantly decreased in 25,000 and 50,000 ppm males. No significant differences were observed between exposed and control groups, for sperm parameters of male mice, for estrous cyclicity of female mice, or for reproductive organ weights of male or female mice, when mice were administered milk thistle extract in feed at 12,500, 25,000, or 50,000 ppm. No exposure-related histopathologic lesions were observed. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were fed diets containing 0, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 570, 1,180, or 2,520 mg/kg to males and 630, 1,300, or 2,750 mg/kg to females) for 105 to 106 weeks. Exposure to milk thistle extract had no effect on survival of male or female rats. Mean body weights of all exposed groups were similar to those of the controls throughout the study. Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study. Significantly decreased incidences of mammary gland fibroadenoma, adenoma, or carcinoma (combined) occurred in females exposed to 25,000 or 50,000 ppm. Significantly increased incidences of clear cell and mixed cell focus of the liver occurred in 25,000 and 50,000 ppm females. The incidences of bile duct hyperplasia were significantly decreased in 50,000 ppm males and in all exposed groups of females, and the incidence of mixed inflammatory cell infiltration was significantly decreased in 50,000 ppm males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were fed diets containing 0, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average daily doses of approximately 1,610, 3,530, or 7,770 mg/kg to males and 1,500, 3,175, or 7,180 mg/kg to females) for 105 to 106 weeks. Exposure to milk thistle extract had no effect on survival of male or female mice. The mean body weights of the 25,000 ppm groups were less than those of controls after week 25; mean body weights of 50,000 ppm groups were less than those of controls after week 12. Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study. Significantly decreased incidences of hepatocellular adenoma and hepatocellular carcinoma occurred in 50,000 ppm males, and decreased incidences of hepatocellular adenoma or carcinoma (combined) occurred in 25,000 and 50,000 ppm males. GENETIC TOXICOLOGY: Five milk thistle extracts were tested independently in bacterial mutagenicity studies using a variety of S. typhimurium tester strains and one E. coli strain. Results were negative in three of the five studies, with and without exogenous metabolic activation. In two studies, milk thistle extract was mutagenic in S. typhimurium strain TA98 in the presence of exogenous metabolic activation enzymes. Silymarin, a major constituent of milk thistle extract, was positive in S. typhimurium strains TA98 and TA100, when testing occurred in the presence of exogenous metabolic activation enzymes. Silybin, another component of milk thistle extract, was negative in a S. typhimurium gene mutation assay, with and without liver S9 activation enzymes. Administration of milk thistle extract in feed for 3 months did not increase the frequencies of micronucleated normochromatic erythrocytes, an indication of chromosomal abnormalities, in the peripheral blood of male or female B6C3F1 mice. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of milk thistle extract in male or female F344/N rats or B6C3F1 mice exposed to 12,500, 25,000, or 50,000 ppm. Exposure to milk thistle extract resulted in increased incidences of clear cell and mixed cell foci in the liver of female rats and decreases in body weights of exposed groups of male and female mice. Decreased incidences of mammary gland neoplasms occurred in exposed groups of female rats, and decreased incidences of hepatocellular neoplasms occurred in exposed groups of male mice.     

