NTP technical report on the toxicology and carcinogenesis studies of Elmiron (Cas No. 37319-17-8) in F344/N rats and B6C3F1 mice (Gavage Studies)

[structure--see text] Elmiron, a white powder, is the sodium salt of pentosan polysulfate, a semisynthetic sulfated polyanion composed of beta-D-xylopyranose residues with biological properties similar to heparin. Elmiron is used in the United States for the relief of urinary bladder pain associated with interstitial cystitis. Because of its stimulating effect on fibrinolysis, Elmiron has been used clinically in the treatment and prevention of thrombotic disorders. The United States Food and Drug Administration nominated Elmiron for toxicology and carcinogenicity testing by the National Toxicology Program because of its orphan drug status. Male and female F344/N rats and B6C3F1 mice received Elmiron, which met product specifications provided by the manufacturer, in deionized water by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were administered 0, 33, 111, 333, 1,000, or 3,000 mg Elmiron/kg body weight in deionized water by gavage, 5 days per week, for 16 days. Elmiron administration had no effect on survival or body weight gain. Activated partial thromboplastin time was significantly increased in 3,000 mg/kg rats. Liver weights of 3,000 mg/kg rats were significantly greater than those of the vehicle controls. Hepatocellular cytoplasmic vacuolization occurred in all 3,000 mg/kg females. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were administered Elmiron in deionized water by gavage at doses of 0, 33, 111, 333, 1,000, or 3,000 mg/kg, 5 days per week, for 16 days. All mice survived to the end of the study. Mean body weight gains of male mice administered 333 mg/kg or greater were significantly greater than that of the vehicle control group. Liver weights of 1,000 and 3,000 mg/kg males were significantly increased. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered Elmiron in deionized water by gavage at doses of 0, 63, 125, 250, 500, or 1,000 mg/kg, 5 days per week, for 14 weeks. No deaths were attributed to administration of Elmiron. Mean body weights of 125 mg/kg males were less than those of vehicle controls and the mean body weights of all dosed groups of females were greater. Hematology results indicated that Elmiron, at the doses selected, induced a minimal erythron decrease and leukocyte and platelet count increases that may have been secondarily related to the inflammatory lesions observed in various tissues of rats. Liver and spleen weights of males administered 250 mg/kg or greater were significantly increased. Liver weights of all dosed groups of females, and kidney, lung, and spleen weights of 1,000 mg/kg females were significantly increased. Histiocytic cellular infiltration, chronic active inflammation, and ulcers of the rectum occurred in most 500 and 1,000 mg/kg rats. Administration of Elmiron was associated with the presence of vacuolated histiocytes in the mandibular and mesenteric lymph nodes, lung, kidney, and liver of male and female rats. Histochemical investigations of the vacuolated histiocytes indicated the presence of neutral and acidic mucins and lipid material within the vacuoles. Transmission electron microscopy identified these vacuoles as lysosomal structures that exhibited a variety of contents. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered Elmiron in deionized water by gavage at doses of 0, 63, 125, 250, 500, or 1,000 mg/kg, 5 days per week, for 14 weeks. One 250 mg/kg female mouse was sacrificed moribund on day 84; all other mice survived to the end of the study. Mean body weights of dosed groups were similar to those of the vehicle control groups. Hematology results indicated that Elmiron, at the doses selected, induced a minimal erythron decrease and leukocyte and platelet count increases that may have been secondarily related to the inflammatory lesions observed in various tissues of mice. in various tissues of mice. Liver weights of 500 mg/kg males and 1,000 mg/kg males and females, and spleen weights of 1,000 mg/kg males were significantly increased. Histiocytic cellular infiltration and chronic active inflammation of the rectum occurred in most 1,000 mg/kg mice. Administration of Elmiron was associated with the presence of vacuolated histiocytes in the mandibular and mesenteric lymph nodes, liver, and spleen of males and females. Histochemical investigations of the vacuolated histiocytes indicated the presence of neutral and acidic mucins within the vacuoles. Transmission electron microscopy identified these vacuoles as lysosomal structures that exhibited a variety of contents. 2-YEAR STUDY IN RATS: Groups of 50 males and 50 females were administered Elmiron in deionized water by gavage at doses of 0, 14, 42, or 126 mg/kg to males and 0, 28, 84, or 252 mg/kg to females, 5 days per week, for 104 or 105 weeks. Survival of all dosed groups of rats was similar to that of the vehicle control groups. Mean body weights of all dosed groups were similar to those of the vehicle controls throughout the 2-year study. Microscopically, myxomatous changes were present in the rectum of 56% of 126 mg/kg males and 83% of 252 mg/kg females. The incidences of chronic active focal alveolar inflammation of the lung were increased in all dosed groups. The incidences of histiocytic cellular infiltration of the mesenteric lymph nodes were increased in 42 and 126 mg/kg males and in 84 and 252 mg/kg females, and lymphohistiocytic hyperplasia was present in the spleen of 126 mg/kg males and 252 mg/kg females. 2-YEAR STUDY IN MICE: Groups of 50 males and 50 females were administered Elmiron in deionized water by gavage at doses of 0, 56, 168, or 504 mg/kg, 5 days per week, for 104 or 105 weeks. Survival of all dosed groups of mice was similar to that of the vehicle control groups. Mean body weights of males were similar to those of vehicle controls. Mean body weights of 504 mg/kg females were progressively less than those of the vehicle controls during the second year of the study. Increased incidences of hemangiosarcomas of the liver and hepatocellular neoplasms were observed in male and female mice. The incidences of hemangiosarcomas in the 504 mg/kg groups exceeded the historical control ranges for males and females; both the trend and the incidence in the 504 mg/kg groups were significant for males. Hemangiosarcomas in males and females were attributed to Elmiron administration. The incidence of hepatocellular adenoma in 504 mg/kg females was significantly increased and exceeded the historical control range; the trends for hepatocellular adenoma and for hepatocellular adenoma or carcinoma (combined) were also significant in females and were attributed to Elmiron administration. There was also a marginal increase in the incidences of hepatocellular neoplasms in male mice, which may have been associated with Elmiron administration. Malignant lymphomas occurred with a positive trend in female mice; the incidence in the 504 mg/kg group was also significantly increased and matched the upper limit of the historical control range. These malignant lymphomas may have been associated with Elmiron administration. Nonneoplastic lesions related to the administration of Elmiron occurred in the liver, rectum, mesenteric lymph node, and spleen of 504 mg/kg mice and to a lesser extent in 168 mg/kg mice. These lesions were similar to those observed in the 3-month study. GENETIC TOXICOLOGY: Elmiron was not mutagenic in S. typhimurium strains TA97, TA98, TA100, or TA1535 with or without induced hamster or rat liver S9 enzymes. No increases in the frequency of micronucleated polychromatic erythrocytes were seen in bone marrow cells of rats or mice administered Elmiron by gavage three times at 24-hour intervals. No significant alterations in the frequency of micronucleated normochromatic erythrocytes were seen in peripheral blood samples from male or female mice administered Elmiron for 3 months by gavage. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of Elmiron in male F344/N rats administered 14, 42, or 126 mg/kg or in female F344/N rats administered 28, 84, or 252 mg/kg. There was some evidence of carcinogenic activity of Elmiron in male B6C3F1 mice based on increased incidences of liver hemangiosarcoma. The increased incidences of hepatocellular neoplasms in male mice may have been related to Elmiron administration. There was some evidence of carcinogenic activity of Elmiron in female B6C3F1 mice based on the increased incidences of liver hemangiosarcoma and hepatocellular neoplasms. The increased incidences of malignant lymphomas in female mice may have been related to Elmiron administration. Elmiron administration caused increased incidences of nonneoplastic lesions (presence of vacuolated histiocytes) of the rectum, lung, mesenteric lymph node, and spleen (males) in rats and of the liver, rectum, mesenteric lymph node, and spleen in mice.     

Toxicology and carcinogenesis studies of androstenedione (CAS No. 63-05-8) in F344/N rats and B6C3F1 mice (gavage studies)

Androstenedione is an androgen steroid that is normally synthesized within men and women and may be metabolized to a more potent androgen or estrogen hormone. It was nominated to the National Toxicology Program for study due to concern for adverse health effects associated with its chronic use as a dietary supplement by athletes (prior to the banning of its over the counter sales). In order to evaluate its subchronic and chronic toxicity, male and female F344/N rats and B6C3F1 mice were administered androstenedione (98% pure) by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, rat bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: groups of five male and five female rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 12 days. All rats survived to the end of the study, and the mean body weights of dosed groups were similar to those of the vehicle control groups. The development of cytoplasmic vacuoles within centrilobular hepatocytes in male rats was the only treatment-related effect observed. 2-WEEK STUDY IN MICE: groups of five male and five female mice were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 12 days. One vehicle control female, one 20 mg/kg female, and one 50 mg/kg female died early due to gavage accidents. There were no significant chemical-related histopathological or mean body weight changes. 3-MONTH STUDY IN RATS: groups of 10 male and 10 female core study rats were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats received the same doses for 23 days. All rats survived to the end of the study. The mean body weights of the 20 mg/kg female group was significantly greater than those of the vehicle control group and there was significant increased weight gain in the 1, 20, and 50 mg/kg female groups. Female thymus weights were significantly increased in the 20 and 50 mg/kg groups, which may be related to the increase in mean body weight. The numbers of sperm per mg cauda epididymis in the 10, 20, and 50 mg/kg male groups and the total number of sperm per cauda epididymis in 50 mg/kg males were significantly less than those of the vehicle controls. No treatment-related histological lesions were observed in males or females. 3-MONTH STUDY IN MICE: groups of 10 male and 10 female mice were administered 0, 1, 5, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for 14 weeks. Except for one 10 mg/kg female that died early due to a dosing accident, all mice survived to the end of the study. The mean body weights of dosed groups were similar to those of the vehicle control groups. The number of spermatids per mg testis and the total number of spermatids per testis in 20 mg/kg males were significantly greater than those of the vehicle controls. Sperm motility in 50 mg/kg males was significantly lower than that in the vehicle controls. The incidences of x-zone atrophy of the adrenal cortex, an androgen-sensitive endpoint, were significantly increased in females administered 5 mg/kg or greater. There were also significant decreases in the incidences of x-zone cytoplasmic vacuolization in 20 and 50 mg/kg females. The incidences of bone marrow hyperplasia were significantly increased in 5 and 50 mg/kg males. 2-YEAR STUDY IN RATS: groups of 50 male and 50 female rats were administered 0, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for at least 104 weeks. Survival of 10 mg/kg males was significantly greater than that of the vehicle controls. The mean body weights of 20 and 50 mg/kg females were greater than those of the vehicle controls after weeks 17 and 9, respectively. The incidences of mononuclear cell leukemia were significantly increased in 20 and 50 mg/kg females and significantly decreased in 20 and 50 mg/kg males. Incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 20 mg/kg males. The incidence of testicular interstitial cell adenoma (including bilateral) was significantly decreased in 50 mg/kg males. In females, the incidences of mammary gland fibroadenoma were significantly decreased in the 20 and 50 mg/kg groups, the incidences of mammary gland hyperplasia were significantly decreased in all dosed groups, and the incidences of mammary gland cyst were significantly decreased in the 10 and 50 mg/kg groups. In the liver of males, the incidences of basophilic focus in all dosed groups, the incidence of clear cell focus in the 20 mg/kg group, and the incidence of eosinophilic focus in the 50 mg/kg group were significantly increased. The incidences of pancreatic islet hyperplasia and atrophy of the exocrine pancreas were significantly increased in 50 mg/kg females. 2-YEAR STUDY IN MICE: groups of 50 male and 50 female mice were administered 0, 2 (females only), 10, 20 (males only), or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for at least 104 weeks. Survival of dosed groups was similar to that of the vehicle control groups. Mean body weights of 10 and 50 mg/kg females were generally less than those of the vehicle controls after weeks 81 and 17, respectively. The incidences of hepatocellular adenoma in males and females were significantly increased in the 50 mg/kg groups. In females, the incidences of hepatocellular carcinoma were significantly increased in all dosed groups. Incidences of hepatocellular adenoma or carcinoma (combined) in males and females were significantly increased in the 50 mg/kg groups. Incidences of hepatoblastoma were marginally increased in dosed males. Incidences of multiple hepatocellular adenomas and carcinomas were significantly increased in 10 and 50 mg/kg males, and there was an increased incidence of multiple hepatocellular adenomas in 50 mg/kg females. The incidence of eosinophilic focus was significantly increased in 50 mg/kg males, and the incidences of mixed cell focus and cytoplasmic vacuolization were significantly increased in 50 mg/kg females. There was a marginally increased incidence of pancreatic islet adenoma in 50 mg/kg males and in 10 and 50 mg/kg females, with an earlier day of first incidence in males. The incidences of clitoral gland hyperplasia and clitoral gland duct dilatation were significantly increased in 10 and 50 mg/kg females. The incidence of glomerular metaplasia of the kidney was significantly increased in 50 mg/kg females, and the incidences of cytoplasmic alteration of the submandibular salivary gland were significantly increased in all dosed female groups. The increased incidences of cytoplasmic alteration of the submandibular salivary gland and glomerular metaplasia of the kidney in female mice indicated a masculinizing effect from androstenedione treatment. In 50 mg/kg females, the incidence of malignant lymphoma was significantly decreased. GENETIC TOXICOLOGY: androstenedione was not mutagenic in either of two independent bacterial mutation assays conducted with and without exogenous metabolic activation. No significant increases in the frequencies of micronucleated polychromatic erythrocytes, indicators of chromosomal damage, were observed in bone marrow of male rats administered androstenedione by gavage once daily for 3 consecutive days. Results of a peripheral blood erythrocyte micronucleus test in mice, in which androstenedione was administered by gavage for 3 months, were negative in males but judged to be equivocal in females due to a small increase (twofold over background) in micronucleated normochromatic erythrocytes observed at the highest dose administered (50 mg/kg). CONCLUSIONS: under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of androstenedione in male F344/N rats based on increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity of androstenedione in female F344/N rats based on increased incidences of mononuclear cell leukemia. There was clear evidence of carcinogenic activity of androstenedione in male B6C3F1 mice based on increased incidences of multiple hepatocellular adenoma and hepatocellular carcinoma and increased incidence of hepatoblastoma. There was clear evidence of carcinogenic activity of androstenedione in female B6C3F1 mice based on increased incidences of hepatocellular adenoma and hepatocellular carcinoma. Increased incidences of pancreatic islet adenoma in male and female mice were also considered chemical related. Androstenedione administration caused increased incidences in nonneoplastic lesions of the liver in male and female rats and mice; pancreatic islets and exocrine pancreas of female rats; and clitoral gland, kidney, and submandibular salivary gland of female mice. Decreases in the incidences of testicular interstitial cell adenoma in male rats, mammary gland fibroadenoma, cysts, and hyperplasia in female rats, and malignant lymphoma in female mice were considered related to androstenedione administration. Synonyms: Andro; androst-4-ene-3,17-dione; 4-androstene-3,17-dione; delta-4-androstene-3,17-dione; delta-4-androstenedione; 3,17-dioxoandrost-4-ene; 17-ketotestosterone; SKF 2170 Trade names: Androtex, Fecundin.     

