Nitrapyrin: a scientific advisory group review of the mode of action and carcinogenicity in B6C3F1 mice.

Nitrapyrin has been registered as a nitrogen stabilizer in the United States for many years based on a robust set of regulatory data. These data demonstrated that nitrapyrin was not genotoxic and that there were no tumors elicited in rats or mice that were relevant for human risk assessment. A repeat carcinogenicity study in B6C3F1 mice, conducted at two substantially higher-dose levels (0, 125 or 250 mg/kg/day) than the original study (0, 5, 25 or 75 mg/kg/day) identified liver, stomach, epididymal and Harderian gland tumors. In order to assess the relevance of these findings for human risk assessment, a Scientific Advisory Group (SAG) examined relevant microscopic changes in these tissues and also evaluated genotoxicity and mechanistic data. The SAG determined that the maximum tolerated dose had been exceeded in mice given 125 or 250 mg/kg/day, based on 26-33% decreased body weight gains (males-250 mg/kg/day), hepatocellular necrosis and compensatory hepatocellular proliferation (males and females-125 and 250 mg/kg/day). The SAG believed that the increased incidences of hepatocellular foci of alteration and hepatocellular neoplasms represented an epigenetic response to hepatocellular necrosis and increased mitogenesis. Increased incidences of proliferative lesions in the forestomach mucosa were likely secondary to the irritant effects of nitrapyrin. Neither the liver nor forestomach effects were interpreted to be a direct carcinogenic effect. Higher incidences of Harderian gland adenomas (females) and undifferentiated sarcomas in the epididymis represented normal biological variations in incidence and were unrelated to nitrapyrin. Therefore, it was the SAG's opinion that nitrapyrin exposure that does not produce target organ toxicity in exposed individuals would not be expected to increase the risk of cancer.

Two-generation reproductive toxicity study of inhaled tertiary amyl methyl ether (TAME) vapor in CD rats.

Under Office of Prevention, Pesticides and Toxic Substances draft guidelines, CD weanling F0 rats (30 of each gender per group) inhaled tertiary amyl methyl ether vapor at 0, 250, 1500 or 3000 ppm 5 days a week and 6 h a day for 10 weeks, with vaginal cytology evaluated for weeks 8-10. The F0 animals then produced F1 offspring, with exposure 7 days a week from mating through to lactation. During the F1 prebreed exposure period, vaginal patency, preputial separation (PPS) and vaginal cytology were evaluated. The F1 animals were mated, with F2 anogenital distance measured on postnatal day zero. At F2 weaning 30 of each gender per group were selected for postwean retention, with no exposures, through vaginal patency and PPS. Body weights, feed consumption and clinical signs were recorded throughout the study. Adult F0 and F1 systemic toxicity was present at 1500 and 3000 ppm. Minor adult male reproductive toxicity was present at 3000 ppm. There were no adult effects on vaginal cyclicity, estrous cycle length, mating, fertility, pregnancy, gestational length or ovarian and uterine weights. There were no treatment-related gross or histopathologic findings in parental male or female systemic or reproductive organs. The F1 and F2 offspring toxicity was present at 1500 and 3000 ppm. The no-observable-adverse-effect level for adult systemic and offspring toxicity was 250 ppm and 1500 ppm for male reproductive toxicity (females at >3000 ppm).

Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats.

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.

Beneficial effects of NTP-2000 diet on growth, survival, and kidney and heart diseases of Fischer 344 rats in chronic studies.