Toxicology and carcinogenesis studies of goldenseal root powder (Hydrastis Canadensis) in F344/N rats and B6C3F1 mice (feed studies)

Goldenseal root powder is used in folk medicine for the treatment of gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth, and eye infections, and inflammation. The major alkaloids in goldenseal are berberine, hydrastine, and canadine. Goldenseal root powder was nominated for study by the National Institute of Environmental Health Sciences based on the potential for human exposure and the lack of carcinogenicity data, and because it is one of the most widely used herbs in the United States. Male and female F344/N rats and B6C3F1 mice were exposed to ground goldenseal root powder in feed for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were fed diets containing 0, 1,560, 3,121, 6,250, 12,500, 25,000, or 50,000 ppm goldenseal root powder (equivalent to average daily doses of approximately 155, 315, 630, 1,190, 2,465, and 4,815 mg goldenseal root powder/kg body weight for males and 150, 290, 640, 1,240, 2,370, and 4,870 mg/kg for females) for 15 days. All rats survived to the end of the study. Mean body weights and feed consumption of all exposed groups of males and females were similar to those of the control groups throughout the study. Liver weights of males exposed to 6,250 ppm or greater and females exposed to 12,500 ppm or greater were significantly greater than those of the controls. Minimal to moderate hepatocellular hypertrophy occurred in three males and all females exposed to 25,000 ppm and in all 50,000 ppm males and females. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were fed diets containing 0, 1,560, 3,121, 6,250, 12,500, 25,000, or 50,000 ppm goldenseal root powder (equivalent to average daily doses of approximately 380, 840, 1,760, 3,435, 6,700, and 15,170 mg/kg body weight for males and 330, 670, 1,240, 2,375, 4,760, and 8,475 mg/kg for females) for 15 days. All mice survived to the end of the study. Mean body weights and feed consumption of all exposed groups of males and females were similar to those of the control groups throughout the study. Significant increases in liver weights occurred in males exposed to 25,000 and 50,000 ppm and in females exposed to 50,000 ppm. Absolute and relative thymus weights of 12,500 and 50,000 ppm males were significantly decreased. Minimal hypertrophy of centrilobular hepatocytes occurred in all males and females exposed to 50,000 ppm. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 3,121, 6,250, 12,500, 25,000, or 50,000 ppm goldenseal root powder (equivalent to average daily doses of approximately 255, 500, 1,000, 2,020, and 4,060 mg/kg for males and 260, 500, 1,030, 2,070, and 4,100 mg/kg for females) for 14 weeks. Additional groups of 10 male and 10 female clinical pathology study rats were given the same concentrations for 23 days. All rats survived to the end of the study. None of the body weights or mean body weight gains were significantly different from those of the controls. Feed consumption by exposed groups was generally similar to that by controls throughout the study. Liver weights were significantly increased in males exposed to 6,250 ppm or greater and in all exposed groups of females. The incidences of hepatocyte hypertrophy were significantly increased in the liver of males and females exposed to 12,500 ppm or greater; cytoplasmic vacuolization of hepatocytes occurred in all exposed males. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were fed diets containing 0, 3,121, 6,250, 12,500, 25,000, or 50,000 ppm goldenseal root powder (equivalent to average daily doses of approximately 680, 1,360, 2,260, 5,370, and 10,550 mg/kg for males and 590, 1,250, 2,345, 4,790, and 10,740 mg/kg for females) for 14 weeks. All mice survived to the end of the study. Mean body weights of males exposed to 50,000 ppm and females exposed to 25,000 or 50,000 ppm were significantly less than those of the controls. Feed consumption by 3,121, 6,250, 12,500, 25,000, and 50,000 ppm males was similar to that by controls. Liver weights were significantly increased in males exposed to 12,500 ppm or greater and in females exposed to 25,000 or 50,000 ppm. The left epidydimal weight in male mice was significantly decreased relative to controls. The incidences of hepatocyte hypertrophy were significantly increased in males and females exposed to 12,500 ppm or greater. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were fed diets containing 0, 3,000, 9,000, or 25,000 ppm goldenseal root powder (equivalent to average daily doses of approximately 135, 400, and 1,175 mg/kg for males and 150, 470, and 1,340 mg/kg for females) for 105 to 106 weeks. Survival of 9,000 ppm females was significantly greater than that of the controls. Mean body weights of females exposed to 9,000 ppm were 6% less than those of the controls after week 37, and those of 25,000 ppm females were 6% less than those of the controls after week 8. Feed consumption by exposed groups of males and females was generally similar to that by the controls throughout the study. The incidences of hepatocellular adenoma were significantly increased in males and females exposed to 25,000 ppm, and the incidence of hepatocellular adenoma or carcinoma (combined) was significantly increased in 25,000 ppm males. All exposed groups of males and females had significantly increased incidences of hepatocyte hypertrophy. The incidences of