Toxicology and carcinogenesis studies of formamide (Cas No. 75-12-7) in F344/N rats and B6C3F1 mice (gavage studies)

Formamide is used as a softener for paper, gums, and animal glues; as an ionizing solvent; and in the manufacture of formic esters and hydrocyanic acid. Formamide was nominated for reproductive and genetic toxicity evaluation by the Environmental Defense Fund and for carcinogenicity evaluation by the National Cancer Institute because of the potential for human exposure associated with its widespread industrial use, the absence of data adequately characterizing its potential for reproductive and genetic toxicity, and the fact that acetamide, a compound structurally related to form-amide, is hepatocarcinogenic in rats when administered in feed. Male and female F344/N rats and B6C3F1 mice were administered formamide (approximately 100% pure) in deionized water by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, Drosophila melanogaster, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 10, 20, 40, 80, or 160 mg formamide/kg body weight in deionized water by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) and five male and five female rats (plasma concentration study) were administered the same doses, 5 days per week for up to 14 weeks. All core study rats survived to the end of the study. Mean body weights of females in the 40 mg/kg group and males and females in the 80 and 160 mg/kg groups were significantly less than those of the vehicle controls. On day 23 and at week 14, there was a dose-related increase in the erythron, evidenced by increases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. The incidences of degeneration of the germinal epithelium of the testes and epididymis were significantly increased in 160 mg/kg males. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 10, 20, 40, 80, or 160 mg formamide/kg body weight in deionized water by gavage, 5 days per week for 14 weeks. Additional groups of five male and five female mice (plasma concentration study) were administered the same doses, 5 days per week for 14 weeks. All mice survived to the end of the study. Final mean body weights of the 80 and 160 mg/kg males and mean body weight gains of 40, 80, and 160 mg/kg males were significantly less than those of the vehicle controls. Dosed females differed significantly from vehicle controls in the relative amount of time spent in the estrous stages. All 160 mg/kg males had abnormal residual bodies in the testes. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 20, 40, or 80 mg formamide/kg body weight, 5 days per week for 104 to 105 weeks in deionized water by gavage. Survival of all dosed groups of rats was similar to that of the vehicle controls. Mean body weights of 80 mg/kg males were less than those of the vehicle controls throughout most of the study. Mean body weights of 40 and 80 mg/kg females were somewhat less than those of the vehicle controls during the second year of the study. A significant increase in the incidence of bone marrow hyperplasia occurred in 80 mg/kg males. No neoplasms were attributed to exposure to formamide. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 0, 20, 40, or 80 mg formamide/kg body weight, 5 days per week for 104 to 105 weeks in deionized water by gavage. Survival of all dosed groups of mice was similar to that of the vehicle controls. Mean body weights of 80 mg/kg males and females were generally less than those of the vehicle controls throughout the study; mean body weights of 40 mg/kg females were generally less after week 13 of the study. The incidences of hemangiosarcoma of the liver occurred with a positive trend in males, and the incidences were significantly increased in the 40 and 80 mg/kg groups. The incidence of hepatocellular adenoma or carcinoma (combined) in 80 mg/kg females was significantly increased. The incidences of mineralization of the testicular arteries and testicular tunic were significantly increased in 80 mg/kg males. The incidence of hematopoietic cell proliferation of the spleen was significantly increased in 80 mg/kg males. GENETIC TOXICOLOGY: Formamide gave no evidence for mutagenicity in a series of short-term assays. In three independent Ames assays, formamide was not mutagenic in any of several strains of S. typhimurium tested with and without rat or hamster liver S9 activation enzymes or in E. coli strain WP uvrA pKM101 tested with and without 10% rat liver S9. Negative results were obtained in a test for induction of sex-linked recessive lethal mutations in germ cells of male D. melanogaster treated with formamide either by feeding or injection. Formamide did not induce increases in micronucleated erythrocytes in male or female mice treated by gavage for 3 months. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of form-amide in male or female F344/N rats administered 20, 40, or 80 mg/kg. There was clear evidence of carcinogenic activity of formamide in male B6C3F1 mice based on increased incidences of hemangiosarcoma of the liver. There was equivocal evidence of carcinogenic activity of formamide in female B6C3F1 mice based on increased incidences of hepatocellular adenoma or carcinoma (combined). An increased incidence of bone marrow hyperplasia occurred in male rats. Mineralization of the testicular arteries and tunic and hematopoietic cell proliferation of the spleen in male mice were also associated with administration of formamide.     

Toxicology and carcinogenesis studies of methylene blue trihydrate (Cas No. 7220-79-3) in F344/N rats and B6C3F1 mice (gavage studies)

Methylene blue trihydrate has a variety of biomedical and biologically therapeutic applications. Methylene blue trihydrate was nominated by the National Cancer Institute (NCI) for carcinogenicity testing based on the numerous uses of this compound and the lack of long-term toxicity data, including epidemiological studies of methylene blue trihydrate, as well as the inadequate animal data on this compound. Male and female F344/N rats and B6C3F1 mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose by gavage for 1 month, 3 months, or 2 years. Genetic toxicology studies were conducted using Salmonella typhimurium, Escherichia coli, cultured Chinese hamster ovary cells, mouse bone marrow cells, and mouse peripheral blood erythrocytes. 1-MONTH STUDY IN RATS: Groups of 10 male and 10 female core study rats and groups of 10 male and 10 female clinical pathology study rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week for 5 weeks. In the 500 mg/kg groups, one male died the first week of the study and one male and four females died the second week of the study. All rats in the 1,000 mg/kg group died by study day 10, and all rats in the 2,000 mg/kg group died by study day 6. Final mean body weights of male and female rats in the 250 and 500 mg/kg groups were significantly less than those of the vehicle controls. Dosed rats developed methemoglobinemia and a regenerative Heinz body anemia. Significant increases in spleen weights occurred in all surviving dosed groups. There were also significant decreases in the thymus weights of 250 and 500 mg/kg males and 125 and 250 mg/kg females. Spleen lesions associated with methylene blue trihydrate administration included hematopoietic cell proliferation, pigmentation, lymphoid depletion of the lymphoid follicles, and capsular fibrosis. Hyperplasia of the bone marrow occurred in all dosed groups of rats. Liver lesions associated with methylene blue exposure included centrilobular necrosis in rats dying early, hematopoietic cell proliferation, and Kupffer cell pigmentation with erythrophagocytosis. 1-MONTH STUDY IN MICE: Groups of 10 male and 10 female core study mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week for 5 weeks. None of the mice in the 500, 1,000, and 2,000 mg/kg groups survived to the end of the study. In the 250 mg/kg groups, two females died on days 16 and 18 and two males died on days 6 and 13. Mean body weights of surviving dosed mice were similar to those of the vehicle controls. Thinness, abnormal respiration, hypothermia, lethargy, ataxia, and ruffled fur were observed in a few surviving animals in the 250 mg/kg groups. Hypothermia and abnormal posture were observed in mice in the 500, 1,000, and 2,000 mg/kg groups. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia. Significant increases in spleen weights occurred in all surviving dosed groups of mice compared to vehicle controls. Significant decreases occurred in the thymus weights of 250 mg/kg males and females. The heart weights of 125 and 250 mg/kg females were significantly increased. Lesions in the spleen associated with methylene blue trihydrate administration included hematopoietic cell proliferation, pigmentation, and congestion. Liver lesions associated with methylene blue trihydrate administration included periportal degeneration, hematopoietic cell proliferation, and Kupffer cell pigmentation with erythrophagocytosis. The incidences of bone marrow pigmentation were significantly increased in all dosed groups of mice. Forestomach lesions that were related to methylene blue trihydrate administration included focal ulcer, inflammation, and squamous hyperplasia. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female core study rats and groups of 20 male and 20 female clinical pathology study rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 25, 50, 100 or 200 mg/kg, 5 days per week for 14 weeks. Mean body weights of males in the 200 mg/kg group were significantly less than those of the vehicle controls. Dosed rats developed methemoglobinemia and a regenerative Heinz body anemia. Significant increases in spleen weights occurred in males and females administered 50 mg/kg or greater. Thymus and lung weights of 50, 100, and 200 mg/kg males (except relative lung weight at 100 mg/kg) were significantly less than those of the vehicle controls. Spleen lesions in dosed rats included hematopoietic cell proliferation, congestion, lymphoid depletion of the lymphoid follicles, and capsular fibrosis. The incidences of bone marrow hyperplasia were significantly increased in groups administered 50 mg/kg or greater. There were no consistent effects of methylene blue trihydrate administration on reproductive system measures in male or female rats. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female core study mice and groups of 20 male and 20 female clinical pathology study mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 25, 50, 100, or 200 mg/kg, 5 days per week for 14 weeks. Mean body weights of all dosed groups were similar to or only slightly less than those of the vehicle control groups. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia. Spleen weights of 100 and 200 mg/kg males and 50 mg/kg or greater females were significantly greater than those of the vehicle control groups. Heart weights were significantly increased in 200 mg/kg males. In females, there were significant decreases in thymus weights at 50 mg/kg or greater. Males had decreased sperm motility and increased epididymal sperm counts at 200 mg/kg. In all dosed groups, the incidences of hematopoietic cell proliferation and pigmentation in the spleen were significantly greater than those in the vehicle controls. In the liver, the incidences of hematopoietic cell proliferation were significantly increased in males and females in the 100 and 200 mg/kg groups, and the incidences of Kupffer cell pigmentation were significantly increased in groups administered 50 mg/kg or greater. The incidences of bone marrow pigmentation were significantly increased in all dosed groups of mice except 25 mg/kg females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 5, 25, or 50 mg/kg, 5 days per week for 2 years. Additional groups of 10 male and 10 female rats were administered the same doses for up to 18 months and were evaluated at 2 weeks and 3, 12, and 18 months for hematology. Survival of all dosed groups of rats was similar to that of the vehicle controls. Mean body weights of 25 and 50 mg/kg male rats were less than those of the vehicle controls after weeks 29 and 21, respectively. In the 25 and 50 mg/kg females, mean body weights were less after weeks 73 and 53. Dosed male and female rats developed methemoglobinemia, and females developed a regenerative Heinz body anemia. The incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined) were increased in all dosed groups of males, were significantly increased in 25 mg/kg males, and exceeded the historical range in controls (all routes). The incidence of pancreatic islet cell hyperplasia was significantly increased in the 50 mg/kg males. In the spleen, the incidence of hematopoietic cell proliferation in 50 mg/kg males was significantly increased; the incidences of capsular fibrosis were significantly increased in all dosed groups of males and in 5 and 50 mg/kg females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered methylene blue trihydrate in a 0.5% aqueous methylcellulose solution by gavage at doses of 0, 2.5, 12.5, or 25 mg/kg, 5 days per week for 2 years. Additional groups of 30 male and 30 female mice were administered the same doses for up to 18 months and were evaluated at 2 weeks and 3, 12, or 18 months for hematology. Survival of dosed male and female groups exceeded that of the vehicle controls in a generally dose-related manner. Mean body weights of dosed female mice began to increase after weeks 29, 61, and 85, reaching final values that were 113%, 111%, and 106% of vehicle controls for the 2.5, 12.5, and 25 mg/kg groups, respectively. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia. The incidences of carcinoma and of adenoma or carcinoma (combined) of the small intestine occurred with a positive trend in males. The incidences of malignant lymphoma occurred with a positive trend in females, and the incidence in 25 mg/kg males exceeded the historical control range. The incidences of hematopoietic cell proliferation of the spleen were significantly increased in 12.5 and 25 mg/kg males and in 25 mg/kg females. The incidences of inflammation of the nose were significantly increased in 12.5 and 25 mg/kg females. GENETIC TOXICOLOGY: Methylene blue trihydrate was mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without rat or hamster liver S9 activation enzymes; mutagenicity was also observed in Escherichia coli strain WP2 uvrA/pKM101 with and without rat liver S9. In cytogenetic tests with cultured Chinese hamster ovary cells, methylene blue trihydrate induced sister chromatid exchanges and chromosomal aberrations with and without S9. (ABSTRACT TRUNCATED).     