Diet is one of the most important environmental factors influencing growth, survival, and appearance of age-associated diseases in rodents. NIH-07 open formula rodent diet was the selected diet for the National Toxicology Program studies from 1980 to 1994. After a number of experimental diets were evaluated, a new one designated as NTP-2000 was selected for rodents in NTP studies beginning in 1994. This report summarizes the results of dosed feed and inhalation studies for differences in growth, survival, and severity of kidney and heart lesions in Fischer 344 rats fed NTP-2000 or NIH-07 diets. In the dosed feed studies, male rats group housed and fed the NTP-2000 diet grew slightly slower, attained maximum body weight later, and lost less body weight by the end of the 2-year studies compared to the groups fed NIH-07. Female rats group housed and fed the NTP-2000 diet in dosed feed studies had significantly slower growth, with lower maximum body weight compared to the groups fed the NIH-07 diet. In the inhalation studies, male rats individually housed and fed the NTP-2000 diet had slightly higher maximum body weight and significantly higher final body weight, with lower loss of weight when compared to similarly housed groups fed the NIH-07 diet. In inhalation studies, female rats fed the NTP-2000 diet and individually housed had significantly slower growth. The NTP-2000 diet significantly increased the survival of male and female rats, with a dramatic increase in survival of males in inhalation studies. This diet also caused significant decreases in severity of nephropathy and cardiomyopathy, and the decrease was marked in males. These observations indicate that diets balanced for nutrients, such as the NTP-2000, could markedly improve the health and increase survival of the rats used in chronic studies.

Inhalation toxicity studies of the alpha,beta-unsaturated ketones: ethyl vinyl ketone.

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of alpha,beta-unsaturated ketones. Ethyl vinyl ketone (EVK) was selected for study because it is a representative straight-chain aliphatic alpha,beta-unsaturated ketone with extensive use and widespread exposure. Short-term inhalation studies of EVK were conducted to provide toxicity data for comparison with the related alpha,beta-unsaturated ketones 2-cyclohexene-1-one (CHX) and methyl vinyl ketone (MVK). These data will be used in designing chronic toxicity and carcinogenicity studies of these ketones. Male and female F344 rats and B6C3F1 mice were exposed to 0, 2, 4, or 8 ppm EVK 6 h/day, 5 days/wk for 13 wk. The nasal cavity was the major target organ of EVK in both rats and mice. Pathologic findings in both the olfactory and respiratory epithelium were observed. Lesions consisted primarily of olfactory epithelial necrosis, atrophy and regeneration, and/or hyperplasia and squamous metaplasia of the respiratory epithelium. Squamous metaplasia of the respiratory epithelium was present in all rats and mice exposed to 4 and 8 ppm EVK, and these lesions were more severe in rats than in mice. Few systemic effects were observed in rats and mice exposed to EVK. A transient decrease in total leukocytes due to decrements in lymphocyte and monocyte populations was present in male rats after exposure to 8 ppm for 3 and 21 days; however, this effect was not present after exposure for 13 wk. There were no chemical-related effects on micronucleus formation in mice, or on sperm motility and vaginal cytology in either species. EVK, like other alpha,beta-unsaturated ketones, is a reactive, direct-acting gaseous irritant with toxicity limited primarily to the upper respiratory tract.

Mammary tumor induction and premature ovarian failure in ApcMin mice are not enhanced by Brca2 deficiency.

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.

Upper respiratory tract toxicity of inhaled methylvinyl ketone in F344 rats and B6C3F1 mice.

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of alpha,beta-unsaturated ketones. Methylvinyl ketone (MVK) was selected for study because it is a representative straight-chain aliphatic alpha,beta-unsaturated ketone and because of its extensive use and widespread exposure. Short-term inhalation studies of MVK were conducted to provide toxicity data for comparison with other alpha,beta-unsaturated ketones and for use in designing chronic toxicity and carcinogenicity studies. In 2-week studies, rats and mice were exposed to 0, 0.25, 0.5, 1, 2, 4, or 8 ppm MVK 6 h/day, 5 days/week for 12 exposures. Morbidity and early deaths occurred in all male and female rats after 1 exposure and in 2 male mice after 10 exposures to 8 ppm. Rats exhibited nasal cavity toxicity and lung necrosis at 4 ppm. No toxicity was observed in animals exposed to less than 2 ppm. Based on these results a 13-week study was conducted at 0, 0.5, 1, and 2 ppm MVK. As observed in the 2-week study, the nasal cavity was the main target organ and rats were more sensitive than mice. Respiratory and olfactory epithelial necrosis were prominent by day 21 in the rat. At study termination these lesions were still evident but not as severe as noted earlier. Additionally, changes such as olfactory epithelial regeneration and metaplasia (respiratory) as well as respiratory epithelial hyperplasia and metaplasia (squamous) were clearly evident. Nasal lesions in mice were limited to a subtle squamous metaplasia of transitional and/or respiratory epithelium covering predominantly the tips of naso- and maxilloturbinates in Levels I and II. A transient, leukopenia was observed in rats exposed to 2 ppm, however, this effect was not present after 13 weeks of exposure. In mice, leukocyte counts were significantly decreased at all exposure concentrations after 13 weeks of exposure. Absolute testicular and epididymal weights and sperm counts were decreased at the high dose only. MVK can be characterized as a reactive, direct-acting gaseous irritant. MVK exposure causes the same nasal cavity lesions as the cyclic alpha,beta-unsaturated ketone, 2-cyclohexen-1-one, although at lower exposure concentrations.

