Gonadal influence on the toxicity of 2-(2'-hydroxy-3',5'-di-tert-butylphenyl) benzotriazole in rats

Previously, we showed that susceptibility of male rats to the toxicity of an ultraviolet absorber, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole (HDBB), was nearly 25 times higher than that of females. In the current study, we investigated the role of sex steroids in the mediation of the gender-related difference using castrated rats. Male and female castrated CD(SD) rats were given HDBB by gavage at 0, 0.5, 2.5, or 12.5 mg/kg/day for 28 days. No deaths, clinical signs of toxicity, or changes in body weight or food consumption were found at any doses. Blood biochemical changes suggestive of hepatic damage, such as increased levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase, were detected at 12.5 mg/kg/day in males. Absolute and relative liver weight increased at 0.5 mg/kg/day and above in males and at 12.5 mg/kg/day in females. In the liver, histopathological changes, such as nucleolar enlargement, increased mitosis, hypertrophy in hepatocytes, and/or focal necrosis were observed at 0.5 mg/kg/day and above in males, and at 2.5 mg/kg/day and above in females. These findings indicate that castration markedly reduced the gender-related differences in toxicity of HDBB in rats.     

Gonadal Influence on the Toxicity of 2-(2′-Hydroxy-3′,5′-di-tert-butylphenyl) benzotriazole in Rats

Previously, we showed that susceptibility of male rats to the toxicity of an ultraviolet absorber, 2-(2′-hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole (HDBB), was nearly 25 times higher than that of females. In the current study, we investigated the role of sex steroids in the mediation of the gender-related difference using castrated rats. Male and female castrated CD(SD) rats were given HDBB by gavage at 0, 0.5, 2.5, or 12.5 mg/kg/day for 28 days. No deaths, clinical signs of toxicity, or changes in body weight or food consumption were found at any doses. Blood biochemical changes suggestive of hepatic damage, such as increased levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase, were detected at 12.5 mg/kg/day in males. Absolute and relative liver weight increased at 0.5 mg/kg/day and above in males and at 12.5 mg/kg/day in females. In the liver, histopathological changes, such as nucleolar enlargement, increased mitosis, hypertrophy in hepatocytes, and/or focal necrosis were observed at 0.5 mg/kg/day and above in males, and at 2.5 mg/kg/day and above in females. These findings indicate that castration markedly reduced the gender-related differences in toxicity of HDBB in rats.     

Combined repeated dose and reproductive/developmental toxicity screening test of the nitrophenolic herbicide dinoseb, 2-sec-butyl-4,6-dinitrophenol, in rats

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening test, Crj:CD(SD)IGS rats were dosed with dinoseb, 2-sec-butyl-4,6-dinitrophenol, by gavage at 0 (vehicle), 0.78, 2.33, or 7.0 mg/kg bw/day. Six males per group were dosed for a total of 42 days beginning 14 days before mating. Twelve females per group were dosed for a total of 44-48 days beginning 14 days before mating to day 6 of lactation throughout the mating and gestation period. Recovery groups of six males per group and nonpregnant six females per group were dosed for 42 days followed by a 14-day recovery period. No deaths were observed in males of any dose group or in females of the recovery groups. At 7.0 mg/kg bw/day, eight females died and two animals were moribund during late pregnancy, and a significant decrease in body weight gain was found in both sexes. Hematocrit was significantly higher at 0.78 mg/kg bw/day and above in the main group males at the end of administration period. Reduction in extramedullary hematopoiesis in the spleen was significant at 2.33 mg/kg bw/day in the main group females. Sperm analysis revealed a decrease in sperm motility and an increase in the rates of abnormal sperm, abnormal tail, and abnormal head at 7.0 mg/kg bw/day. A number of dams delivered their pups and of dams with live pups at delivery was significantly lowered in the 7.0 mg/kg bw/day group. Based on these findings, the LOAEL for males and NOAEL for females were 0.78 mg/kg bw/day, and the NOAEL for reproductive/developmental toxicity was considered to be 2.33 mg/kg bw/day.     