Toxicology and carcinogenesis studies of pulegone (CAS No. 89-82-7) in F344/N rats and B6C3F1 mice (gavage studies)

Several essential oils contain pulegone and are used for flavoring foods, drinks, and dental products, as fragrance agents, and in herbal medicines. Pulegone was nominated for study by the National Institute of Environmental Health Sciences based on the potential for human exposure and the absence of carcinogenicity data. Male and female F344/N rats and B6C3F1 mice received pulegone (approximately 96% pure) 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 0, 37.5, 75, 150, 300, or 600 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 16 days. All male rats and nearly all female rats in the 300 and 600 mg/kg groups died prior to the end of the study. All moribund sacrifices and early deaths were attributed to liver toxicity. Mean body weight gains of males administered 37.5 or 150 mg/kg were significantly less than that of the vehicle controls. Clinical findings in 300 and 600 mg/kg rats included nasal/eye discharge, thinness, lethargy, and ruffled fur. Liver and kidney weights of dosed groups of females were generally significantly greater than those of the vehicle control group. The incidences of necrosis and cytoplasmic vacuolization of the liver in 300 and 600 mg/kg males and females were significantly greater than those in the vehicle control groups. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were administered 0, 18.75, 37.5, 75, 150, or 300 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 16 days. Four females and one male in the 300 mg/kg groups died by study day 5. All early deaths were attributed to liver toxicity. Mean body weights of the dosed groups were similar to those of the vehicle controls. Clinical findings were observed only in 300 mg/kg mice and included thinness, lethargy, and ruffled fur. Liver weights of 300 mg/kg males were significantly greater than those of the vehicle controls. The incidences of cytoplasmic vacuolization and diffuse fatty change in 300 mg/kg females and necrosis in 300 mg/kg males were significantly greater than those in the vehicle controls. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 9.375, 18.75, 37.5, 75, or 150 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All rats survived until the end of the study except for one female in the 150 mg/kg group that died on day 9. Mean body weights of 75 and 150 mg/kg males and 150 mg/kg females were significantly less than those of the vehicle controls. At the end of the study, there was a small dose-related decrease in the erythron, evidenced by decreases in the hematocrit and hemoglobin values and the erythrocyte counts. An apparent erythroid response to the decreased erythron was evidenced by increased reticulocyte counts. Reduced and oxidized glutathione levels were generally increased in 75 and 150 mg/kg males and in 37.5 mg/kg or greater females. Absolute and relative liver weights of 75 and 150 mg/kg females and relative liver weights of males administered 18.75 mg/kg or greater were significantly greater than those of the vehicle controls. The absolute kidney weight of 150 mg/kg females and the relative kidney weights of all dosed groups, except 9.375 mg/kg males, were significantly greater than those of the vehicle controls. Absolute and relative thymus weights of 150 mg/kg males and females and the absolute thymus weight of 75 mg/kg males were significantly less than those of the vehicle controls. In the kidney, there was hyaline glomerulopathy in 75 mg/kg males and 150 mg/kg males and females. The incidence of renal tubule protein casts was significantly increased in the 150 mg/kg females. In the liver, incidences of bile duct hyperplasia and hepatocyte hypertrophy in 75 and 150 mg/kg males and 150 mg/kg females, hepatocyte focal necrosis in 150 mg/kg males, and oval cell hyperplasia and periportal fibrosis in 150 mg/kg males and females were increased. Incidences of bone marrow hyperplasia in 37.5 mg/kg males and 75 and 150 mg/kg males and females, heart mineralization in 150 mg/kg males, glandular stomach mineralization in 75 and 150 mg/kg females, and cellular histiocytic infiltration in the lung and ovarian cyst in 150 mg/kg females were significantly increased. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 9.375, 18.75, 37.5, 75, or 150 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All mice survived to the end of the study. Mean body weights of dosed mice were similar to those of the vehicle controls. Reduced and oxidized glutathione levels were generally greater than vehicle control levels in 150 mg/kg males and in 75 and 150 mg/kg females. Liver weights of 150 mg/kg males and 75 and 150 mg/kg females were significantly greater than those of the vehicle controls. No histopathologic lesions were observed that could be attributed to the administration of pulegone. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 18.75 (males only), 37.5, 75, or 150 (females only) mg pulegone/kg body weight in corn oil by gavage, 5 days per week for up to 104 weeks. Due to excessive morbidity and mortality, 75 mg/kg males and 150 mg/kg females were not administered pulegone after week 60 (stop-exposure); these groups were administered the corn oil vehicle until the end of the study. Survival of 37.5 mg/kg males was significantly less than that of the vehicle controls; only two 75 mg/kg stop-exposure males survived, and no 150 mg/kg stop-exposure females survived to the end of the study. Compared to those of the vehicle controls, mean body weights were less in 75 mg/kg stop-exposure males after week 13 and in 75 mg/kg and 150 mg/kg stop-exposure females after weeks 21 and 9, respectively. Clinical findings included thinness, lethargy, and ruffled fur in the 75 mg/kg stop-exposure males and 150 mg/kg stop-exposure females. The incidences of urinary bladder papilloma and of papilloma or carcinoma (combined) were significantly increased in 150 mg/kg stop-exposure females. In the kidney, incidences of hyaline glomerulopathy were significantly increased in 37.5 mg/kg and 75 mg/kg stop-exposure males and in all dosed groups of females. The severity of chronic progressive nephropathy was increased in 37.5 mg/kg and 75 mg/kg stop-exposure males and in 75 mg/kg and 150 mg/kg stop-exposure females; the incidences of nephropathy were significantly increased in 75 mg/kg and 150 mg/kg stop-exposure females. The incidence of renal cyst was significantly increased in 75 mg/kg stop-exposure males. In the liver, incidences of diffuse hepatocyte cellular alteration were significantly increased in 37.5 mg/kg and 75 mg/kg stop-exposure males and 75 mg/kg and 150 mg/kg stop-exposure females. There were significant increases in the incidences of other liver lesions including fatty change, bile duct cyst, hepatocyte necrosis, oval cell hyperplasia, bile duct hyperplasia, and portal fibrosis. In the nose, 37.5 mg/kg and 75 mg/kg stop-exposure males and all dosed groups of females had significantly increased incidences of olfactory epithelium degeneration. All dosed groups of females had significantly increased incidences of respiratory metaplasia of the olfactory epithelium and nasal inflammation. In the forestomach, incidences of inflammation and ulcer were significantly increased in 37.5 mg/kg and 75 mg/kg stop-exposure males, and incidences of epithelial hyperplasia and perforation were increased in 75 mg/kg stop-exposure males. In the glandular stomach, the incidence of inflammation was significantly increased in 75 mg/kg stop-exposure males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 0, 37.5, 75, or 150 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 105 weeks. Survival of all dosed groups was similar to that of the vehicle controls. Mean body weights of 150 mg/kg males and females were less than those of the vehicle controls after weeks 25 and 33, respectively. The incidences of multiple hepatocellular adenoma were significantly increased in all dosed groups of males, and the incidences of hepatocellular adenoma (includes multiple) and hepatoblastoma (includes multiple) were significantly increased in the 75 mg/kg males. The combined incidences of hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma occurred with positive trends and were significantly increased in 75 mg/kg males and 150 mg/kg females. The incidence of hepatocellular adenoma was significantly increased in 150 mg/kg females. The incidences of several nonneoplastic liver lesions were significantly increased, primarily in the 75 and 150 mg/kg groups. These nonneoplastic lesions included clear cell, eosinophilic, and mixed cell foci; focal fatty change; centrilobular hepatocyte hypertrophy; intravascular hepatocyte; necrosis; pigmentation; bile duct cyst and hyperplasia; and oval cell hyperplasia. In the kidney, incidences of hyaline glomerulopathy were significantly increased in all dosed groups of males and 75 and 150 mg/kg females. The incidence of mineralization was significantly increased in 150 mg/kg females, and the incidence of nephropathy in 150 mg/kg females and severity of nephropathy in 150 mg/kg males were increased. Incidences of congestion of the glomerulus were increased in 150 mg/kg males and females. The incidence of osteoma or osteosarcoma (combined) in all organs of 75 mg/kg females exceeded the historical control ranges. One 150 mg/kg male and one 75 mg/kg female had nasal osteoma; no nasal osteomas have been observed in historical control mice. (ABSTRACT TRUNCATED)     

Toxicology and carcinogenesis studies of isoeugenol (CAS No. 97-54-1) in F344/N rats and B6C3F1 mice (gavage studies)

Isoeugenol is one of several structurally similar phenylpropenoid compounds produced by plants. It has been extracted from calamus, savory, basil, ylang-ylang, clove, tuberose, jonquil, nutmeg, tobacco, sandalwood, dill seed, mace, gardenia, petunia, and other flowers. Isoeugenol can also be produced by isomerization of eugenol, which occurs naturally in clove, pimento, bay leaf, and cinnamon. As a fragrance with a spicy, carnation-like odor, isoeugenol is incorporated into numerous household and personal hygiene products, including perfumes, cream lotions, soaps, and detergents. As a flavoring agent, isoeugenol is added to nonalcoholic drinks, baked foods, and chewing gums. Isoeugenol was nominated by the National Cancer Institute and was selected for carcinogenicity testing because of widespread human exposure through its use as a flavoring and fragrance agent and because of its structural similarity to phenylpropenoids such as safrole, isosafrole, eugenol, methyleugenol, estragole, and anethole, most of which are known rodent carcinogens. Male and female F344/N rats and B6C3F1 mice were administered isoeugenol (99% or greater pure) in corn oil by gavage for 3 months or 2 years. Genetic toxicity tests were conducted in Salmonella typhimurium, Escherichia coli, 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 to isoeugenol in corn oil by gavage at doses of 0, 37.5, 75, 150, 300, or 600 mg/kg, 5 days per week for 14 weeks. All rats survived to the end of the study except one 600 mg/kg male and one 37.5 mg/kg female that were killed in dosing accidents. Mean body weights of all exposed groups of males were significantly less than that of the vehicle control group; however, only the decrease for the 600 mg/kg group exceeded 10% and was considered related to isoeugenol exposure. Liver weights were significantly increased in 300 and 600 mg/kg females. The incidences of minimal atrophy of the olfactory epithelium of the nose were significantly increased in 150 mg/kg or greater males and in 300 or 600 mg/kg females. The incidence of atrophy of olfactory nerve bundles was significantly increased in 600 mg/kg females. Minimal to mild periportal hepatocellular cytoplasmic alteration occurred in all 300 or 600 mg/kg females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed to isoeugenol in corn oil by gavage at doses of 0, 37.5, 75, 150, 300, or 600 mg/kg, 5 days per week for 14 weeks. All mice survived to the end of the study. The mean body weight of 600 mg/kg males was significantly less (12%) than that of the vehicle controls. Liver weights of 300 and 600 mg/kg males were significantly greater than those of the vehicle controls. Minimal to moderate atrophy of olfactory epithelial tissue and nerve bundles was observed in 600 mg/kg males and females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to isoeugenol in corn oil by gavage at doses of 0, 75, 150, or 300 mg/kg, 5 days per week for 105 weeks. Survival rates of exposed male and female rats were similar to those of vehicle controls. Mean body weights of 300 mg/kg male rats were 9% greater than the vehicle controls at the end of the study. The general lack of toxicity and nonneoplastic lesions indicates that rats might have been able to tolerate higher doses. Two male rats in the 300 mg/kg group had rare benign or malignant thymomas, while two other males in this group had rare mammary gland carcinomas. Low incidences of minimal atrophy and minimal to mild respiratory metaplasia of the olfactory epithelium were increased in 150 mg/kg males and 300 mg/kg males and females. Similar incidences of minimal to mild olfactory epithelial degeneration in 300 mg/kg males were also increased. Incidences of keratoacanthoma of the skin were decreased in 150 and 300 mg/kg males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed to isoeugenol in corn oil by gavage at doses of 0, 75, 150, or 300 mg/kg, 5 days per week for 104 (females) or 105 (males) weeks. Survival of 300 mg/kg males was significantly decreased compared to the vehicle controls. Mean body weights of 300 mg/kg male and female groups were less than those of vehicle controls at the end of the study, 10% and 15% less, respectively. In all groups of exposed males, the incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) were significantly greater than those in the vehicle control group; incidences of multiple hepatocellular adenoma were also significantly increased. Incidences of clear cell focus were significantly increased in 75 and 150 mg/kg male mice. There was a significant positive trend in the incidences of histiocytic sarcoma in females, and this neoplasm occurred in multiple tissues. Incidences of respiratory metaplasia in olfactory epithelium in all exposed groups and of atrophy and hyaline droplet accumulation in all exposed groups except 75 mg/kg females were significantly greater than those in corresponding vehicle control groups. Incidences of minimal to marked hyperplasia of Bowman's gland were increased significantly in all exposed groups. Incidences of minimal to mild necrosis of renal papilla and mild to moderate necrosis of renal tubules were increased significantly in 300 mg/kg females. Incidences of forestomach squamous hyperplasia, inflammation, and ulceration (males only) increased with exposure and were significant in the 300 mg/kg groups. The incidence of glandular stomach ulcers was low but significantly increased in the 300 mg/kg groups. GENETIC TOXICOLOGY: Isoeugenol was not mutagenic in two independent assays in bacteria (S. typhimurium and E. coli) conducted with and without exogenous metabolic activation (S9 liver enzymes). Neither did it induce chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9 activation. Frequencies of micronucleated erythrocytes were not increased in peripheral blood of male mice exposed to isoeugenol by gavage for 3 months; however, an increasing trend and a threefold increase in the 600 mg/kg group indicate a positive result for this test in female mice. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of isoeugenol in male F344/N rats based on increased incidences of rarely occurring thymoma and mammary gland carcinoma. There was no evidence of carcinogenic activity of isoeugenol in female F344/N rats administered 75, 150, or 300 mg/kg. There was clear evidence of carcinogenic activity of isoeugenol in male B6C3F1 mice based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity of isoeugenol in female B6C3F1 mice based on increased incidences of histiocytic sarcoma. Exposure to isoeugenol resulted in nonneoplastic lesions of the nose in male and female rats; of the nose, forestomach, and glandular stomach in male and female mice; and of the kidney in female mice.     