Mice heterozygous for a Brca1 or Brca2 mutation display distinct mammary gland and ovarian phenotypes in response to diethylstilbestrol.

Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to the development of breast and ovarian cancer. We used mice with a Brca1 mutation on a BALB/cJ inbred background (BALB/cB1+/- mice) or a Brca2 genetic alteration on the 129/SvEv genetic background (129B2+/- mice) to investigate potential gene-environment interactions between defects in these genes and treatment with the highly estrogenic compound diethylstilbestrol (DES). Beginning at 3 weeks of age, BALB/cB1+/-, 129B2+/-, and wild-type female mice were fed a control diet or a diet containing 640 ppb DES for 26 weeks. DES treatment caused vaginal epithelial hyperplasia and hyperkeratosis, uterine inflammation, adenomyosis, and fibrosis, as well as oviductal smooth muscle hypertrophy. The severity of the DES response was mouse strain specific. The estrogen-responsive 129/SvEv strain exhibited an extreme response in the reproductive tract, whereas the effect in BALB/cJ and C3H/HeN(MMTV-) mice was less severe. The Brca1 and Brca2 genetic alterations influenced the phenotypic response of BALB/cJ and 129/SvEv inbred strains, respectively, to DES in the mammary gland and ovary. The mammary duct branching morphology was inhibited in DES-treated BALB/cB1+/- mice compared with similarly treated BALB/cB1+/+ littermates. In addition, the majority of BALB/cB1+/- mice had atrophied ovaries, whereas wild-type littermates were largely diagnosed with arrested follicular development. The mammary ductal architecture in untreated 129B2+/- mice revealed a subtle inhibited branching phenotype that was enhanced with DES treatment. However, no significant differences were observed in ovarian pathology between 129B2+/+ and 129B2+/- mice. These data suggest that estrogenic compounds may modulate mammary gland or ovarian morphology in BALB/cB1+/- and 129B2+/- mice. These observations are consistent with the hypothesis that compromised DNA repair processes in cells harboring Brca1 or Brca2 mutations lead to inhibited growth and differentiation compared with the proliferative response of wild-type cells to DES treatment.

Two-generation reproduction study with para-tert-octylphenol in rats.

Octylphenol (OP) is a commercial intermediate used primarily for the production of octylphenol polyethoxylate surfactants. To determine potential reproductive toxicity of OP, a two-generation reproduction study was conducted according to U.S. EPA OPPTS Guideline 870.3800 (draft 1996). Additional measurements were made on retained F2 offspring. OP was administered ad libitum to five groups of rats (30/sex) at dietary concentrations of 0, 0.2, 20, 200, or 2000 ppm. The 0.2 ppm concentration was included to evaluate potential low dose effects. Effects were observed only at 2000 ppm, including decreased body weights in adults and during the latter portion of lactation in offspring and minor body weight-related delays in acquisition of vaginal opening and preputial separation. No effects on reproductive parameters, testes, prostate, or ovary weights or morphology, on sperm counts, motility, morphology, production, or on estrous cyclicity were observed. No estrogen-like effects were evident. The NOAELs for systemic and postnatal toxicity were 200 ppm and at or above 2000 ppm for reproductive toxicity. This study supports the increasing evidence that screening assays for estrogenic activity or studies with limited numbers of animals and/or unrealistic dose regimens are inappropriate for use in the assessment of human health and environmental risk. It does not support previous preliminary data on low dose effects of OP.