Reproductive and developmental toxicity screening test of basic rubber accelerator, 1,3-di-o-tolylguanidine, in rats

Twelve male and female rats per group were exposed to the rubber accelerator 1,3-di-o-tolylguanidine (DTG) by gavage at 0, 8, 20 or 50 mg/kg bw/day. Males were dosed for a total of 49 days beginning 14 days before mating. Females were dosed for a total of 40-49 days beginning 14 days before mating to day 3 of lactation throughout the mating and gestation period. At 50 mg/kg bw/day, deaths were observed in two males and three females. Lowered body weight gain and food consumption were noted in males at 50 mg/kg bw/day and females at 20 and 50 mg/kg bw/day. Mydriasis, decreased locomotor activity, bradypnea, prone position, tremor and/or salivation were observed in males and females at 20 and 50 mg/kg bw/day. No effects of DTG were found on the estrous cyclicity, precoital interval, copulation, fertility and gestational indices, numbers of corpora lutea and implantations, or gestation length. A significant decrease in the number, body weight and viability of offspring and increase in the incidence of fetuses with external malformations were found at 50 mg/kg bw/day. Oligodactyly, anal atresia and tail anomalies were observed. These data suggest that DTG may be teratogenic. The NOAELs of DTG for general and developmental toxicity in rats are 8 and 20 mg/kg bw/day, respectively.     

A repeated 28-day oral dose toxicity study of genistein in rats,based on the 'Enhanced OECD Test Guideline 407' for screening endocrine-disrupting chemicals

In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of genistein, which is known as a phytoestrogen. Attention was paid to the sensitivity of certain additional parameters, such as histopathology observations and organ weights of endocrine related organs, sperm characteristics, serum hormone levels and estrous cycle, for detecting endocrine-related effects of endocrine-disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were assigned to one of four groups, each consisting of ten males and ten females, and genistein was administered once daily by gavage at doses of 0 (control), 120, 400 or 1000 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during the 4 days after the 28th administration. Endocrine-disrupting effects of genistein were detected in females by histopathology. The changes included vacuolation and mucinification of the vaginal epithelium in the 400 and 1000 mg/kg groups; however, the incidences of the lesion were very low. Although increased serum prolactin levels were recorded in the males of the 1000 mg/kg group, we could not determine whether this was indeed induced by genistein. General toxicological effects of genistein were detected in blood chemistry, such as increased triglycerides and total protein and a decreased albumin/globulin ratio, as well as increased liver weight and glycogen deposition in the periportal hepatocytes. Based on these results, the no-observed-adverse-effect level (NOAEL) in the present study was estimated to be 120 mg/kg per day. In particular, endocrine-related effects were most sensitively detected by histopathology examination of sexual organs. However, the findings indicate that chemicals with weak endocrine-disrupting potential like genistein must be evaluated taking into consideration the results of other test systems.     

Reproductive and developmental toxicity screening study of 2,4-dinitrophenol in rats

Rats were treated by gavage once daily with 2,4-dinitrophenol (DNP) at 0 (control), 3, 10, or 30 mg/kg bw. Males were dosed for 46 days, beginning 14 days before mating, and females were dosed for 40-47 days, from 14 days before mating to day 3 of lactation. No deaths were observed in males and females of any group. A significant decrease in body weight gain and significant increase in liver weight were found in males and females at 30 mg/kg bw/day. The number of live pups on postnatal days (PNDs) 0 and 4, live birth index, and body weight of live male and female pups on PNDs 0 and 1 were significantly lowered at 30 mg/kg bw/day. External and internal examinations of pups revealed no increased incidence of malformations in DNP-treated groups. On the basis of these findings, we concluded that DNP has general and reproductive/developmental toxicity, but not teratogenicity, under the present conditions. The NOAEL of DNP is considered to be 10 mg/kg bw/day in rats.     

Short term toxicity study in rats dosed with isoeugenol benzyl ether

Isoeugenol benzyl ether was given to rats by gavage for 28 days at 0, 60, 120, and 240 mg/kg body weight/day. For both sexes at the highest dose and females at the intermediate dose statistically significantly decreased values were found for body weight, blood glucose (also for males at intermediate dose), blood urea and relative liver weights. No dose-related histopathological changes were seen in any organs. The no effect level was 60 mg/kg body weight/day.     