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 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 diisopropylcarbodiimide (Cas No. 693-13-0) in F344/N rats and B6C3F1 mice (dermal studies)

Diisopropylcarbodiimide is used as a reagent for peptide syntheses and as a chemical intermediate. The National Cancer Institute nominated diisopropylcarbodiimide for study as a representative chemical in the alkylcarbodiimide class because of its acute toxicity; its use in chemical, pharmaceutical, and recombinant DNA industries; and the absence of data on potential health effects. Male and female F344/N rats and B6C3F1 mice were administered diisopropylcarbodiimide (greater than 99% pure) dermally for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female F344/N rats were dermally administered 0.3 mL ethanol containing 0, 3, 9, 27, or 81 mg diisopropylcarbodiimide or 0.3 mL of the neat chemical containing 242 mg per animal, 5 days a week for 2 weeks. All rats in the 27, 81, and 242 mg groups died before the end of the study. Of the surviving groups, final body weights were similar to those of the vehicle controls. Clinical findings included convulsions/seizures, nasal/eye discharge, tremors, and comatose conditions in 81 and 242 mg rats and lethargy, ataxia, and abnormal breathing in 27 mg rats. The incidences of epidermal hyperplasia at the site of application in 9 and 27 mg males and 27 mg females were significantly greater than those in the vehicle controls; the incidences of hyperkeratosis in 3 and 9 mg males and 9 mg females were also significantly increased. 2-WEEK STUDY IN MICE: Groups of five male and five female B6C3F1 mice were dermally administered 0.1 mL ethanol containing 0, 1, 3, 9, or 27 mg diisopropylcarbodiimide or 0.1 mL of the neat chemical containing 81 mg per animal, 5 days a week for 2 weeks. All 9, 27, and 81 mg mice died before the end of the study. Final body weights of the surviving groups were similar to those of the vehicle controls. Clinical findings in 9, 27, and 81 mg mice included comatose conditions, convulsions/seizures, tremors, abnormal breathing, nasal/eye discharge, lethargy, and irritation at the site of application. Incidences of chronic active inflammation at the site of application in 9 mg males and females were significantly greater than those in the vehicle control groups. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female core study F344/N rats were dermally administered 0, 10, 20, 40, 80, or 160 mg diisopropylcarbodiimide/kg body weight in ethanol, 5 days per week for 3 months. Groups of 10 male and 10 female clinical pathology rats were administered the same doses for 22 days. All 160 mg/kg core study rats were sacrificed moribund or died within the first week of the study. All 80 mg/kg rats died or were found moribund by day 59. Significant decreases in body weight gain occurred in 40 mg/kg males and females, and a significant decrease in final mean body weight occurred in 40 mg/kg females. Clinical findings in groups administered 40 mg/kg or more generally included irritation of the skin at the site of application, seizures, ataxia, abnormal breathing, ruffled fur, thinness, and lethargy. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in all dosed groups of males (except 160 mg/kg) and 40 mg/kg or greater females, epidermal necrosis in 160 mg/kg males and females, and chronic active inflammation in 80 and 160 mg/kg males and females. Significantly increased incidences of nonneoplastic lesions occurred in the brain, lung, and liver (males only) of rats administered 80 or 160 mg/kg. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female B6C3F1 mice were dermally administered 0, 17.5, 35, 70, 140, or 280 mg/kg diisopropylcarbodiimide in ethanol, 5 days per week for 3 months. All mice in the 280 mg/kg group and nine males and nine females in the 140 mg/kg group died before the end of the study. The final mean body weight gain of 70 mg/kg males was significantly less than that of the vehicle control group. Clinical findings observed in 140 and 280 mg/kg mice included abnormal breathing, ataxia, comatose conditions, convulsions/seizures, irritation at the site of application, lethargy, ruffled fur, and thinness. Significant increases in kidney weights occurred in 17.5 and 35 mg/kg males. Significant decreases in total spermatid heads per testis and average spermatid count occurred in 17.5 mg/kg males. At the site of application, the incidences of epidermal hyperplasia in males and females administered 70 mg/kg or greater, chronic inflammation in 140 and 280 mg/kg males and 70 mg/kg or greater females, and sebaceous gland hyperplasia in 140 mg/kg males were significantly increased. Thymic atrophy was significantly increased in 140 and 280 mg/kg males and females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were dermally administered 0, 10, 20, or 40 mg/kg diisopropylcarbodiimide in anhydrous ethanol 5 days per week for 2 years. Survival of 20 mg/kg males was significantly greater than that of the vehicle controls; survival of all dosed groups of females was similar to that of the vehicle controls. Body weights of 40 mg/kg rats were generally less than those of the vehicle controls after week 13. Clinical findings frequently observed in 40 mg/kg males included ataxia, excitability, impaired gait, low muscle tone, abnormal breathing, lethargy, vocalization, and seizures. Because of severe neurological signs exhibited by the 40 mg/kg males, a neuropathological review of these animals was performed. The principal pathological findings of the brain included neuronal necrosis, hemorrhage, and/or fibrinoid arteriole necrosis. Incidences of hemorrhage in the lung of 40 mg/kg males, chronic lung inflammation in 10 and 20 mg/kg females, and alveolar epithelium hyperplasia in 20 mg/kg females were significantly greater than those of the vehicle controls. At the site of application, the incidences of epidermal hyperplasia in all dosed groups of males and 20 and 40 mg/kg females and chronic inflammation in all dosed groups of males and 40 mg/kg females were significantly increased. There was no increased incidences of neoplasms related to diisopropylcarbodiimide administration. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were dermally administered 0, 10, 20, or 40 mg/kg diisopropylcarbodiimide in anhydrous ethanol, 5 days per week for 2 years. Survival of all dosed groups was similar to that of the vehicle control groups. Mean body weights of dosed groups of mice were generally similar to those of the vehicle control groups throughout the study. There were no increased incidences of neoplasms that were attributed to the administration of diisopropylcarbodiimide. Significantly increased incidences of epidermal hyperplasia and focal dermal inflammation of the skin at the site of application occurred in 20 mg/kg male mice. GENETIC TOXICOLOGY: Diisopropylcarbodiimide was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 with or without liver S9 activation enzymes. In vivo, the frequency of micronucleated normochromatic erythrocytes was significantly increased in male and female mice after 3 months of dermal exposure to diisopropylcarbodiimide. In addition, significantly elevated frequencies of micronucleated polychromatic erythrocytes (reticulocytes) and micronucleated normochromatic erythrocytes were seen in male mice during a 4-month dermal exposure to diisopropylcarbodiimide. Negative results were obtained, however, in an acute three-injection rat bone marrow micronucleus study. A three-treatment acute micronucleus test in male mice also showed no increase in micronucleated erythrocytes, but results of a single injection micronucleus test in male mice were concluded to be equivocal, due to an increase in micronucleated erythrocytes seen in peripheral blood but not in bone marrow preparations. CONCLUSIONS: Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of diisopropylcarbodiimide in male or female F344/N rats or B6C3F1 mice administered 10, 20, or 40 mg/kg. Clinical and histological signs of neurotoxicity in male rats were associated with diisopropylcarbodiimide administration.     

Toxicology and carcinogenesis studies of bis(2-chloroethoxy)methane (CAS No. 111-91-1) in F344/N rats and B6C3F1 mice (dermal studies)

Bis(2-chloroethoxy)methane is used as a solvent and the starting agent in the production of fungicides and polysulfide polymers. Bis(2-chloroethoxy)methane was nominated for study by the National Institute of Environmental Health Sciences because of its widespread use as a starting material to produce polysulfide elastomers, and because there were no 2-year carcinogenicity studies reported in the literature. Male and female F344/N rats and B6C3F1 mice received dermal applications of bis(2-chloroethoxy)-methane in ethanol (greater than 98% pure) for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, rat bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were dermally administered 0, 12.5, 25, 50, 100, or 200 mg bis(2-chloroethoxy)methane/kg body weight in ethanol, 5 days per week for 16 days. All rats survived to the end of the study. Mean body weights of dosed rats were similar to those of the vehicle control groups. There were no histopathologic lesions related to bis(2-chloroethoxy)methane administration. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were dermally administered 0, 12.5, 25, 50, 100, or 200 mg bis(2-chloroethoxy)methane/kg body weight in ethanol, 5 days per week for 17 days. All mice survived to the end of the study. Mean body weights of dosed mice were similar to those of the vehicle control groups. There were no histopathologic lesions related to bis(2-chloroethoxy)methane administration. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were dermally administered 0, 50, 100, 200, 400, or 600 mg bis(2-chloroethoxy)methane/kg body weight in ethanol, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were administered the same doses for 23 days. All core study 600 mg/kg males and females and two 400 mg/kg females died before the end of the study. The cause of death was considered to be related to the cardiotoxic effect of bis(2-chloroethoxy)methane. There were no significant differences between final mean body weights of dosed rats and those of the vehicle control groups; the mean body weight gain of 400 mg/kg males was significantly less than that of the vehicle controls. Clinical findings included prostration and ataxia in 600 mg/kg rats during the first week of the study and nasal/eye discharge, lethargy, ataxia, and abnormal breathing in 400 and 600 mg/kg females beginning week 5. An enlarged heart was noted in one 100 mg/kg female rat. Relative kidney weights of 100, 200, and 400 mg/kg males were significantly greater than that of the vehicle control group. Increased incidences and severities of myofiber cytoplasmic vacuolization and interstitial mononuclear cell infiltration in the heart occurred in 400 and 600 mg/kg male and female rats and in 200 mg/kg females. Increased incidences and severities of myofiber necrosis occurred in 600 mg/kg males and females; one female each in the 200 and 400 mg/kg groups also had this lesion. Three 600 mg/kg males had atrial thrombosis. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were dermally administered 0, 50, 100, 200, 400, or 600 mg bis(2-chloroethoxy)methane/kg body weight in ethanol, 5 days per week for 14 weeks. Except for three 600 mg/kg females, all mice survived to the end of the study. Mean body weights of dosed and vehicle control mice were similar. One 600 mg/kg female that died early exhibited lethargy, abnormal breathing, and tremors, and one animal had clonic seizures. One 600 mg/kg female that died early had focal erosion of the glandular stomach and a focus in the duodenum found to consist of acute suppurative inflammation and thrombosis. Absolute and relative kidney weights of 400 and 600 mg/kg males and 600 mg/kg females were significantly greater than those of the vehicle control groups. Absolute liver weights of 400 and 600 mg/kg females were also significantly increased. Significantly increased incidences of myofiber cytoplasmic vacuolization occurred in 400 and 600 mg/kg females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were dermally administered 0, 75, 150, or 300 mg bis(2-chloroethoxy)methane/kg body weight in ethanol, 5 days per week for 105 weeks. Survival of all dosed groups of rats was generally similar to that of the vehicle controls. Mean body weights of dosed rats were similar to those of the vehicle controls throughout the study. Clinical findings in 300 mg/kg females that died during the first year of the study included abnormal breathing, lethargy, thinness, nasal discharge, and ataxia. Significantly increased incidences of degeneration of the olfactory epithelium in the nose occurred in all dosed groups of males and in 150 and 300 mg/kg females. The incidences of inflammation of the forestomach were significantly increased in 150 and 300 mg/kg males, and the incidence of ulcers was significantly increased in 300 mg/kg males. Increased incidences of cystic degeneration of the liver occurred in 150 and 300 mg/kg male rats; the incidence was significantly increased in the 300 mg/kg group. 2-YEAR STUDY IN MICE: Groups of 50 male mice were dermally administered 0, 150, 300, or 600 mg bis(2-chloroethoxy)methane/kg body weight in ethanol, 5 days per week for 105 weeks. Groups of 50 female mice were dermally administered 0, 100, 200, or 400 mg/kg in ethanol, 5 days per week for 104 weeks. Survival of 600 mg/kg male mice was significantly less than that of the vehicle control group. Mean body weights of dosed mice were generally similar to those of the vehicle controls throughout the study. Clinical findings observed in 600 mg/kg male mice that died during the first year of the study included lethargy and thinness. Myocardial heart changes were recorded according to the characteristic lesions of cardiomyopathy syndrome (necrosis, mononuclear cell infiltration, myocardial cell vacuolization, and interstitial fibrosis) separately, and in addition, where appropriate, they were also categorized as cardiomyopathy. Increased incidences of cardiomyopathy and mononuclear cell infiltration occurred in 600 mg/kg males and 400 mg/kg females; the incidences were significantly increased in 600 mg/kg males compared to the vehicle controls. Significantly increased incidences of cardiomyocyte vacuolization and interstitial fibrosis occurred in 600 mg/kg males. A few early deaths in the 600 mg/kg males were considered to be due, at least in part and probably exclusively, to bis(2-chloroethoxy)methane-induced cardiotoxicity. The incidence of ulceration of the forestomach was significantly increased in 600 mg/kg males. Significantly increased incidences of dermal inflammation and fibrosis and epidermal hyperplasia at the site of application occurred in 600 mg/kg male mice. GENETIC TOXICOLOGY: Bis(2-chloroethoxy)methane was mutagenic in S. typhimurium strains TA100 and TA1535 in the presence of exogenous metabolic activation enzymes (S9) in one study; results from a second bacterial mutagenicity test were judged to be equivocal based on responses observed in TA100 and in E. coli strain WP2 uvrA/pKM101 in the presence of S9. No mutagenicity was observed in other tester strains or in the absence of S9. Bis(2-chloroethoxy)methane did not increase the frequency of micronucleated reticulocytes in bone marrow of male F344/N rats following three daily treatments by gavage or micronucleated erythrocytes in peripheral blood of male or female mice after 3 months of dermal exposure. CONCLUSIONS: Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of bis(2-chloroethoxy)methane in male or female F344/N rats administered 75, 150, or 300 mg/kg. There was no evidence of carcinogenic activity of bis(2-chloroethoxy)methane in male B6C3F1 mice administered 150, 300, or 600 mg/kg or in female B6C3F1 mice administered 100, 200, or 400 mg/kg. The administration of bis(2-chloroethoxy)methane for 2 years resulted in increased incidences of nonneoplastic lesions in the nose of male and female rats, the forestomach of male rats, the heart of male and female mice, and the forestomach and skin of male mice.     