Two-generation reproductive toxicity study of dietary tributyl phosphate in CD rats.

Tributyl phosphate (TBP) was tested for reproductive toxicity in rats. Thirty weanlings/sex (F0) were exposed to TBP in the diet ad libitum at 0, 200, 700, or 3000 ppm for 10 weeks and then randomly mated within groups for 3 weeks with continued exposure. F0 parents and 10 F1 weanlings/sex/dose were necropsied, and adult reproductive organs, urinary bladders (both sexes), kidneys (males), and livers (females) were evaluated histologically. Thirty F1 weanlings/sex/dose continued exposure for 11 weeks and were bred as described above. F1 parents and F2 weanlings, 10/sex/dose, were then necropsied as described above. Adult toxicity was observed in both sexes and generations at 700 and 3000 ppm; observations included reduced body weights, weight gain and feed consumption, urinary bladder epithelial hyperplasia (both sexes), renal pelvis epithelial hyperplasia only at 3000 ppm (male kidneys), and centrilobular hypertrophy (female livers). At 200 ppm, transient reductions in body weight were observed in F0 and F1 females, with urinary bladder epithelial hyperplasia in F0 males and females and in F1 males. There was no evidence of reproductive toxicity, of reproductive organ pathology, or of effects on gestation or lactation at any dose tested. Postnatal toxicity was evidenced by consistent reductions in F1 and F2 pup body weights at 3000 ppm and by occasional weight reductions in F2 litters at 700 ppm, and was associated with maternal toxicity observed at these doses and times. Under the conditions of this study, a NOAEL was not determined for adult toxicity; the NOAEL for reproductive toxicity was at least 3000 ppm and the NOAEL for postnatal toxicity was approximately 200 ppm.

Alterations in the reproductive patterns of female mice exposed to xenobiotics.

Chemicals, by virtue of their varied interactions with biological molecules, are expected to differ in the way they may alter female reproduction. Reproductive toxicity may reflect effects either on the female germ cells or on various maternal processes such as ovulation, implantation, pregnancy, and parturition. In either case, the ultimate manifestation of chemical toxicity on female reproduction is a decrease in the number of normal young born. Very little information is available on the effects of chemicals that are nonhormonal in nature on the long-term ability of treated females to produce offspring. This report presents the results of long-term female total reproductive capacity (TRC) tests on 29 chemicals, including pharmaceuticals, pesticides, and alkylating and industrial agents. For each chemical, the minimum test involved an evaluation of the maximum tolerated dose administered as a single intraperitoneal injection. Females were single-pair mated with an untreated male for most of the female's reproductive life span (a minimum of 347 days posttreatment) and scored for the number of live births produced during this period. Confirmatory dominant lethal experiments or histological examinations for numbers of small follicles were carried out when mutagenic effects or cytotoxicity, respectively, were suspected as the basis for reduced fertility. Of the 29 chemicals studied, 17 had reproductive effects which may be grouped into one of three classes: (1) those that reduced the total number of young and litters per female, (2) those that reduced the total number of young but not of litters, and (3) those that had no significant effect on the total number of young produced but reduced the size of the first and/or second litters. The TRC provides a capacity for detecting a range of toxic insults upon female reproduction. Many of the chemicals were indeed shown to affect the reproductive performance of females through mutagenic and/or cytotoxic effects on follicles. In some cases, however, no causative mechanism could be identified for the observed reduction in reproductive performance. Nevertheless, with this report the number of chemicals tested by this TRC procedure has been quadrupled and the categories of chemicals tested have been substantially broadened.

Phenolphthalein induces thymic lymphomas accompanied by loss of the p53 wild type allele in heterozygous p53-deficient (+/-) mice.