hepatocyte degeneration were significantly increased in all exposed groups of males and in 9,000 and 25,000 ppm females. The incidences of eosinophilic focus were significantly increased in 9,000 and 25,000 ppm males and all exposed groups of females. The incidences of cardiomyopathy were significantly decreased in all exposed groups of males and in 25,000 ppm females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were fed diets containing 0, 3,000, 9,000, or 25,000 ppm goldenseal root powder (equivalent to average daily doses of approximately 375, 1,120, and 3,275 mg/kg for males and 330, 1,000, and 2,875 mg/kg for females) for 105 to 106 weeks. Survival of 9,000 ppm females was significantly less than that of the controls. Mean body weights of females exposed to 25,000 ppm were 3% to 9% less than those of the controls after week 13, 6% less for weeks 14 to 52, and 5% less for weeks 53 to 101. Feed consumption by exposed groups of males and females was generally similar to that of the controls throughout the study. The incidences of hepatocellular adenoma occurred with a positive trend in males, and the incidences of multiple hepatocellular adenoma were significantly increased in 9,000 and 25,000 ppm males. The incidences of hepatoblastoma occurred with a positive trend in males with a marginal increase in the 25,000 ppm group. Significantly increased incidences of eosinophilic focus or mixed cell focus occurred in all exposed groups of males. GENETIC TOXICOLOGY: Goldenseal root powder was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains, with or without liver S9 metabolic activation enzymes. In addition, no increases in the frequencies of micronucleated erythrocytes were observed in peripheral blood samples from mice exposed to goldenseal root powder in feed for 3 months. Berberine chloride was also tested for mutagenicity in standard screening assays. No mutagenicity was observed in several tester strains of Salmonella typhimurium, with or without rat or hamster liver S9 metabolic activation enzymes. In an acute exposure assay, no increase in the frequency of micronucleated polychromatic erythrocytes was seen in bone marrow of male mice administered three intraperitoneal injections of berberine chloride at 24-hour intervals. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenic activity of goldenseal root powder in male F344/N rats based on the increased incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined). There was clear evidence of carcinogenic activity of goldenseal root powder in female F344/N rats based on the increased incidence of hepatocellular adenoma. There was some evidence of carcinogenic activity of goldenseal root powder in male B6C3F1 mice based on the increased incidences of hepatoblastoma and multiple hepatocellular adenoma. There was no evidence of carcinogenic activity of goldenseal root powder in female B6C3F1 mice exposed to 3,000, 9,000, or 25,000 ppm goldenseal root powder in feed for 2 years. Administration of goldenseal root powder resulted in increased incidences of nonneoplastic lesions in the liver of male and female rats and male mice.     

Toxicology and carcinogenesis studies of dipropylene glycol in rats and mice

Dipropylene glycol (DPG) is a component of many commercial products such as antifreeze, air fresheners, cosmetic products, solvents, and plastics. Male and female F344/N rats and B6C3F1 mice were exposed to DPG in the drinking water for 2 weeks, 3 months, or 2 years. In the 2-week and 3-month studies, rats and mice were exposed to 0, 5000, 10,000, 20,000, 40,000, or 80,000 ppm DPG. There was no mortality in the 2-week studies. In the 3-month rat study, all animals survived to the end of the study. Liver weights of rats exposed to 10,000 ppm or greater and kidney weights of rats exposed to 40,000 and 80,000 ppm were greater than those of the controls. The incidences of liver and kidney lesions were significantly increased in males exposed to 20,000 ppm or greater and females exposed to 80,000 ppm. Focal olfactory epithelial degeneration was present in all rats exposed to 80,000 ppm. In males, the incidences of testicular atrophy, epididymal hypospermia, and preputial gland atrophy were significantly increased in the 80,000 ppm group. In the 3-month mouse study, three males and one female exposed to 80,000 ppm died. Liver weights were increased, as was the incidence of centrilobular hypertrophy in males exposed to 40,000 ppm and males and females exposed to 80,000 ppm. In the 2-year studies, exposure groups were 0, 2500 (rats only), 10,000, 20,000 (mice only) or 40,000 ppm DPG. Survival of male rats exposed to 40,000 ppm and mean body weights of males and females exposed to 40,000 ppm were significantly less than controls. In male rats, exposure to DPG resulted in increased incidences and severities of nephropathy and secondary lesions in the parathyroid and forestomach. Increased incidences of focal histiocytic and focal granulomatous inflammation of the liver were also observed. In male and female rats, there were increased incidences of bile duct hyperplasia and changes in the olfactory epithelium of the nose. In mice, survival of males and females was similar to controls. Mean body weights and water consumption of males exposed to 40,000 ppm were less than that of the controls. Treatment-related nonneoplastic lesions did not occur in mice. Treatment-related neoplastic lesions did not occur in rats or mice.     