Toxicology and carcinogenesis studies of benzophenone (CAS No. 119-61-9) in F344/N rats and B6C3F1 mice (feed studies)

Benzophenone is used as a photoinitiator, a fragrance enhancer, an ultraviolet curing agent, and occasionally as a flavor ingredient; it is also used in the manufacture of insecticides, agricultural chemicals, and hypnotics, antihistamines, and other pharmaceuticals; and it is used as an additive in plastics, coatings, and adhesive formulations. Benzophenone was nominated for study by the National Institute of Environmental Health Sciences based on its potential for occupational and consumer exposure and the lack of long-term toxicity data. Male and female F344/N rats and B6C3F1 mice were exposed to benzophenone (greater than 99% pure) in feed for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse bone marrow cells, and mouse peripheral blood erythrocytes. Results of 14-week toxicity studies in F344/N rats and B6C3F1 mice were reported earlier (NTP, 2000). 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were fed diets containing 0, 312, 625, or 1,250 ppm benzophenone (equivalent to average daily doses of approximately 15, 30, and 60 mg benzophenone/kg body weight to males and 15, 30, and 65 mg/kg to females) for 105 weeks. Survival of 1,250 ppm males was significantly less than that of controls. Mean body weights of 1,250 ppm males were markedly less than those of the controls during year 2 of the study, and weights of exposed females were consistently less than controls throughout the study. Feed consumption by 1,250 ppm males was less than that by the controls after week 70; feed consumption by 1,250 ppm females was generally less than that by the controls throughout the study. There was a positive trend in the incidences of renal tubule adenoma in males, and the incidences in 625 and 1,250 ppm males exceeded the historical control range for all routes; these neoplasms were accompanied by significantly increased incidences of renal tubule hyperplasia. Due to these findings, additional kidney sections were evaluated; results indicated additional renal tubule adenomas in all groups of males and renal tubule hyperplasia in all groups of males and females. The incidences of pelvic transitional epithelium hyperplasia and the severity of nephropathy were significantly increased in all exposed groups of male rats. Increased incidences of mononuclear cell leukemia in all exposed groups of females exceeded the historical control range from feed studies, and the incidence in 625 ppm females was significantly greater than that in the controls. Male rats exposed to 312 or 625 ppm had significantly increased incidences of mononuclear cell leukemia. One 625 ppm female and two 1,250 ppm females had histiocytic sarcomas, and the incidence in the 1,250 ppm group exceeded the range in the historical controls. Liver lesions included significantly increased incidences of hepatocytic centrilobular hypertrophy in all exposed groups of males and females, cystic degeneration in 625 and 1,250 ppm males, and bile duct hyperplasia in all exposed groups of females. Incidences of mammary gland fibroadenoma in females exposed to 625 or 1,250 ppm were lower than expected after adjusting for body weight. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were fed diets containing 0, 312, 625, or 1,250 ppm benzophenone (equivalent to average daily doses of approximately 40, 80, and 160 mg/kg body weight to males and 35, 70, and 150 mg/kg to females) for 105 weeks. Survival of all exposed groups of mice was generally similar to that of the control groups. Mean body weights of exposed females were less than vehicle controls. Feed consumption by exposed males and females was similar to that by the controls. In male mice, there were significantly increased incidences of hepatocellular adenoma in the 625 and 1,250 ppm groups, and these incidences exceeded the historical control range. All hepatocellular neoplasms combined occurred with a positive trend. In female mice, the incidences of hepatocellular adenoma in the 625 and 1,250 ppm groups were higher than expected after adjusting for the lower body weights in these groups. Incidences of centrilobular hepatocyte hypertrophy were significantly increased in all exposed groups of males and females. All exposed groups of male mice had significant increases in the incidences of multinucleated hepatocytes and chronic active inflammation. The incidences of cystic degeneration of hepatocytes in 625 and 1,250 ppm males were significantly increased. The incidence of histiocytic sarcoma in 625 ppm females was significantly increased and exceeded the historical control range. The incidences of kidney nephropathy and mineralization in exposed groups of females and the severity of nephropathy in exposed groups of males were significantly increased. The incidences of metaplasia of the olfactory epithelium were significantly increased in 1,250 ppm males and females. The incidences of hyperplasia of lymphoid follicles in the spleen were significantly increased in all exposed groups of males and in 312 and 625 ppm females. GENETIC TOXICOLOGY: Benzophenone was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537, with or without hamster or rat liver activation enzymes. No significant increases in the frequencies of micronucleated polychromatic erythrocytes were seen in bone marrow samples from male mice administered benzophenone three times by intraperitoneal injection. In addition, no increases in micronucleated normochromatic erythrocytes were noted in peripheral blood of male or female mice administered benzophenone for 14 weeks in dosed feed. CONCLUSIONS: Under the conditions of these 2-year studies, there was some evidence of carcinogenic activity of benzophenone in male F344/N rats based on increased incidences of renal tubule adenoma; mononuclear cell leukemia in male F344/N rats may have been related to benzophenone exposure. There was equivocal evidence of carcinogenic activity of benzophenone in female F344/N rats based on the marginally increased incidences of mononuclear cell leukemia and histiocytic sarcoma. There was some evidence of carcinogenic activity of benzophenone in male B6C3F1 mice based on increased incidences of hepatocellular neoplasms, primarily adenoma. There was some evidence of carcinogenic activity of benzophenone in female B6C3F1 mice based on increased incidences of histiocytic sarcoma; the incidences of hepatocellular adenoma in female B6C3F1 mice may have been related to benzophenone exposure. Administration of benzophenone in feed resulted in increased incidences and/or severities of nonneoplastic lesions in the kidney and liver of male and female rats and in the liver, kidney, nose, and spleen of male and female mice. Decreased incidences of mammary gland fibroadenoma in female rats were related to benzophenone exposure.     

NTP technical report on the toxicology and carcinogenesis studies of beta-myrcene (CAS No. 123-35-3) in F344/N rats and B6C3F1 mice (Gavage studies)

Beta-myrcene, an acyclic unsubstituted monoterpene, and the essential oils which contain it are used as intermediates in the production of terpene alcohols (geraniol, nerol, and linalool), which, in turn, serve as intermediates in the production of aroma and flavor chemicals. Thus beta-myrcene is used widely in cosmetics, soaps, and detergents and as a flavoring additive in food and beverages. Beta-myrcene is also the major constituent of hop and bay oils, which are used in the manufacture of alcoholic beverages. Beta-myrcene was nominated for study by the National Institute of Environmental Health Sciences based on its high production volume, high level of human exposure, and structural relationship to d-limonene, which induced neoplasms in the kidneys of male rats in association with hyaline droplet nephropathy (NTP, 1990). Male and female F344/N rats and B6C3F1 mice were administered beta-myrcene (greater than 90% pure) by gavage for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 0.25, 0.5, 1, 2, or 4 g beta-myrcene/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses for 23 days. All core study rats in the 4 g/kg groups died during the first week of the study except one male that died on day 11. One to three rats in the 1 and 2 g/kg groups and one 0.5 g/kg male died by week 10 of the study. One 2 g/kg female died during the last week of the study. Except for lesion incidence data in groups administered 2 g/kg or less, data from rats that died early were excluded from the analysis and summary tables. Mean body weights were significantly decreased in male rats in the 0.5, 1, and 2 g/kg groups. Special study rats in the 4 g/kg groups died by the end of the first week. Dose-related clinical findings in animals that died early included thinness, lethargy, abnormal breathing, and ruffled fur. Right kidney and liver weights of dosed males and females were generally significantly greater than those of the vehicle controls. In special study rats evaluated on day 23, the incidences and severities of chronic progressive nephropathy (CPN) and renal tubule degeneration were increased in 2 g/kg males. At the end of the 3-month study, the incidences of renal tubule necrosis were significantly increased in all dosed groups of males and females. At 3 months, the incidences of olfactory epithelium degeneration in 2 g/kg males and females were significantly increased, and the severities were increased. The incidences of chronic inflammation in 1 and 2 g/kg males and females were significantly increased. All 2 g/kg males and females had splenic atrophy. In the mesenteric lymph node, significantly increased incidences of atrophy occurred in 2 g/kg males and 1 and 2 g/kg females. Acute inflammation of the forestomach occurred in four 2 g/kg females. The incidences of porphyrin pigmentation in the Harderian gland of males administered 0.5 g/kg or greater were significantly increased. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 0.25, 0.5, 1, 2, or 4 g beta-myrcene/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All 4 g/kg male and female mice died during week 1; nine 2 g/kg males and eight 2 g/kg females died by week 4. The mean body weights of 1 g/kg males were significantly less than those of the vehicle controls. Clinical findings in animals that did not survive to the end of the study included thinness, lethargy, and abnormal breathing. The right kidney weights of 1 g/kg females and the liver weights of females administered 0.5 or 1 g/kg were significantly increased. No histopathology changes were observed in mice administered 1 g/kg or less. The 2 and 4 g/kg mice were not evaluated due to early deaths. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 0.25, 0.5, or 1 g beta-myrcene/kg body weight in corn oil by gavage, 5 days per week for 105 weeks. All 1 g/kg male rats died before the end of the study due to renal toxicity. Compared to vehicle controls, the mean body weights of 0.25 and 0.5 g/kg males were slightly greater, and mean body weights of 1 g/kg males and females were at least 8% less than those of vehicle controls after 11 weeks and 13 weeks, respectively. In the standard evaluation of the kidney, the incidence of renal tubule adenoma was significantly increased in 0.5 g/kg male rats, and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in 0.25 and 0.5 g/kg males. In both the extended evaluation and the combined standard and extended evaluations, the incidences of renal tubule adenoma and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in the 0.25 and 0.5 g/kg groups of males. The incidences of renal tubule nephrosis (nephrosis) were markedly increased in all dosed groups of both sexes except in 0.25 g/kg females. The incidences of papillary mineralization in 0.25 and 0.5 g/kg males were significantly increased. Significantly increased incidences of nephropathy occurred in dosed females, and the severity was increased in the 0.5 and 1 g/kg males and females. The incidences of hyperplasia of the transitional epithelium lining the pelvis and overlying the renal papilla were significantly increased in all dosed groups of males and females. In male rats, the incidences of focal suppurative inflammation were significantly increased in the 0.25 and 0.5 g/kg groups. A significantly increased incidence of chronic active inflammation of the nose occurred in 0.5 g/kg males. Also in 0.5 g/kg males, the incidence of chronic active inflammation of the forestomach was increased. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 0, 0.25, 0.5, or 1 g beta-myrcene/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 weeks. Survival of 1 g/kg mice was significantly less than that of the vehicle controls; the cause of the deaths was uncertain. Mean body weights of 1 g/kg males were at least 8% less than those of the vehicle controls between week 8 and week 56. Mean body weights of 0.5 g/kg females were at least 7% less than those of the vehicle controls after week 17, and those of 1 g/kg females were at least 8% less from week 11 to week 96. The incidences of liver neoplasms were significantly increased in 0.25 and/or 0.5 g/kg males and 0.25 g/kg females. Liver neoplasms included hepatocellular adenoma and hepatocellular carcinoma in males and females and hepatoblastoma in males. The incidences of hepatocellular hypertrophy were significantly increased in 0.5 g/kg males and females, as was the incidence of mixed cell focus in 0.5 g/kg females. The incidences of bone marrow atrophy and lymph node follicle atrophy in the spleen were significantly increased in 0.5 g/kg females. In the forestomach, there were significantly increased incidences of inflammation and epithelial hyperplasia in 0.5 g/kg females. GENETIC TOXICOLOGY: beta-myrcene did not show evidence of genotoxicity in assays conducted by the NTP. No mutagenicity was observed in any of several strains of Salmonella typhimurium or Escherichia coli in two independent Ames assays conducted with and without exogenous metabolic activation. In addition, no significant increase in frequency of micronucleated normochromatic erythrocytes, biomarkers of chromosomal damage, was observed in male or female mice administered beta-myrcene for 3 months by gavage. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity of beta-myrcene in male F344/N rats based on increased incidences of renal tubule neoplasms. There was equivocal evidence of carcinogenic activity of beta-myrcene in female F344/N rats based on increased incidences of renal tubule adenoma. There was clear evidence of carcinogenic activity of beta-myrcene in male B6C3F1 mice based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma. There was equivocal evidence of carcinogenic activity of beta-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma and carcinoma. Administration of beta-myrcene induced nonneoplastic lesions in the kidney of male and female rats, nose of male rats, and liver of male and female mice. Synonyms: 2-Methyl-6-methylene-2,7-octadiene; 7-methyl-3-methylene-1,6-octadiene; myrcene.     

NTP toxicology and carcinogensis Studies of 2,4-hexadienal (89% trans,trans isomer, CAS No. 142-83-6; 11% cis,trans isomer) (Gavage Studies)