Epidemiology studies have indicated that many human cancers are influenced by environmental factors. Genetically altered mouse model systems offer us the opportunity to study the interaction of chemicals with genetic predisposition to cancer. Using the heterozygous p53-deficient (+/-) mouse, an animal model carrying one wild type p53 gene and one p53 null allele, we studied the effects of phenolphthalein on tumor induction and p53 gene alterations. Earlier studies showed that phenolphthalein caused carcinogenic effects in Fisher 344 rats and B6C3F1 mice after a 2-yr dosing period (Dunnick and Hailey, Cancer Res. 56: 4922-4926, 1996). The p53 (+/-) mice received phenolphthalein in the feed at concentrations of 200, 375, 750, 3,000, or 12,000 ppm (approximately 43, 84, 174, 689, or 2,375 mg/kg body weight/day or 129, 252, 522, 2,867, or 7,128 mg/m2 body surface area/day) for up to 6 mo. A target organ cancer site that accumulated p53 protein in the B6C3F1 mouse (i.e., thymic lymphoma) was also a target site for cancer in the p53 (+/-) mouse. In the p53 (+/-) mouse, treatment-related atypical hyperplasia and malignant lymphoma of thymic origin were seen in the control and dosed groups at a combined incidence of 0, 5, 5, 25, 100, and 95%, respectively. Twenty-one of the thymic lymphomas were examined for p53 gene changes, and all showed loss of the p53 wild type allele. Chemical-induced ovarian tumors in the B6C3F1 mouse showed no evidence for p53 protein accumulation and did not occur in the p53 (+/-) mouse. The p53-deficient (+/-) mouse model responded to phenolphthalein treatment with a carcinogenic response in the thymus after only 4 mo of dosing. This carcinogenic response took 2 yr to develop in the conventional B6C3F1 mouse bioassay. The p53-deficient (+/-) mouse is an important model for identifying a carcinogenic response after short-term (< 6 mo) exposures. Our studies show that exposure to phenolphthalein combined with a genetic predisposition to cancer can potentiate the carcinogenic process and cause p53 gene alterations, a gene alteration found in many human cancers.

The developmental toxicity of boric acid in rabbits.

Boric acid (BA), an ingredient of many pharmaceutical, cosmetic, and pesticide products, was previously shown to induce reproductive and developmental toxicity in laboratory rodents. In this study, BA (0, 62.5, 125, or 250 mg/kg/day, po) was administered on Gestational Days (GD) 6-19 to New Zealand White rabbits (18-23 pregnant/group). Maternal body weight, food consumption, and clinical condition were monitored at regular intervals throughout gestation. At termination (GD 30), the numbers of uterine implantations, resorptions, dead fetuses, and live fetuses were determined. Fetuses were weighed, and live fetuses examined for external, visceral, and skeletal defects. Maternal food intake decreased during treatment at 250 mg/kg/day and increased at >/=125 mg/kg/day after treatment. Maternal body weight (GD 9-30), weight gain during treatment, gravid uterine weight, and number of ovarian corpora lutea decreased at 250 mg/kg/day. In contrast, maternal corrected gestational weight gain increased at >/=125 mg/kg/day. Maternal liver weight was not affected. Relative (but not absolute) maternal kidney weight increased at 250 mg/kg/day, and microscopic evaluation revealed no treatment-related renal pathology. At 250 mg/kg/day, prenatal mortality was increased (90% resorptions/litter vs 6% for controls), the proportion of pregnant females with no live fetuses was increased (73% vs 0%), and live litter size was reduced (2.3 fetuses/litter vs 8.8). As a result, there were only 14 live fetuses (6 live litters) available for evaluation in the high-dose group, compared to 153-175 live fetuses (18-23 live litters) in the other groups. The percentage malformed fetuses/litter was increased at 250 mg/kg/day, primarily due to cardiovascular defects in 72% of high-dose fetuses vs 3% of controls. The most prevalent cardiovascular malformation (interventricular septal defect) was observed in 57% of high-dose fetuses compared to 0.6% among controls. At 250 mg/kg/day, average fetal body weight/litter was 92% of the average control weight (not statistically significant). In summary, no definitive maternal or developmental toxicity was observed at 62.5 or 125 mg/kg/day BA. Mild maternal effects and severe developmental toxicity were observed at 250 mg/kg/day.