Combined repeated dose and reproductive/developmental toxicities of copper monochloride in rats

This study investigated the combined repeated dose and reproductive/developmental toxicity of copper monochloride in rats. The test substance was administered once daily by gavage at 0, 1.3, 5, 20, or 80 mg/kg/day. Male rats were dosed for a total of 30 days beginning 14 days before mating. Female rats were dosed from 2 weeks before mating to day 3 of lactation throughout the mating and gestation period. At 80 mg/kg/day, deaths were observed in 3 out of 12 females. There was a dose-dependent increase in the incidence of clinical signs and a reduction in the food consumption. Hematological and serum biochemical investigations revealed a decrease in the red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), and serum total protein levels and an increase in the white blood cell and platelets in males, and a decrease in the MCH and an increase in the platelets in females. Histopathological examination showed an increased incidence of squamous cell hyperplasia of the stomach in both genders as well as increased hematopoiesis of the femur in males. There was an increase in the number of icteric and runt pups at birth. At 20 mg/kg/day, there was an increase in the incidence of clinical signs and squamous cell hyperplasia of the stomach in both genders. At 5 mg/kg/day, an increase in the incidence of squamous cell hyperplasia of the stomach was observed in females. There were no adverse effects in the lowest group in both genders. Based on these findings, the no-observed-adverse-effect levels of copper monochloride were concluded to be 5 mg/kg/day in male rats and 1.3 mg/kg/day in female rats for general toxicity and 20 mg/kg/day for reproductive/developmental toxicity.     

A 52-Week Repeated Dose Toxicity Study of Ultraviolet Absorber 2-(2′-Hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole in Rats

A 52-week repeated dose toxicity study of an ultraviolet absorber, 2-(2′-hydroxy-3′,5′ -di-tert-butylphenyl)benzotriazole (HDBB), was conducted according to OECD TG 452 under GLP. CD(SD)IGS rats were given HDBB by gavage at 0, 0.1, 0.5, or 2.5 mg/kg/day in males and 0, 0.5, 2.5, or 12.5 mg/kg/day in females. No substance-related deaths or clinical signs of toxicity were observed in any group; however, a lowered body weight was found from day 36 to the end of the 52-week administration period at 2.5 mg/kg in males. At the completion of the dosing period, a decrease in red blood cells at 0.5 mg/kg and higher, and in hematocrit at 2.5 mg/kg, was detected in males. Blood biochemical changes, including increases in the levels of alkaline phosphatase and glucose and the A/G ratio, were also found at 0.5 mg/kg and higher in males and at 12.5 mg/kg in females. At necropsy, absolute and relative liver weight was increased at 0.5 mg/kg and higher in males and at 12.5 mg/kg in females. Histopathological changes were observed in the liver; centrilobular hypertrophy of hepatocytes at 0.5 mg/kg and higher in males, and at 12.5 mg/kg in females, and altered hepatocellular foci at 0.5 mg/kg and higher, and cystic degeneration and lipofuscin deposition in hepatocytes at 2.5 mg/kg in males. Based on these findings, the no observed adverse effect level was concluded to be 0.1 mg/kg/day in male rats and 2.5 mg/kg/day in female rats.     

A 52-week repeated dose toxicity study of ultraviolet absorber 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole in rats

A 52-week repeated dose toxicity study of an ultraviolet absorber, 2-(2'-hydroxy-3',5' -di-tert-butylphenyl)benzotriazole (HDBB), was conducted according to OECD TG 452 under GLP. CD(SD)IGS rats were given HDBB by gavage at 0, 0.1, 0.5, or 2.5 mg/kg/day in males and 0, 0.5, 2.5, or 12.5 mg/kg/day in females. No substance-related deaths or clinical signs of toxicity were observed in any group; however, a lowered body weight was found from day 36 to the end of the 52-week administration period at 2.5 mg/kg in males. At the completion of the dosing period, a decrease in red blood cells at 0.5 mg/kg and higher, and in hematocrit at 2.5 mg/kg, was detected in males. Blood biochemical changes, including increases in the levels of alkaline phosphatase and glucose and the A/G ratio, were also found at 0.5 mg/kg and higher in males and at 12.5 mg/kg in females. At necropsy, absolute and relative liver weight was increased at 0.5 mg/kg and higher in males and at 12.5 mg/kg in females. Histopathological changes were observed in the liver; centrilobular hypertrophy of hepatocytes at 0.5 mg/kg and higher in males, and at 12.5 mg/kg in females, and altered hepatocellular foci at 0.5 mg/kg and higher, and cystic degeneration and lipofuscin deposition in hepatocytes at 2.5 mg/kg in males. Based on these findings, the no observed adverse effect level was concluded to be 0.1 mg/kg/day in male rats and 2.5 mg/kg/day in female rats.     