2,4-Hexadienal, a colorless to yellow liquid with a pungent "green" or citrus odor, is used as a food additive for flavor enhancement, as a fragrance agent, as a starting material or intermediate in synthetic reactions in the chemical and pharmaceutical industries, as a fumigant, and as a corrosion inhibitor for steel. 2,4-Hexadienal was nominated for study by the National Cancer Institute because of the potential for carcinogenicity based on its alpha,beta-unsaturated aldehyde structure and the potential link between exposure to lipid peroxidation products in the diet and human malignancies. The commercial product is a mixture containing chiefly trans,trans-2,4-hexadienal in equilibrium with cis,trans-2,4-hexadienal. Male and female F344/N rats and B6C3F1 mice received 2,4-hexadienal (89% trans,trans; 11% cis,trans) in corn oil by gavage for 16 days, 14 weeks, or 2 years. Tissues and plasma from dosed rats were examined for malondialdehyde and glutathione concentrations, and DNA adducts were characterized in liver and forestomach samples from dosed rats and mice. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 16-DAY STUDY IN RATS: Groups of five male and five female rats were administered 0, 3, 9, 27, 80, or 240 mg 2,4-hexadienal/kg body weight in corn oil by gavage, 5 days per week, for 16 days. Three male and three female 240 mg/kg rats died before the end of the study. Mean body weight gains of 240 mg/kg rats were significantly less than those of the vehicle controls. Clinical findings included diarrhea, ataxia, lethargy, and nasal/eye discharge in males, and lethargy, paleness, and abnormal breathing in females in the 240 mg/kg groups. Liver weights of 240 mg/kg females were significantly greater than those of the vehicle controls. Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in most 240 mg/kg rats, and forestomach epithelial hyperplasia was microscopically evident in most 80 mg/kg rats. 16-DAY STUDY IN MICE: Groups of five male and five female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 3, 9, 27, 80, or 240 mg/kg, 5 days per week, for 16 days. Chemical-related deaths occurred in one male and one female in the 240 mg/kg groups. Female mice in the 240 mg/kg group lost weight during the study. Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in all 240 mg/kg mice, and forestomach epithelial hyperplasia and hyperkeratosis were microscopically evident in 80 mg/kg mice. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks. All rats survived to the end of the study. Mean body weights of 30, 60, and 120 mg/kg males were significantly less than those of the vehicle controls. The only clinical finding attributed to 2,4-hexadienal administration was hypersalivation in 30 and 120 mg/kg males and females. The incidences of forestomach hyperplasia and nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg rats. Nasal lesions occurred in most 120 mg/kg male rats. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks. No deaths were attributed to administration of 2,4-hexadienal. Mean body weights of males and females were similar to those of the vehicle controls throughout the study. Clinical findings included salivation and anal wetness in males and females. Kidney weights of 60 and 120 mg/kg males and liver weights of 60 mg/kg males and females were significantly greater than those of the vehicle controls. The incidences of forestomach hyperplasia and/or nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg mice. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 2,4-hexministered 2,4-hexadienal in corn oil by gavage at doses of 0, 22.5, 45, or 90 mg/kg, 5 days per week, for up to 105 weeks. Survival of all dosed groups of rats was similar to that of the vehicle control groups. The mean body weights of 90 mg/kg males were generally less than those of the vehicle controls throughout the study. The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female rats. This neoplasm was found in 58% of males and 34% of females in the 90 mg/kg groups. In the forestomach of male rats, papilloma multiplicity was increased in the 90 mg/kg group, and squamous cell carcinomas were found in one 45 mg/kg male and two 90 mg/kg males. Epithelial hyperplasia of the forestomach occurred in most 45 and 90 mg/kg rats. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 30, 60, or 120 mg/kg, 5 days per week, for up to 105 weeks. Survival of dosed mice was similar to that of the vehicle controls. The mean body weights of all dosed groups were generally similar to those of the vehicle controls throughout the study. The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female mice; squamous cell carcinomas were present in 120 mg/kg males and females. Epithelial hyperplasia of the forestomach occurred in many 120 mg/kg mice. Two 120 mg/kg males had uncommon squamous cell carcinoma of the oral cavity (tongue). GENETIC TOXICOLOGY: 2,4-Hexadienal was mutagenic in S. typhimurium strain TA100 with and without induced hamster or rat liver enzymes; no mutagenic activity was detected with strains TA1535 or TA98, with or without S9. Results of bone marrow tests in male rats and male mice given intraperitoneal injections of 2,4-hexadienal showed a small increase in the induction of micronucleated erythrocytes. However, neither test was repeated, and the test results were judged to be inconclusive. Results of peripheral blood micronucleus tests in male and female mice treated with 2,4-hexadienal by gavage for 14 weeks were negative. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity* of 2,4-hexadienal in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of squamous cell neoplasms of the forestomach. The occurrence of squamous cell carcinoma of the oral cavity (tongue) in male B6C3F1 mice may have been related to the administration of 2,4-hexadienal. Hyperplasia of the forestomach in male and female rats and mice was associated with administration of 2,4-hexadienal. Synonyms: Hexa-2,4-dienal; 2,4-hexadienal; 2,4-hexadien-1-al; 2,4-Hx; 1,3-pentadiene-1-carboxaldehyde; 2-propylene acrolein; sorbaldehyde; sorbic aldehyde     

Toxicology and carcinogenesis studies of 3,3',4,4'-tetrachloroazobenzene (TCAB) (CAS No. 14047-09-7) in Harlan Sprague-Dawley rats and B6C3F1 mice (gavage studies)

3,3',4,4'-Tetrachloroazobenzene (TCAB) is not commercially manufactured but is formed as an unwanted by-product in the manufacture of 3,4-dichloroaniline and its herbicidal derivatives Propanil, Linuron, and Diuron. It occurs from the degradation of chloroanilide herbicides (acylanilides, phenylcarbamates, and phenylureas) in soil by peroxide-producing microorganisms; and is formed by the photolysis and biolysis of 3,4-dichloroaniline. Humans may be exposed to TCAB during the manufacture as well as the application of herbicides containing TCAB as a contaminant. TCAB was nominated by the United States Environmental Protection Agency for toxicity and carcinogenicity testing based on its structural and biological similarity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the potential for human exposure from the consumption of crops contaminated with 3,4-dichloroaniline-derived herbicides. Male and female Harlan Sprague-Dawley rats and B6C3F1 mice were administered TCAB (at least 97.8% pure) in corn oil:acetone (99:1) by gavage for 3 months (rats only) or 2 years. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female Harlan Sprague-Dawley rats were administered 0.1, 0.3, 1, 3, 10, 30, or 100 mg TCAB/kg body weight in corn oil:acetone (99:1) by gavage, 5 days a week, for 14 weeks; groups of 10 male and 10 female rats received the corn oil:acetone vehicle alone. Special study groups of 30 (dosed groups) or 6 (vehicle control group) female Harlan Sprague-Dawley rats were administered 0.1, 3, or 100 mg TCAB/kg body weight in corn oil:acetone (99:1) by gavage, 5 days a week, for 13 weeks; vehicle controls received the corn oil:acetone vehicle alone. All male and female rats survived to the end of the study. Terminal mean body weights of males were not significantly different from vehicle controls in any group. Terminal mean body weights of females administered 10 mg/kg or greater were significantly less than those of the vehicle controls. Mean body weight gains of all dosed groups of females were significantly less than those of the vehicle controls. The hematology results indicate that TCAB induced a microcytic normochromic responsive anemia in male Sprague-Dawley rats. Serum concentrations of total thyroxine (T4) and free T4 were significantly decreased in a dose-related manner in all dosed groups in both sexes compared to their respective vehicle controls; total triiodothyronine (T3) and thyroid stimulating hormone (TSH) concentrations were generally unaffected. There were no statistically significant differences in the BrdU labeling indices in the liver of males or females exposed to TCAB compared to their respective vehicle controls. Significant induction of hepatic 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-deethylase activities was observed in all dosed groups of males and females. Significant induction of hepatic acetanilide-4-hydroxylase activity was observed in males exposed to 3 mg/kg or greater and all treated groups of females. EROD activities in the lung generally increased with increasing dose and were significantly greater in all treated groups of males and females compared to their respective vehicle controls. The highest concentrations of TCAB were observed in fat tissue with lower concentrations in the liver and lung. TCAB concentrations were significantly increased in a dose-dependent manner in all tissues from dosed groups relative to vehicle controls. At the end of the 3-month study, absolute and relative liver weights were significantly greater than those of the vehicle controls in all dosed groups of males and in females administered 10 mg/kg or greater. Absolute and relative lung weights were significantly greater in 100 mg/kg males and 3 mg/kg or greater females. Absolute and relative right kidney and spleen weights were generally significantly greater for all dosed groups of males. Absolute thymus weights of 10 mg/kg or greater males and absolute and relative thymus weights of 1 mg/kg or greater females were significantly less than those of the vehicle controls. In the liver, the incidences of midzonal to diffuse hepatocytic hypertrophy in males administered 1 mg/kg or greater and in females administered 10 mg/kg or greater were significantly greater than the vehicle control incidences. Hematopoietic cell proliferation occurred in most males administered 3 mg/kg or greater and most females administered 10 mg/kg or greater. The incidences of midzonal hepatocytic cytoplasmic fatty vacuolization were significantly increased in males administered 3 mg/kg or greater. In the lung, significantly increased incidences of bronchiolar metaplasia of the alveolar epithelium and interstitial mononuclear cell infiltration occurred in 10, 30, and 100 mg/kg males. The incidence of interstitial mononuclear cell infiltration was also significantly increased in 100 mg/kg females. Significantly increased incidences of hematopoietic cell proliferation of the spleen occurred in males administered 10 mg/kg or greater. The incidences of hemosiderin pigment of the spleen were significantly increased in 10 mg/kg or greater females. Atrophy in the thymus was significantly increased in all dosed groups of females, except the 0.1 mg/kg group, and in males administered 10 mg/kg or greater. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female Harlan Sprague-Dawley rats were administered 10, 30, or 100 mg TCAB/kg body weight in corn oil:acetone (99:1) by gavage, 5 days a week, for 2 years; groups of 50 male and 50 female rats received the corn oil:acetone vehicle alone. The survival of all dosed groups of males was significantly less than that of the vehicle controls. Mean body weights of 100 mg/kg males were less than those of the vehicle control group throughout the study. Mean body weights of 30 mg/kg males were 6% less than those of the vehicle control group after week 24, and those of 10 mg/kg males were 7% less than the vehicle control group after week 80. Mean body weights of 100 mg/kg females were less than those of the vehicle control group throughout the study, and those of 30 mg/kg females were 6% less than the vehicle control group after week 36. In the lung, the incidences of multiple cystic keratinizing epithelioma and single or multiple cystic keratinizing epithelioma (combined) in males and females were significantly increased in all dosed groups (except multiple epithelioma in 10 mg/kg females). Significantly increased incidences of pigmentation, alveolar epithelium squamous metaplasia (except 10 mg/kg females), and alveolar epithelium bronchiolar metaplasia occurred in all dosed groups of males and females. The incidences of histiocytic cellular infiltration in all dosed groups of males were significantly increased. In the liver, the incidences of cholangiocarcinoma (single or multiple) occurred in a positive trend in males and were significantly greater than that in the vehicle control group; the incidence in 100 mg/kg females was also increased. A significant dose-related increase in hepatic toxicity was observed in dosed rats and was characterized by increased incidences of numerous lesions including hepatocyte hypertrophy, centrilobular degeneration, hepatocellular necrosis, pigmentation, fatty change, bile duct hyperplasia, oval cell hyperplasia, nodular hyperplasia, hematopoietic cell proliferation, eosinophilic focus, mixed cell focus, multinucleated hepatocytes, bile duct cyst, toxic hepatopathy, and cholangiofibrosis. Significantly increased incidences of gingival squamous cell carcinoma within the oral mucosa occurred in 10 mg/kg males and 100 mg/kg males and females. The incidences of gingival squamous hyperplasia and cystic keratinizing hyperplasia in dosed groups of males and females were generally significantly increased. The incidences of follicular cell adenoma (single or multiple) of the thyroid gland in 30 and 100 mg/kg males were significantly greater than that in the vehicle control group. The incidences of follicular cell hypertrophy, follicular cell hyperplasia, and inflammation were significantly increased in 30 and 100 mg/kg males. Three incidences of single or multiple squamous cell papilloma of the forestomach occurred in 100 mg/kg females, and single incidences of squamous cell carcinoma of the forestomach occurred in 10 and 100 mg/kg females. Significantly increased incidences of epithelial hyperplasia occurred in all dosed groups of males and females. There were three incidences of malignant schwanomma in the thoracic cavity in 100 mg/kg males and a single incidence in 30 mg/kg males. In the adrenal cortex of 30 and 100 mg/kg females, there were slightly increased incidences of adenoma. In all dosed groups of males, the incidences of degeneration, cytoplasmic vacuolization, and hyperplasia of the zona fasciculata were significantly increased. Increased incidences and severities of necrosis occurred in 30 and 100 mg/kg males. Incidences of cytoplasmic vacuolation in 10 and 100 mg/kg females and hyperplasia of the zona fasciculata in 30 mg/kg females were significantly greater than those in the vehicle controls. Numerous nonneoplastic effects were seen in other organs including atrophy, acinar cytoplasmic vacuolization, and inflammation of the pancreas; blood vessel inflammation; lymphoid follicle atrophy and pigmentation of the spleen; pigmentation and atrophy of the mesenteric lymph node; germinal epithelial degeneration of the testes; and inflammation of the nose. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 3, 10, or 30 mg TCAB/kg body weight in corn oil:acetone (99:1) by gavage, 5 days a week, for 2 years; groups of 50 male and 50 female rats received the corn oil:acetone vehicle alone. Survival of 10 and 30 mg/kg males and 30 mg/kg females was significantly less than that of vehicle controls. All 30 mg/kg males died before the end of the study. (ABSTRACT TRUNCATED)     

Toxicology and carcinogenesis studies of kava kava extract (CAS No. 9000-38-8) in F344/N rats and B6C3F1 mice (Gavage Studies)