Reproductive toxicity evaluation of methylethyl ketoxime by gavage in CD rats.

Methylethyl ketoxime (CAS No. 96-29-7; MEKO; 2-butanone oxime), an antioxidant agent used in paints, resins, and adhesives, was tested for reproductive toxicity in a two-generation study with CD (Sprague-Dawley) rats. Thirty-eight-week-old rats/sex/group (F0) were administered MEKO in water, by gavage, at 0, 10, 100, or 200 mg/kg/day (at a dosing volume of 2 ml/kg), 5 days/week for 10 weeks with vaginal cytology evaluation (VCE) of F0 females during the last 3 weeks of the prebreed period. Animals were mated within groups for 3 weeks with dosing during mating, gestation, and lactation for 7 days/week. F0 parents and F1 weanlings, 10/sex/dose, were necropsied (after a 2-week postwean VCE in F0 females) with hematologic evaluation (including methemoglobin) and histology of adult livers, spleens, and reproductive organs. F1 weanlings, 30/sex/dose, were dosed for 11 weeks and mated as described above. Because of poor reproductive performance, not treatment related, F1 animals with no F2a litters were rebred to produce F2b litters. F1 parents and F2a weanlings, 10/sex/dose, were necropsied and evaluated as described above. Inguinal mammary glands were examined histologically from all nonselected F1 and F2 (a and b) female weanlings. Adult toxicity was observed in both generations and both sexes at all doses. Treatment-related parental deaths occurred at 200 mg/kg/day. At 100 and 200 mg/kg/day, parents exhibited dose-related reduced body weights and weight gains, reduced feed consumption, clinical signs of toxicity, and anemia with concomitant extramedullary hematopoiesis and hemosiderosis in livers and spleens (and increased spleen weights). At 10 mg/kg/day, only adult liver and spleen histologic effects were present. There was no evidence of reproductive organ or mammary glad pathology or of reproductive or postnatal toxicity at any dose tested. There was no adult "no observable adverse effect level" (NOAEL) established; the NOAEL for reproductive and postnatal toxicity was at least 200 mg/kg/day for rats in this study.

Potentiation of carbon tetrachloride hepatotoxicity by inhaled methanol: time course of injury and recovery.

Increases in the use of methanol (MeOH) as a transportation fuel would result in greater potential for inhalation exposure. Because oral exposure to MeOH potentiates the hepatotoxicity of carbon tetrachloride (CCl4), we examined the ability of inhaled MeOH to potentiate CCl4 hepatotoxicity and the time course of injury and recovery. Adult male F-344 rats were exposed to 0 or to 10,000 ppm MeOH by inhalation for 6 h and gavaged with 0.075 ml CCl4/kg 24 h later. Hepatotoxicity was assessed 0.5, 1, 1.5, 2, 3, 7, 15, 30, and 61 d after CCl4 exposure. For CCl4 alone, hepatotoxicity was most severe at 0.5 and 1 d, when minimal centrilobular hepatocellular necrosis and predominately mild centrilobular hepatocellular vacuolar degeneration occurred. By d 3, the livers from the CCl4 rats were histologically normal. For MeOH+CCl4, peak severity of hepatic injury was at 1 and 1.5 d, when moderate centrilobular necrosis and moderate/marked centrilobular degeneration occurred. MeOH+CCl4 resulted in serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) that were increased, relative to CCl4 alone, 171- and 113-fold, respectively, on d 1, and 166- and 140-fold, respectively, on d 1.5. Significant serum elevations in MeOH+CCl4 rats, relative to CCl4 alone rats, were present until d 7 and d 15 for AST and ALT, respectively. By d 3 and d 7, degeneration and necrosis, respectively, due to MeOH+CCl4 were essentially resolved. On d 7, the MeOH+CCl4 hepatic injury consisted mainly of chronic inflammation and centrilobular fibrosis. By d 30, the livers of MeOH+CCl4 rats were histologically normal. These data demonstrate that inhaled MeOH potentiates the hepatotoxicity of orally ingested CCl4, increasing the severity of CCl4 hepatotoxicity as well as the time required for recovery.