Comparative toxicity of ethylene dichloride in F344/N, Sprague-Dawley and Osborne-Mendel rats

Studies were conducted to compare the toxicity of ethylene dichloride (EDC) in F344/N rats, Sprague-Dawley rats, and Osborne-Mendel rats. Ten rats/sex/group were exposed to EDC in drinking-water at 0, 500, 1000, 2000, 4000 and 8000 ppm for 13 wk. The highest concentration was limited by the maximum solubility of EDC in water (about 9000 ppm). In addition, F344/N rats (10/sex/group) were administered EDC in corn oil by gavage to compare toxicity resulting from bolus administration with that of continuous exposure in drinking-water. Gavage doses of EDC were within the range of total daily doses (in mg/kg body weight/day) resulting from exposure in drinking-water. EDC administered by gavage resulted in greater toxicity to F344/N rats than did administration of similar doses in drinking-water. All males receiving 240 and 480 mg/kg body weight and 9/10 females receiving 300 mg/kg body weight by gavage died before the end of the study. Necrosis of the cerebellum was observed in the brains of 3 males receiving 240 mg/kg body weight and 3 females receiving 300 mg/kg body weight. Hyperplasia and inflammation of the forestomach mucosa were observed in 8 male and 3 female rats that died or were killed in moribund condition. EDC caused minimal toxicity to F344/N, Sprague-Dawley and Osborne-Mendel rats at the drinking-water concentrations used in these studies; only female F344/N rats had EDC-related renal lesions. Based on mortality and EDC-related lesions, the no-effect levels for EDC administered by gavage to F344/N rats were 120 mg/kg body weight for males and 150 mg/kg body weight for females.     

Reproductive and developmental toxicity studies of S-1090, cefmatilen hydrochloride hydrate (1)--A study on oral administration prior to and in the early stages of pregnancy in rats

Cefmatilen hydrochloride hydrate (S-1090) was administered daily by gavage to rats at doses of 100, 300 or 1000 mg potency/kg/day prior to and in the early stage of pregnancy to assess its adverse effects on parental reproductive ability and embryo-fetal development. Loose and/or reddish brown feces were observed in both males and females of all the S-1090 dosing groups, and abdominal distention was also observed in males throughout the dosing period. No drug-related deaths occurred in either males or females. In males, body weight and food consumption were increased at a dose of 1000 mg potency/kg/day throughout the dosing period. In females, body weight gain was restrained during late pregnancy, and food consumption was decreased transiently following the initiation of dosing, and then remained high on the day before parturition in all the S-1090 dosing groups. Necropsy of male and female rats revealed an increase in the cecum weight. The reproductive ability of males and females was normal in all the S-1090 dosing groups. No effects of S-1090 were observed in the implantation ratio, embryo-fetal viability, fetal body weight, and incidence of external, skeletal and visceral anomalies. Based on these results, the no observed adverse effect levels of S-1090 are estimated to be less than 100 mg potency/kg/day for parental general toxicity, 1000 mg potency/kg/day for reproductive toxicity, and 1000 mg potency/kg/day for developmental toxicity in embryo-fetuses under the conditions of the present study.     