Kava beverages, made from dried roots of the shrub Piper methysticum, have been used ceremonially and socially in the South Pacific and in Europe since the 1700s. The drink is reported to have pleasant mild psychoactive effects, similar to alcoholic beverages. In the United States, kava kava is an herbal product used extensively as an alternative to anti-anxiety drugs such as Xanax and Valium. It has also been reported as being used to help children with hyperactivity and as a skin-conditioning agent in cosmetics. Kava kava was nominated by the National Cancer Institute for study because of its increasing use as a dietary supplement in the mainstream United States market and reports of liver toxicity among humans. Male and female F344/N rats and B6C3F1 mice received kava kava extract in corn oil 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 kava kava extract in corn oil by gavage at doses of 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/kg body weight, 5 days per week for 16 days. One female rat administered 2.0 g/kg kava kava extract died on day 3 of the study. Mean body weights of all dosed groups of rats were similar to those of the vehicle controls. Clinical findings included abnormal breathing, ataxia, and lethargy in the 2.0 g/kg groups of males and females and ataxia and lethargy in the 1.0 g/kg group of females. Liver weights were significantly increased in 1.0 and 2.0 g/kg males and in 0.5 g/kg or greater females compared to the vehicle controls. Minimal hepatocellular hypertrophy occurred in all 2.0 g/kg males and in all females administered 0.25 g/kg or greater. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were administered kava kava extract in corn oil by gavage at doses of 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/kg body weight, 5 days per week for 17 days. In the 2.0 g/kg group of males, one died on day 2 and one died on day 3. Mean body weights of all dosed groups of mice were similar to those of the vehicle controls. Clinical findings included abnormal breathing, ataxia, and lethargy in males and females in the 1.0 and 2.0 g/kg groups. Liver weights of 2.0 g/kg males and females were significantly increased. The incidence of hepatocellular hypertrophy in 2.0 g/kg female mice was significantly greater than that in the vehicle control group. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered kava kava extract in corn oil by gavage at doses of 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/kg, 5 days per week for 14 weeks. Deaths attributed to kava kava extract administration included three males and four females in the 2.0 g/kg groups and one female in the 1.0 g/kg group. One 0.25 g/kg male and one vehicle control female also died before the end of the study. The mean body weights of males in the 1.0 and 2.0 g/kg groups and females in the 2.0 g/kg group were significantly less than those of the vehicle controls. Ataxia and lethargy were observed in males and females in the 1.0 g/kg groups during week 1 and in the 2.0 g/kg groups throughout the study. Increased -glutamyltransferase activity in 1.0 g/kg females and 2.0 g/kg males and females may represent enzyme induction. However, the hepatocellular hypertrophy observed in the 2.0 g/kg females may have contributed to the increased -glutamyltransferase activity. The liver weights of 0.25 g/kg or greater males and 0.5 g/kg or greater females were significantly increased compared to the vehicle controls. The kidney weights of 0.5 g/kg or greater males and females were significantly increased compared to the vehicle controls. The incidence of hepatocellular hypertrophy in 2.0 g/kg females was significantly greater than that in the vehicle controls. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered kava kava extract in corn oil by gavage at doses of 0, 0.125, 0.25, 0.5, 1.0, or 2.0 g/kg, 5 days per week for 14 weeks. Four male and three female 2.0 g/kg mice died during week 1; these deaths were attributed to kava kava extract administration. One additional 2.0 g/kg female died during week 6 due to a gavage accident. The mean body weights of dosed males and females were similar to those of the vehicle controls. Ataxia and lethargy occurred in males and females in the 1.0 and 2.0 g/kg groups during week 1. The liver weights of 2.0 g/kg males and 1.0 and 2.0 g/kg females were significantly increased compared to those of the vehicle control groups. The incidences of centrilobular hypertrophy in the liver of 0.5 g/kg or greater males and 1.0 and 2.0 g/kg females were significantly greater than those in the vehicle controls. 2-YEAR STUDY IN RATS: Groups of 49 or 50 male and 50 female rats were administered kava kava extract in corn oil by gavage at doses of 0, 0.1, 0.3, or 1.0 g/kg, 5 days per week for 104 (males) or 105 (females) weeks. Survival of dosed groups of males and females was similar to that of the vehicle controls. Mean body weights of males administered 1.0 g/kg were less than those of the vehicle controls after week 65, and those of the 1.0 g/kg females were less than those of the vehicle controls after week 41. Clinical findings included ataxia and lethargy that occurred in 21 males and 14 females in the 1.0 g/kg groups during the first 4 weeks of the study. After week 5, ataxia and lethargy were noted in 10 males and eight females in the 1.0 g/kg groups and these findings were observed randomly and intermittently throughout the study. At approximately 1 year into the study, twitching and seizures were observed in males and females in all dosed groups but mainly in the 1.0 g/kg groups. There was a dose-related increase in the incidences of interstitial cell adenoma in the testis with increased incidences of bilateral neoplasms. The incidences of hepatocellular hypertrophy in 1.0 g/kg males and females were significantly greater than those in the vehicle controls. Increased -glutamyltransferase activity and/or bile salt concentrations in males and females may represent a cholestatic event related to the hepatocellular hypertrophy observed in rats. Enzyme induction may have played a role in the increased -glutamyltransferase activity. Significantly increased incidences of centrilobular fatty change occurred in 0.1 and 1.0 g/kg males. The incidences of inflammation, ulcer, and epithelial hyperplasia in the forestomach were significantly increased in 1.0 g/kg males and females. The severity of nephropathy was increased in 1.0 g/kg male rats, and the incidence of nephropathy was significantly increased in 1.0 g/kg females. Incidences of transitional epithelial hyperplasia of the pelvis of the kidney were significantly increased in 1.0 g/kg males and 0.3 and 1.0 g/kg females. The incidences of retinal degeneration in the eye were significantly increased in 1.0 g/kg males and females. The incidences of metaplasia of pancreatic acinar cells to a hepatocytic morphology increased in 1.0 g/kg males and females, and the increase in males was significant. Significantly decreased incidences of pars distalis adenoma in the pituitary gland occurred in 1.0 g/kg males and in 0.1 and 1.0 g/kg females. The incidence of fibroadenoma of the mammary gland in 1.0 g/kg females was significantly less than that in the vehicle control group. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice received kava kava extract in corn oil by gavage at doses of 0, 0.25, 0.5, or 1.0 g/kg, 5 days per week for 105 weeks. Survival of dosed groups of males and females was similar to that of the vehicle controls. Mean body weights of males administered 1.0 g/kg were generally similar to those of the vehicle controls until the end of the study; however, those of 1.0 g/kg females were less than those of the vehicle controls after week 21. Clinical findings included ataxia and lethargy that occurred in 13 males and 31 females in the 1.0 g/kg groups during the first week of the study. Decreasing numbers of animals exhibited ataxia or lethargy during the remainder of the study, but these findings were observed in 1.0 g/kg females as late as week 101. The incidences of hepatoblastoma in 0.5 and 1.0 g/kg males were significantly increased compared to the vehicle controls. The incidences of hepatocellular carcinoma or hepatoblastoma (combined) were significantly increased in 0.5 g/kg males. Incidences of hepatocellular carcinoma were increased in all dosed groups of females, and the increase was significant in the 0.25 g/kg group. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in 0.25 and 0.5 g/kg females. In the liver, the incidences of centrilobular hypertrophy in all dosed groups of males and females were significantly greater than those in the vehicle control groups. Significantly increased incidences of eosinophilic foci occurred in 0.5 g/kg males and in 1.0 g/kg males and females, and the incidence of angiectasis was significantly increased in the 1.0 g/kg males. The incidences of hepatocellular necrosis were significantly increased in 0.25 and 1.0 g/kg males. In the forestomach, the incidences of chronic inflammation, epithelial hyperplasia, and erosion were significantly increased in 0.5 and 1.0 g/kg females, and the incidence of ulceration was significantly increased in 1.0 g/kg females. GENETIC TOXICOLOGY: Kava kava extract was tested for bacterial mutagenicity over a broad range of concentrations in two independent assays using several strains of bacteria (S. typhimurium tester strains TA97, TA98, TA100, and TA1535 and E. coli strain WP2 uvrA/pKM101), with and without exogenous metabolic activation. No increase in mutant colonies was seen in any of the tester strains, under any activation condition. (ABSTRACT TRUNCATED)     

Toxicology and carcinogenesis studies of 4-methylimidazole (Cas No. 822-36-6) in F344/N rats and B6C3F1 mice (feed studies)

4-Methylimidazole is used in the manufacture of pharmaceuticals, photographic chemicals, dyes and pigments, cleaning and agricultural chemicals, and rubber. It has been identified as a by-product of fermentation in foods and has been detected in mainstream and sidestream tobacco smoke. 4-Methylimidazole was nominated by the National Cancer Institute for a long-term study because of the high potential for human exposure. Male and female F344/N rats and B6C3F1 mice were exposed to 4-methylimidazole (99.5% pure) in feed for 2 years. Fifteen-day and 14-week toxicity studies of 4-methylimidazole in F344/N rats and B6C3F1 mice are reported in NTP Toxicity Report No. 67. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were fed diets containing 0, 625, 1,250, or 2,500 ppm 4-methylimidazole (males) or 0, 1,250, 2,500, or 5,000 ppm 4-methylimidazole (females) (equivalent to average daily doses of approximately 30, 55, or 115 mg 4-methylimidazole/kg body weight to males and 60, 120, or 260 mg/kg to females) for 106 weeks. Survival of all exposed groups of male and female rats was similar to that of the control groups. Mean body weights of males in the 1,250 and 2,500 ppm groups and females in the 2,500 and 5,000 ppm groups were less than those of the control groups throughout the study; mean body weights of 1,250 ppm females were less after week 41. Feed consumption by 5,000 ppm females was less than that by the controls. Clonic seizures, excitability, hyperactivity, and impaired gait were observed primarily in 2,500 and 5,000 ppm females. The incidence of mononuclear cell leukemia in 5,000 ppm females was significantly greater than that in the controls, and the incidence exceeded the historical range in feed study controls. The incidences of hepatic histiocytosis, chronic inflammation, and focal fatty change were generally significantly increased in all exposed groups of male and female rats. The incidences of hepatocellular eosinophilic and mixed cell focus were significantly increased in 2,500 ppm males and 5,000 ppm females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were fed diets containing 0, 312, 625, or 1,250 ppm 4-methylimidazole (equivalent to average daily doses of approximately 40, 80, and 170 mg 4-methylimidazole/kg body weight to males and females) for 106 weeks. Survival of all exposed groups of male and female mice was similar to that of the control groups. Mean body weights of males and females in the 1,250 ppm groups were less than those of the control groups after weeks 17 and 12, respectively. Mean body weights of 312 and 625 ppm females were less after weeks 85 and 65, respectively. Feed consumption by exposed groups of male and female mice was generally similar to that by the controls. The incidences of alveolar/bronchiolar adenoma in all exposed groups of females, alveolar/bronchiolar carcinoma in 1,250 ppm males, and alveolar/bronchiolar adenoma or carcinoma (combined) in 1,250 ppm males and 625 and 1,250 ppm females were significantly greater than those in the control groups. The incidence of alveolar epithelium hyperplasia was significantly increased in 1,250 ppm females. GENETIC TOXICOLOGY: 4-Methylimidazole was not mutagenic in the S. typhimurium mutation assay when tested in strains TA97, TA98, TA100, and TA1535, with and without hamster or rat liver metabolic activation enzymes. No consistent or significant increases in the frequencies of micronucleated erythrocytes were seen in the bone marrow of male rats or mice treated with 4-methylimidazole by intraperitoneal injection, or in peripheral blood samples from male and female mice administered the compound in dosed feed for 14 weeks. CONCLUSIONS: Under the conditions of these 2-year studies, there was no evidence of carcinogenic activity of 4-methylimidazole in male F344/N rats exposed to 625, 1,250, or 2,500 ppm. There was equivocal evidence of carcinogenic activity of 4-methylimidazole in female F344/N rats based on increased incidences of mononuclear cell leukemia. There was clear evidence of carcinogenic activity of 4-methylimidazole in male and female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Exposure to 4-methylimidazole resulted in nonneoplastic lesions in the liver of male and female rats and the lung of female mice and in clinical findings of neurotoxicity in 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.     

NTP technical report on the toxicology and carcinogenesis studies of Stoddard Solvent IIC (Cas No. 64742-88-7) in F344/N rats and B6C3F1 mice (inhalation studies)

Stoddard solvent (white spirit/mineral spirit) is the most widely used solvent in the paint industry. It is used as a dry cleaning agent; as an extraction, cleaning, and degreasing solvent; and as a solvent in aerosols, paints, wood preservatives, asphalt products, lacquers, and varnishes. Stoddard solvent IIC was nominated by the International Union, United Auto Workers, for carcinogenicity testing because of the large volume used in industrial and other settings. Male and female F344/N rats and B6C3F1 mice were exposed to Stoddard solvent IIC (greater than 99% pure) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m3, 6 hours per day, 5 days per week for 16 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. Liver weights of males exposed to 550 mg/m3 or greater and of females exposed to 275 mg/m3 or greater were increased. Minimal diffuse cytoplasmic vacuolization of hepatocytes of the liver occurred in all females exposed to 2,200 mg/m3. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m3, 6 hours per day, 5 days per week for 17 days. All mice survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Liver weights of males and females exposed to 275 mg/m3 or greater were significantly increased. Cytomegaly of the liver occurred in all males and females exposed to 2,200 mg/m3. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m3, 6 hours per day, 5 days per week for 14 weeks. All rats survived to the end of the study, and the final mean body weight of females exposed to 275 mg/m3 was greater than that of the chamber controls. The relative kidney, liver, and testis weights of all exposed groups of males and the absolute kidney weights of males exposed to 550 mg/m3 or greater were increased. The sperm motility of 550 mg/m3 or greater males was significantly decreased. The incidences of renal tubule granular casts were significantly increased in males exposed to 550 mg/m3 or greater, and the severities of renal tubule hyaline droplet accumulation, granular casts, and regeneration increased with increasing exposure concentration in males. The incidences of goblet cell hypertrophy of the nasal respiratory epithelium in males and females exposed to 2,200 mg/m3 were significantly increased. Sperm motility was decreased in males exposed to 550 mg/m3 or greater. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m3, 6 hours per day, 5 days per week for 14 weeks. Mean body weights of exposed groups were similar to those of the chamber controls, but liver weights of males exposed to 2,200 mg/m3 were significantly increased. The sperm motility of 2,200 mg/m3 males was significantly decreased. This reduction in sperm motility, while statistically significant, is probably of modest importance as studies in mice have found that fertility is unaffected by motility decreases of less than 40%. The incidences of hematopoietic cell proliferation of the spleen in all exposed groups of females were greater than that in the chamber controls. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138 (males), 550, 1,100, or 2,200 (females) mg/m3, 6 hours per day, 5 days per week for 104 to 105 weeks. Additional groups of 10 males and 10 females were exposed to the same concentrations for 3 months for renal toxicity analyses. Survival in the top exposure concentration groups of males and females was significantly less than that of the chamber controls. Mean body weights of exposed males and females were similar to those of the chamber controls. Cell proliferation analyses were performed in the left kidney of males and females after 3 months of exposure. The mean numbers of labeled cells and the labeling indices in males exposed to 550 and 1,100 mg/m3 were significantly increased. The amount of alpha2u-globulin in the right kidney of males increased with increasing exposure concentration. Also, the incidences of granular casts and cortical tubule degeneration and regeneration were generally increased in exposed males, as was the severity of hyaline droplets. These effects did not occur in females. At 2 years, the incidences of benign and benign or malignant pheochromocytoma (combined) of the adrenal medulla occurred with positive trends in males, and the incidences in the 550 and 1,100 mg/m3 groups were significantly increased. Due to increased incidences of renal tubule hyperplasia in males at 2 years, extended kidney evaluations were conducted; a slightly increased incidence of renal tubule adenoma occurred in the 1,100 mg/m3 group. Nonneoplastic lesions related to Stoddard solvent IIC exposure occurred in the kidney of males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 550, 1,100, or 2,200 mg/m3, 6 hours per day, 5 days per week for 105 weeks. Survival of exposed mice was similar to that of the chamber controls. Mean body weights of exposed females were greater than those of the chamber controls. The incidences of hepatocellular adenoma occurred with a positive trend in females, and the incidence of multiple hepatocellular adenoma in females exposed to 2,200 mg/m3 was significantly increased. However, the incidences of hepatocellular adenoma or carcinoma (combined) and hepatocellular carcinoma alone in exposed males and females were not significantly increased. GENETIC TOXICOLOGY: Stoddard solvent IIC was tested for mutagenicity in Salmonella typhimurium strains TA97, TA98, TA100, and TA1535, with and without S9 metabolic activation enzymes; all results were negative. In vivo, the frequency of micronucleated erythrocytes was assessed in peripheral blood samples from male and female B6C3F1 mice after 3 months of inhalation exposure to Stoddard solvent IIC, and results were negative. CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of Stoddard solvent IIC in male F344/N rats based on increased incidences of adrenal medulla neoplasms; the slightly increased incidences of renal tubule adenoma may have been related to Stoddard solvent IIC exposure. There was no evidence of carcinogenic activity of Stoddard solvent IIC in female F344/N rats exposed to 550, 1,100, or 2,200 mg/m3. There was no evidence of carcinogenic activity of Stoddard solvent IIC in male B6C3F1 mice exposed to 550, 1,100, or 2,200 mg/m3. There was equivocal evidence of carcinogenic activity of Stoddard solvent IIC in female B6C3F1 mice based on increased incidences of hepatocellular adenoma; this slight increase was associated with increased body weight in exposed females. Exposure of male rats to Stoddard solvent IIC resulted in nonneoplastic lesions of the kidney characteristic of alpha2u-globulin accumulation.     