Toxicology studies of a chemical mixture of 25 groundwater contaminants: hepatic and renal assessment, response to carbon tetrachloride challenge, and influence of treatment-induced water restriction.

Because groundwater contamination is an important environmental concern, we examined the hepatic and renal effects of repeated exposure to a mixture of 25 chemicals frequently found in groundwater near hazardous-waste disposal sites and the effect of such exposure on carbon tetrachloride (CCI4) toxicity. Adult male F-344 rats received ad libitum deionized water and feed (Ad Lib Water) or ad libitum 10% MIX (referring to 10% of a technically achievable stock mixture) and feed for 14 d. Because exposure to the 25-chemical mixture via the drinking water resulted in decreased water and feed consumption, restricted deionized water and feed controls (Restricted Water) were included. On d 14, rats were gavaged with 0, 0.0375, 0.05, 0.075 or 0.15 ml CCl4/kg, and hepatic and renal toxicity assessed 24 h later. Little or no hepatic and renal toxicity was observed in rats exposed to 10% MIX alone. No hepatic or renal lesions occurred that could be attributed to 10% MIX alone. Slight but statistically significant alterations, of uncertain biological significance, resulted from the water treatments: 10% MIX increased alanine aminotransferase, urea nitrogen (BUN), and BUN/creatinine ratio; Restricted Water increased 5'-nucleotidase and decreased alkaline phosphatase. Relative kidney weight was increased by both 10% MIX and Restricted Water. CCI4 resulted in significant dosage-dependent hepatotoxicity in all three water treatment groups but had little or no effect on renal indicators of toxicity. Relative to Ad Lib Water, significantly greater hepatotoxicity occurred in both 10% MIX and Restricted Water rats. The response to CCI4 in the Restricted Water rats was similar to that of 10% MIX rats, indicating that a substantial portion of the effect of 10% MIX on CCI4 hepatotoxicity is due to decreased water and feed intake.

Toxicity of bromodichloromethane in female rats and mice after repeated oral dosing.

The carcinogenic water disinfection byproduct, bromodichloromethane (BDCM), produces renal and hepatic toxicity in rodents in acute and subchronic studies. In the present investigation, female rats and mice (n = 6) were dosed daily for 5 consecutive days with BDCM (dissolved in an aqueous, 10% Emulphor solution) by gavage. Rats received 75, 150 and 300 mg BDCM/kg body weight/day and mice received 75 and 150 mg BDCM/kg body weight/day. Two rats in the 300 mg/kg/day treatment group died on day 5. On day 6, the animals were sacrificed and serum samples were taken for analysis of indicators of hepatic and renal toxicity. Livers and kidneys were excised and samples taken for histopathological evaluation. Portions of the livers were also utilized to produce microsomes for analysis of cytochrome P450 enzyme activities and total P450 content. Total hepatic cytochrome P450 was decreased in rats dosed with 150 and 300 mg BDCM/kg body weight/day, but was not significantly affected in BDCM-treated mice. Serum lactate (LDH) and sorbitol (SDH) dehydrogenase, aspartate aminotransferase (AST), creatinine and blood urea nitrogen were increased above those of controls in rats dosed with 300 mg BDCM/kg/day. These data suggested that hepatic and renal damage had occurred in this treatment group. This was confirmed by histopathological analyses which revealed that lesions occurred in both hepatic and renal tissues from rats dosed with 150 and 300 mg BDCM/kg/day. The hepatic lesions were centrilobular and primarily consisted of vacuolar degeneration. The hepatotoxicity indicators alanine aminotransferase (ALT) and SDH were increased in mice dosed with 150 mg BDCM/kg/day. However, no histopathological lesions were observed in these animals. This study shows that BDCM is both hepatotoxic and nephrotoxic to female rats after repeated dosing, but is only weakly hepatotoxic to female mice at the administered doses. Also, reduced activities of hepatic cytochrome P450 were observed in rats, but not mice. These species differences in toxicity and xenobiotic metabolizing enzyme inhibition caused by BDCM suggest that an understanding of the mechanism of toxicity of this compound will be critical when extrapolating rodent toxicity data to humans for this environmental pollutant.