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 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)     

A repeated 28-day oral dose toxicity study of nonylphenol in rats,based on the ‘Enhanced OECD Test Guideline 407’ for screening of endocrine-disrupting chemicals

A 28-day repeated oral dose toxicity study of nonylphenol (NP) was performed for an international validation of the ‘Enhanced OECD Test Guideline 407’ paying particular attention to the sensitivity of individual endocrine-related parameters. Sprague-Dawley rats, each group consisting of ten males and ten females, were administered NP once daily by gavage at doses of 0 (control), 10, 50, or 250 mg/kg body weight. At 250 mg/kg, three females died or became moribund during the experiment. At this dose, hepatic and renal toxicity was evident in both sexes with increase of relative liver and kidney weights as well as histopathological changes, such as centrilobular liver cell hypertrophy and a variety of renal tubular lesions, and alteration of serum biochemical parameters, some of them being evident from 50 mg/kg in females (glucose and inorganic phosphates). Hematologically, development of anemia was evident at 250 mg/kg in both sexes. Regarding endocrine-related effects, increase of thyroid weight in males was detected from 50 mg/kg. At 250 mg/kg, males exhibited reduction of relative weights of the ventral prostate and seminal vesicles, and females developed irregular estrous cyclicity and vaginal mucosal hyperplasia. Although changes in serum hormone levels were detected in both sexes, magnitude of the changes was small to be regarded as a low toxicological significance. In summary, repeated oral doses of NP to rats for 28 days resulted in hepato-renal toxicity from 50 mg/kg and anemia at 250 mg/kg. Effects on the endocrine system were observed from 50 mg/kg, and assessment of weights and histopathology of endocrine-related organs and estrous cyclicity may be valid in a battery for detecting endocrine effects of NP. The no-observed-adverse-effect level of NP was estimated to be 10 mg/kg per day.     

Lack of gender-related difference in the toxicity of 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole in preweaning rats

In our previous toxicity studies using young rats, we showed that an ultraviolet absorber, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole (HDBB), principally affected the liver, and male rats had nearly 25 times higher susceptibility to the toxic effects than females. In the present study, the toxicity of HDBB was investigated in preweaning rats. HDBB was administered by gavage to male and female CD(SD) rats from postnatal days 4 to 21 at a dose of 0, 0.1, 0.5, 2.5, or 12.5 mg/kg/day. No substance-related deaths, clinical signs of toxicity, or body-weight changes were observed. Increased levels of albumin, AST and ALP in both sexes, BUN in males, and LDH in females were found at 12.5 mg/kg. Liver weights increased at 2.5 mg/kg and above in both sexes. Histopathologically, hepatocellular findings, such as nucleolar enlargement, anisokaryosis, increased mitosis, and/or hypertrophy, were observed at 2.5 mg/kg and above in both sexes. These results indicate no gender-related differences in the susceptibility to the toxic effects of HDBB in preweaning rats.     

Lack of Gender-Related Difference in the Toxicity of 2-(2′-Hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole in Preweaning Rats

In our previous toxicity studies using young rats, we showed that an ultraviolet absorber, 2-(2′-hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole (HDBB), principally affected the liver, and male rats had nearly 25 times higher susceptibility to the toxic effects than females. In the present study, the toxicity of HDBB was investigated in preweaning rats. HDBB was administered by gavage to male and female CD(SD) rats from postnatal days 4 to 21 at a dose of 0, 0.1, 0.5, 2.5, or 12.5 mg/kg/day. No substance-related deaths, clinical signs of toxicity, or body-weight changes were observed. Increased levels of albumin, AST and ALP in both sexes, BUN in males, and LDH in females were found at 12.5 mg/kg. Liver weights increased at 2.5 mg/kg and above in both sexes. Histopathologically, hepatocellular findings, such as nucleolar enlargement, anisokaryosis, increased mitosis, and/or hypertrophy, were observed at 2.5 mg/kg and above in both sexes. These results indicate no gender-related differences in the susceptibility to the toxic effects of HDBB in preweaning rats.     