Toxicology and carcinogenesis studies of methyl trans-styryl ketone (CAS NO 1896-62-4) in F344/N rats and B6C3F1 mice (feed and dermal studies)

Methyl trans-styryl ketone is used as a synthetic flavoring agent and a fragrance additive in food and personal care products. Methyl trans-styryl ketone was nominated for study by the National Cancer Institute due to widespread human exposure as a flavoring and fragrance additive, positive results in the Ames/Salmonella assay and the mouse lymphoma L5178Y/tk+/- assay, and as a representative of the α,β-unsaturated ketone chemical class. Male and female F344/N rats and B6C3F1 mice received methyl trans-styryl ketone (98.6% pure) in feed for 3 months and dermally for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. Two-year studies were conducted to provide data for assessment of possible toxicity due to exposure to methyl trans-styryl ketone. The dermal route was chosen since this is the route for highest human exposure and due to studies demonstrating systemic exposure following dermal application to methyl trans-styryl ketone. 3-MONTH FEED STUDY IN RATS Groups of 10 male and 10 female rats were fed diets containing 0%, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% methyl trans-styryl ketone (equivalent to average daily doses of approximately 18, 36, 72, 145, or 290 mg methyl trans-styryl ketone/kg body weight to males and 19, 38, 77, 150, or 300 mg/kg to females) for 14 weeks. Groups of 10 male and 10 female clinical pathology rats were fed the same concentrations for 24 days. All core study rats survived to the end of the study. Final mean body weights of males and females receiving 0.4% and mean body weight gains of males receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Clinical findings included diarrhea and hyperactivity in males and females. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male rats; however, it exhibits potential for reproductive toxicity in female rats based upon an increased probability of extended diestrus at the highest exposure concentration. In all exposed groups of males, there were treatment-related increased incidences of goblet cell hyperplasia of the respiratory epithelium of the nose and nephropathy of the kidney. In females, there was an increased incidence of goblet cell hyperplasia of the respiratory epithelium of the nose in the group receiving 0.4%. 3-MONTH FEED STUDY IN MICE: Groups of 10 male and 10 female mice were fed diets containing 0%, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% methyl trans-styryl ketone (equivalent to average daily doses of approximately 55, 110, 220, 400, or 750 mg/kg to males and 50, 100, 200, 350, or 600 mg/kg to females) for 14 weeks. One male receiving 0.2% and one control female died before the end of the study. Mean body weights of males and females receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Hyperactivity in both sexes was the only clinical finding. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male mice; however, it exhibits potential for reproductive toxicity in female mice based upon an increased probability of extended diestrus at the lowest and the highest exposure concentrations. There were significantly increased incidences of olfactory epithelial atrophy of the nose in males and females receiving 0.4%. 3-MONTH DERMAL STUDY IN RATS: Groups of 10 male and 10 female rats were dermally administered 0, 22, 44, 87.5, 175, or 350 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 14 weeks. Groups of 10 male and 10 female clinical pathology rats were administered the same doses for 23 days. All rats survived to the end of the study. Mean body weights of 175 and 350 mg/kg males were significantly less than that of the vehicle controls. Clinical findings in groups administered 175 or 350 mg/kg included dermal irritation, thickened skin, and ulceration at the site of application. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male or female rats at the doses used in this study. Histologically, there were significantly increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, and sebaceous gland hypertrophy in the skin at the site of application in males and/or females. There were significantly increased incidences of goblet cell hyperplasia of the nose in 350 mg/kg males and 22, 175, and 350 mg/kg females. 3-MONTH DERMAL STUDY IN MICE Groups of 10 male and 10 female mice were dermally administered 0, 87.5, 175, 350, 700, or 1,400 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 13 weeks. All mice in the 700 and 1,400 mg/kg groups were sacrificed moribund before the end of the study. The final mean body weights of surviving groups of dosed males and females were similar to those of the vehicle controls; however, the mean body weight gains of the 175 mg/kg groups were significantly less than those of the vehicle controls. Clinical findings at the site of application included dermal irritation in 350 mg/kg males and crust formation in all 700 and 1,400 mg/kg mice except one female. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male or female mice at the doses used in this study. There were treatment-related increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, sebaceous gland hypertrophy, and hair follicle hyperplasia in the skin at the site of application in males and females. There were increased incidences of olfactory epithelial atrophy of the nose in groups of males and females administered 350 mg/kg or greater. 2-YEAR DERMAL STUDY IN RATS: Groups of 50 male and 50 female rats were dermally administered 0, 10, 30, or 90 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 105 weeks. Survival of all dosed groups was similar to that of the vehicle controls. Mean body weights of dosed groups were within 10% of those of the vehicle control groups throughout the study. In the skin at the site of application, there were increased incidences of epidermal hyperplasia and hyperkeratosis in males and females administered 30 or 90 mg/kg. 2-YEAR DERMAL STUDY IN MICE: Groups of 50 male and 50 female mice were dermally administered 0, 10, 30, or 90 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 105 weeks. Survival of all dosed groups was similar to that of the vehicle controls. Mean body weights of dosed groups were within 10% of those of the vehicle control groups throughout the study. In the skin at the site of application in males and females, there were treatment-related increased incidences of epidermal hyperplasia, hyperkeratosis, chronic inflammation, and melanocyte hyperplasia. GENETIC TOXICOLOGY: Methyl trans-styryl ketone was mutagenic in Salmonella typhimurium strain TA100 when testing was conducted in the presence of rat liver microsomes (S9). No mutagenic activity was seen with methyl trans-styryl ketone in strain TA98 with or without S9 or in Escherichia coli strain WP2 uvrA/pKM101 in the absence of S9. With S9, inconsistent responses were seen in the E. coli tester strain. No increases in the frequencies of micronucleated normochromatic erythrocytes were seen in peripheral blood samples from male or female mice administered methyl trans-styryl ketone for 3 months via dosed feed or dermal application. CONCLUSIONS: Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of methyl trans-styryl ketone in male or female F344/N rats or in male or female B6C3F1 mice administered 10, 30, or 90 mg/kg. Administration of methyl trans-styryl ketone resulted in nonneoplastic lesions of the skin at the site of application in male and female rats and mice. Synonyms: Acetocinnamone; benzalacetone; benzylideneacetone; methyl 2-phenylvinyl ketone; methyl styryl ketone; methyl β-styryl ketone; MSK; 4-phenyl-3-butene-2-one; 4-phenylbutenone; 2-phenylvinyl methyl ketone; styryl methyl ketone Systematic name: (3E)-4-Phenylbut-3-en-2-one.     

Toxicology and carcinogenesis studies of sodium dichromate dihydrate (Cas No. 7789-12-0) in F344/N rats and B6C3F1 mice (drinking water studies)

Sodium dichromate dihydrate is one of a number of inorganic compounds containing hexavalent chromium (Cr VI) found in drinking water source supplies as a contaminant resulting from various industrial processes including electroplating operations, leather tanning, and textile manufacturing. Because of the lack of adequate experimental data on the toxicity and carcinogenicity of hexavalent chromium ingested orally and because hexavalent chromium has been found in drinking water source supplies, the California Congressional Delegation, the California Environmental Protection Agency, and the California Department of Health Services nominated hexavalent chromium to the National Toxicology Program for study. Results of 3 month toxicity studies in F344/N rats and B6C3F1, BALB/c, and am3-C57BL/6 mice were reported earlier in NTP Toxicity Report 72. In the current study, male and female F344/N rats and B6C3F1 mice were exposed to sodium dichromate dihydrate (greater than 99.7% pure) in drinking water for 2 years. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to drinking water containing 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate (equivalent to 0, 5, 20, 60, or 180 mg/L chromium) for 2 years (equivalent to average daily doses of approximately 0.6, 2.2, 6, or 17 mg sodium dichromate dihydrate/kg body weight for males and 0.7, 2.7, 7, or 20 mg/kg for females). Survival of exposed groups was similar to that of the control groups. Mean body weights of 516 mg/L males and females were less than those of the controls throughout the study. The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption. Water consumption by 172 and 516 mg/L rats was less than that by the controls throughout the study. Exposure to sodium dichromate dihydrate caused a microcytic hypochromic anemia in rats that ameliorated with time. Exposure to sodium dichromate dihydrate resulted in the development of neoplasms of the squamous epithelium that lines the oral mucosa and tongue. The incidences of squamous cell carcinoma in the oral mucosa of 516 mg/L male and female rats were significantly greater than those in the controls. The incidence in 172 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration. The incidences of squamous cell papilloma or squamous cell carcinoma (combined) of the oral mucosa or tongue of 516 mg/L male and female rats were significantly greater than those in the controls. Exposure concentration-related nonneoplastic liver lesions were observed in males and females exposed to 57.3 mg/L or greater. These included histiocytic cellular infiltration, chronic inflammation, fatty change (females), basophilic focus (males), and clear cell focus (females). Increased incidences of histiocytic cellular infiltration also occurred in the small intestine (duodenum), mesenteric lymph node, and pancreatic lymph node of males and/or females exposed to 57.3 mg/L or greater. 2-YEAR STUDY IN MICE: Groups of 50 male mice were exposed to drinking water containing 0, 14.3, 28.6, 85.7, or 257.4 mg/L sodium dichromate dihydrate (equivalent to 0, 5, 10, 30, or 90 mg/L chromium) for 2 years (equivalent to average daily doses of approximately 1.1, 2.6, 7, or 17 mg sodium dichromate dihydrate/kg body weight). Groups of 50 female mice were exposed to drinking water containing 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate (equivalent to 0, 5, 20, 60, or 180 mg/L chromium) for 2 years (equivalent to average daily doses of approximately 1.1, 3.9, 9, or 25 mg/kg). Survival of exposed groups was similar to that of the control groups. Mean body weights of 257.4 mg/L males were less than those of controls from months 2 through 6 of the study, but by the end of the study, the mean body weight of 257.4 mg/L males was only slightly less than that of the control group. Mean body weights of 172 mg/L females were less than those of the controls from months 3 through 12 of the study, and mean body weights of 516 mg/L females were less than those of the controls from month 2 until the end of the study. By the end of the study, the mean body weight of 172 mg/L females was 8% less than that of the controls, and the mean body weight of 516 mg/L females was 15% less than that of the controls. The lower body weights were partly attributed to poor palatability of the dosed water and consequent reductions in water consumption. Water consumption by 85.7 and 257.4 mg/L males and 172 and 516 mg/L females was less than that by the controls throughout the study. A treatment-related microcytosis occurred in exposed mice; the mice were less affected than the rats. The incidences of neoplasms of the small intestine (duodenum, jejunum, or ileum) were increased in exposed groups of male and female mice. The incidences of adenoma of the duodenum in 257.4 mg/L males and 172 and 516 mg/L females were significantly greater than those in the controls. The incidence of carcinoma of the duodenum was significantly increased in 516 mg/L females. The incidence of adenoma of the jejunum in 516 mg/L females was significantly increased compared to that in the controls. When the incidences of adenoma and carcinoma were combined for all sites of the small intestine, the incidences were significantly increased in 85.7 and 257.4 mg/L males and 172 and 516 mg/L females compared to those in the controls. The incidences in 57.3 mg/L females exceeded the historical control ranges for drinking water studies and for all routes of administration. The incidences of diffuse epithelial hyperplasia were significantly increased in the duodenum of all exposed groups of male and female mice. The incidences of histiocytic cellular infiltration were significantly increased in the duodenum of 85.7 and 257.4 mg/L males and in 172 and 516 mg/L females. In the jejunum, the incidences of diffuse epithelial hyperplasia and histiocytic cellular infiltration were significantly increased in 516 mg/L females. The incidences of histiocytic cellular infiltration of the liver in all exposed groups of females, of the mesenteric lymph node in all exposed groups of males and females, and of the pancreatic lymph node of 85.7 and 257.4 mg/L males and 172 and 516 mg/L females were significantly increased. Tissue distribution studies showed that total chromium concentrations tended to increase with increasing exposure concentration and duration of exposure. CONCLUSIONS: Under the conditions of these 2-year drinking water studies, there was clear evidence of carcinogenic activity of sodium dichromate dihydrate in male and female F344/N rats based on increased incidences of squamous cell neoplasms of the oral cavity. There was clear evidence of carcinogenic activity of sodium dichromate dihydrate in male and female B6C3F1 mice based on increased incidences of neoplasms of the small intestine (duodenum, jejunum, or ileum). Exposure to sodium dichromate dihydrate resulted in histiocytic cellular infiltration in the liver, small intestine, and pancreatic and mesenteric lymph nodes of rats and mice and diffuse epithelial hyperplasia in the small intestine of male and female mice.