Reproductive effects of 4-vinylcyclohexene in Swiss mice assessed by a continuous breeding protocol.

4-Vinylcyclohexene (VCH), a dimer of 1,3-butadiene, was evaluated for reproductive toxicity in Swiss (CD-1) mice using the continuous breeding protocol (NTP, 1989). VCH in corn oil was administered by gavage at doses of 0, 100, 250, and 500 mg/kg/day to animals that were housed in same sex pairs for 1 week and then cohabited in breeding pairs for 14 weeks. During cohabitation, newborn litters were euthanized immediately after evaluation on postnatal Day (PND) 0. Litters born after Week 15 were reared until PND 21, when all F0 animals and low- and mid-dose F1 weanlings were humanely killed without a necropsy. At PND 74 +/- 10, control and high-dose F1 animals were cohabited within groups for 1 week and necropsied after delivery of the litters. In F0 breeding pairs, VCH did not affect measures of reproductive competence, including initial fertility, litters per pair, live litter size, or the proportion of pups born alive. Pup weight was decreased (4%) in the high-dose group relative to controls. High-dose F0 females exhibited slight general toxicity, manifested as an 8% difference in body weight compared to controls. VCH did not adversely affect preweaning growth or survival in the F1 generation. VCH had no effect on the reproductive competence of the F1 generation. High-dose F1 adult males and females had decreased body weight. At necropsy, increased relative liver weight (males 9% and females 8%) and sperm motility (although not thought to be biologically significant) were observed in the 500 mg/kg VCH group.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity evaluation of ethylene glycol by gavage in New Zealand white rabbits.

Artificially inseminated New Zealand white (NZW) rabbits were administered ethylene glycol (EG) by gavage on Gestational Days (GD) 6 through 19 at doses of 0, 100, 500, 1000, or 2000 mg/kg/day, with 23-24 inseminated animals per group. Clinical signs were recorded and water consumption was measured daily; does were weighed on GD 0, 6-19, 25, and 30. At necropsy (GD 30), maternal liver, kidney, and gravid uterine weights were recorded. Histopathologic examination was performed on kidneys from 10 does/dose and for all unscheduled deaths. Ovarian corpora lutea were counted and uterine implantation sites (total sites, resorptions, dead and live fetuses) were recorded. All live fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and variations. EG resulted in profound maternal toxicity at 2000 mg/kg/day (42% mortality; three early deliveries and one spontaneous abortion) associated with renal pathology and unaccompanied by any other indicators of maternal toxicity. Renal lesions at 2000 mg/kg/day involved the cortical renal tubules and included intraluminal oxalate crystals, epithelial necrosis, and tubular dilatation and degeneration. No dose-related maternal toxicity occurred at 100-1000 mg/kg/day. There was no indication of developmental toxicity at any dose tested, including no effects on pre- or postimplantation loss, number of fetuses, fetal body weight, or sex ratio (% male fetuses) per litter, and no evidence of teratogenicity. The "no observable adverse effect level" (NOAEL) for maternal toxicity was therefore 1000 mg/kg/day and the NOAEL for developmental toxicity was at least 2000 mg/kg/day in this study. The sensitivity of NZW rabbits relative to that of Sprague-Dawley rats and Swiss mice for maternal and developmental toxicity from gavage administration of EG during organogenesis can be determined for maternal toxicity: rabbits > mice > rats, and for developmental toxicity, mice >> rats >> rabbits.