Reproductive and developmental toxicity screening study of 4-aminophenol in rats

Twelve male and female rats per group were given 4-aminophenol (PAP) by gavage at 0, 20, 100, or 500 mg/kg/day. Males were dosed for a total of 49 days, beginning 14 days before mating. Females were dosed for a total of 40-60 days, from 14 days before mating to Day 3 of lactation throughout the mating and gestation periods. Four males and 2 females died at 500 mg/kg/day, and all surviving males and females showed brown urine at 100 mg/kg/day and above. Body-weight gain was lower in males and females at 500 mg/kg/day, and food consumption was decreased in males at 500 mg/kg/day and in females at 100 and 500 mg/kg/day. Absolute and relative weights of the testes and epididymides were decreased at 500 mg/kg/day. Histopathological examinations revealed decreased spermatocyte and spermatid levels in the testis, debris of germ cell in the epididymis lumen, basophilic tubules in the kidney, and deposits of hemosiderin in the red pulp and extramedullary hematopoiesis in the spleen in males at 500 mg/kg/day. Longer gestation period, decreased delivery index, and lower body weight of pups on postnatal day (PND) 0 and increased number of stillborns at 500 mg/kg/day were also observed. At this dose, the viability of pups on PND 4 was decreased markedly. No adverse effects on reproduction or development were detected at 20 and 100 mg/kg/day. These findings indicate that PAP is general and reproductive/developmental toxic, but is unlikely to be teratogenic, in rats.     

Reproductive and Developmental Toxicity Screening Study of 4-Aminophenol in Rats

Twelve male and female rats per group were given 4-aminophenol (PAP) by gavage at 0, 20, 100, or 500 mg/kg/day. Males were dosed for a total of 49 days, beginning 14 days before mating. Females were dosed for a total of 40–60 days, from 14 days before mating to Day 3 of lactation throughout the mating and gestation periods. Four males and 2 females died at 500 mg/kg/day, and all surviving males and females showed brown urine at 100 mg/kg/day and above. Body-weight gain was lower in males and females at 500 mg/kg/day, and food consumption was decreased in males at 500 mg/kg/day and in females at 100 and 500 mg/kg/day. Absolute and relative weights of the testes and epididymides were decreased at 500 mg/kg/day. Histopathological examinations revealed decreased spermatocyte and spermatid levels in the testis, debris of germ cell in the epididymis lumen, basophilic tubules in the kidney, and deposits of hemosiderin in the red pulp and extramedullary hematopoiesis in the spleen in males at 500 mg/kg/day. Longer gestation period, decreased delivery index, and lower body weight of pups on postnatal day (PND) 0 and increased number of stillborns at 500 mg/kg/day were also observed. At this dose, the viability of pups on PND 4 was decreased markedly. No adverse effects on reproduction or development were detected at 20 and 100 mg/kg/day. These findings indicate that PAP is general and reproductive/developmental toxic, but is unlikely to be teratogenic, in rats.     

A 28-day oral gavage toxicity study of 3-monochloropropane-1,2-diol (3-MCPD) in CB6F1-non-Tg rasH2 mice

3-Monochloro-1,2-propanediol (3-MCPD) is a well-known contaminant of foods containing hydrolyzed vegetable protein. However, limited toxicity data are available for the risk assessment of 3-MCPD and its carcinogenic potential is controversial. To evaluate the potential toxicity and determine the dose levels for a 26-week carcinogenicity test using Tg rasH2 mice, 3-MCPD was administered once daily by oral gavage at doses of 0, 25, 50, and 100 mg/kg body weight (b.w.)/day for 28 days to male and female CB6F1-non-Tg rasH2 mice (N = 5 males and females per dose). The standard toxicological evaluations were conducted during the in-life and post-mortem phase. In the 100 mg/kg b.w./day group, 3 males and 1 female died during the study and showed clinical signs such as thin appearance and subdued behavior accompanied by significant decreases in mean b.w. Microscopy revealed tubular basophilia in the kidneys, exfoliated degenerative germ cells in the lumen of the seminiferous tubule of the testes, vacuolation in the brain, axonal degeneration of the sciatic nerve, and cardiomyopathy in the 100, ≥25, ≥50, 100, and 100 mg/kg b.w./day groups, respectively. In conclusion, 3-MCPD's target organs were the kidneys, testes, brain, sciatic nerve, and heart. The “no-observed-adverse-effect level” (NOAEL) of 3-MCPD was ≤25 and 25 mg/kg b.w./day in males and females, respectively.