Toxicology and carcinogenesis studies of fumonisin B1 (cas no. 116355-83-0) in F344/N rats and B6C3F1 mice (feed studies)

[formula: see text] Fumonisin B1 is a mycotoxin produced by the fungus Fusarium moniliforme, one of the major species found in corn. There are no known commercial or medical uses of fumonisin B1. Fumonisin B1 was nominated by the FDA Center for Food Safety and Applied Nutrition for study because of its occurrence in corn and corn-based products in the United States and its toxicity in field exposure of horses and pigs. Male and female F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV-) mice were exposed to fumonisin B1 (92% pure) in feed for 28 days or (greater than 96% pure) for 2 years. 28-DAY STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 99, 163, 234, or 484 ppm fumonisin B1 for 28 days. There were no exposure-related deaths in rats. The mean body weights of the 484 ppm groups were significantly less (-16%) than those of the controls. Dietary concentrations of 99, 163, 234, and 484 ppm fumonisin B1 resulted in average daily doses of 12, 20, 28, and 56 mg fumonisin B1/kg body weight for males and females. Additional groups of male and female rats were exposed to the same concentrations of fumonisin B1 for 28 days for clinical pathology studies. The concentrations of creatinine, cholesterol, triglycerides, and total bile acids, as well as activities of the enzymes alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and gamma-glutamyltransferase, were generally significantly greater in the 484 ppm groups than in the control groups at all time points, indicating hyperlipidemia and a hepatic effect. Fumonisin B1 is an inhibitor of ceramide synthase, resulting in an interruption of de novo sphingolipid synthesis. This enzyme inhibition results in increased levels of sphinganine (or increased sphinganine:sphingosine ratio) in tissues and urine. Urinary sphinganine was increased in groups of males exposed to 163 ppm or greater, while urinary sphinganine was increased in all exposed groups of females. The kidney weights, relative to body weight, of all exposed groups of rats were less than those of the control groups, decreasing by approximately 11% in the females and 20% in the males. Apoptosis and degeneration of the kidney were observed in all exposed males and in most females exposed to 163 ppm or greater. The incidences of minimal to mild apoptosis, degeneration, and mitotic alteration of the liver were significantly increased in 234 and 484 ppm males and in females exposed to 163 ppm or greater. The incidences of bile duct hyperplasia were significantly increased in males and females in the 484 ppm groups. In the core study, male rats in all exposed groups and females exposed to 163 ppm or greater had significantly increased percentages of hepatocytes in one or more proliferative (non-G0) states. 28-DAY STUDY IN MICE: Groups of 12 male and 12 female mice were fed diets containing 0, 99, 163, 234, or 484 ppm fumonisin B1 for 28 days. There were no exposure-related deaths in mice. The mean body weights of the 484 ppm groups of males were significantly less than those of the controls. Feed consumption by males exposed to 484 ppm was less than that by the controls; dietary concentrations of 99, 163, 234, and 484 ppm fumonisin B1 resulted in average daily doses of approximately 19, 31, 44, and 93 mg/kg for males and 24, 41, 62, and 105 mg/kg for females. Additional groups of male and female mice were exposed to the same concentrations of fumonisin B1 for 28 days for clinical pathology studies. Cholesterol and total bile acid concentrations and alanine aminotransferase and alkaline phosphatase activities were increased at 484 ppm, indicating hyperlipidemia and a hepatic effect. Urinary sphinganine concentrations and sphinganine/sphingosine ratios were increased in 484 ppm male mice. In 484 ppm males and all exposed groups of females, the incidences of hepatocellular necrosis, diffuse periportal hypertrophy, and diffuse centrilobular hyperplasia, as well as hyperplasia of the bile canaliculi and Kupffer cells, were generally significantly greater than those in the controls. Core study males exposed to 99, 163, or 234 ppm had significantly increased incidences of hepatocellular cytoplasmic alteration. Hepatocytes of 484 ppm male mice and all exposed groups of female mice were induced into proliferative (non-G0) states. 2-YEAR STUDY IN RATS: Groups of 48 male and 48 female rats (40 for 5 ppm groups) were fed diets containing 0, 5, 15, 50, or 150 ppm fumonisin B1 (males) or 0, 5, 15, 50, or 100 ppm fumonisin B1 (females) (equivalent to average daily doses of approximately 0.25, 0.76, 2.5, or 7.5 mg/kg to males and 0.31, 0.91, 3.0, or 6.1 mg/kg to females) for 105 weeks. Additional groups of four male and four female rats were exposed to the same concentrations as the core study animals and were evaluated at 6, 10, 14 or 26 weeks. Survival, Body Weights, and Feed Consumption Survival, mean body weights, and feed consumption of exposed male and female rats were generally similar to the controls throughout the study. Clinical Pathology Findings Sphinganine/sphingosine ratios were increased in the urine of 15, 50 and 150 ppm males and 50 and 100 ppm females exposed to fumonisin B1 for up to 26 weeks. The sphinganine/sphingosine ratios were also increased in kidney tissue of 50 and 150 ppm males (85- and 119-fold) and 50 and 100 ppm females (7.8- and 22-fold) at 2 years. Cell Proliferation Analyses Renal tubule epithelial cell proliferation was increased in 50 and 150 ppm male rats exposed to fumonisin B1 for up to 26 weeks. Renal tubule epithelial cell proliferation was marginally increased in 100 ppm females. Organ Weights and Pathology Findings Kidney weights of 50 and 150 ppm males were less than those of the controls at 6, 10, 14, and 26 weeks and at 2 years. Kidney weights of 100 ppm females were less than those of the controls at 26 weeks, and kidney weights of 15, 50, and 100 ppm females were less than those of the controls at 2 years. At 2 years, there was a significant increase in the incidences of renal tubule adenoma from none in the groups receiving 15 ppm or less to five of 48 in 150 ppm males. Renal tubule carcinomas were not present in male rats receiving 15 ppm or less and occurred in seven of 48 and 10 of 48 male rats in the 50 and 150 ppm groups, respectively. Incidences of apoptosis of the renal tubule epithelium were generally significantly increased in males exposed to 15 ppm or greater for up to 26 weeks. The incidences of focal renal tubule epithelial hyperplasia were significantly increased in 50 and 150 ppm males at 2 years. 2-YEAR STUDY IN MICE: Groups of 48 male and 48 female mice were fed diets containing 0, 5, 15, 80, or 150 ppm (males) or 0, 5, 15, 50, or 80 ppm (females) fumonisin B1 (equivalent to average daily doses of approximately 0.6, 1.7, 9.7, or 17.1 mg/kg to males or 0.7, 2.1, 7.1, or 12.4 mg/kg to females) for 105 weeks. Additional groups of four male and four female mice were exposed to the same concentrations as the core study animals and were evaluated at 3, 7, 9, or 24 weeks. Survival, Body Weights, and Feed Consumption Survival of males and females in the 15 ppm groups and of 5 ppm females was significantly greater and survival of 80 ppm males and females was significantly less than that of the control groups. Mean body weights and feed consumption of exposed mice were generally similar to the controls. Organ Weights and Pathology Findings Liver weights, relative to body weight, were increased 1.3- and 2.9-fold in 50 and 80 ppm females at 2 years. At 2 years, the incidences of hepatocellular adenoma in 50 and 80 ppm females were significantly greater than those in the controls and occurred with a positive trend. Similarly, the incidences of hepatocellular carcinoma increased from none in the groups receiving 0, 5, or 15 ppm fumonisin B1 to 10 of 47 females at 50 ppm and nine of 45 females at 80 ppm. The incidences of hepatocellular hypertrophy were significantly increased in 15, 80, and 150 ppm males and in 50 and 80 ppm females at 2 years. The incidences of hepatocellular apoptosis were significantly increased in 50 and 80 ppm females at 2 years. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenic activity of fumonisin B1 in male F344/N rats based on the increased incidences of renal tubule neoplasms. There was no evidence of carcinogenic activity of fumonisin B1 in female F344/N rats exposed to 5, 15, 50, or 100 ppm. There was no evidence of carcinogenic activity of fumonisin B1 in male B6C3F1 mice exposed to 5, 15, 80, or 150 ppm. There was clear evidence of carcinogenic activity of fumonisin B1 in female B6C3F1 mice based on the increased incidences of hepatocellular neoplasms. The sphinganine/sphingosine ratios were increased in the urine and the kidney tissue of rats receiving diets containing fumonisin B1. There was evidence of apoptosis and increased cell proliferation of the renal tubule epithelium in exposed rats, particularly in those groups of males that developed renal tubule neoplasms. Increased incidences of hyperplasia of the renal tubule epithelium also occurred in these groups of male rats. In mice exposed to the higher concentrations of fumonisin B1, males and females had increased incidences of hepatocellular hypertrophy and females had increased incidences of hepatocellular apoptosis.