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.

The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration.

Bisphenol A (BPA) is used to produce polymers for food contact applications, thus there is potential for oral exposure of humans to trace amounts via the diet. BPA was weakly estrogenic in screening assays measuring uterine weight/response, although much higher oral doses of BPA were required to elicit a uterotropic response as compared to other routes of administration. The objective of this study was to determine if a route dependency exists in the pharmacokinetics and metabolism of 14C-labeled BPA following single oral (po), intraperitoneal (ip), or subcutaneous (sc) doses of either 10 or 100 mg/kg to Fischer 344 rats. Results indicated a marked route dependency in the pharmacokinetics of BPA. The relative bioavailability of BPA and plasma radioactivity was markedly lower following oral administration as compared to sc or ip administration. The major fraction of plasma radioactivity following oral dosing was the monoglucuronide conjugate of BPA (68-100% of plasma radioactivity). BPA was the major component in plasma at Cmax following sc or ip administration exceeded only by BPA-monoglucuronide in females dosed ip. Up to four additional unidentified metabolites were present only in the plasma of animals dosed ip or sc. One of these, found only following ip administration, was tentatively identified as the monosulfate conjugate of BPA. The monoglucuronide conjugate was the major urinary metabolite; unchanged BPA was the principal component excreted in feces. These results demonstrated a route dependency of BPA bioavailability in rats, with oral administration resulting in the lowest bioavailability, and offer an explanation for the apparent route differences in estrogenic potency observed for BPA.

Normal reproductive organ development in Wistar rats exposed to bisphenol A in the drinking water.

Bisphenol A (BPA) is a chemical used primarily as a monomer in the manufacture of numerous chemical products, such as epoxy resins and polycarbonate. The objective of this study was to evaluate potential effects of BPA on sexual development of male rats and was designed to clarify low-dose observations reported as preliminary results by Sharpe et al. (1996). The protocol for the present study followed the same treatment schedule as reported by Sharpe et al. (1995, 1996), but included more treatment groups, a greater number of animals per group, and a more comprehensive number of reproductive endpoints. Groups of 28 female Han-Wistar albino rats were exposed to drinking water that contained 0, 0.01, 0.1, 1.0, or 10 ppm BPA or 0.1 ppm diethylstilbestrol (DES), 7 days per week, for a total of 10 weeks. Treatment of the females began at 10 weeks of age and continued throughout a 2-week premating period, 2 weeks of mating (to untreated males), 21-22 days of gestation, and 22 days of lactation. Offspring weanling males were given untreated drinking water and maintained until 90 days of age when evaluations were made of various reproductive organs. Consistent with Sharpe et al. (1996) the female offspring were not evaluated. No treatment-related effects on growth or reproductive endpoints were observed in adult females exposed to any concentration of BPA. Similarly, no treatment-related effects were observed on the growth, survival, or reproductive parameters (including testes, prostate and preputial gland weights, sperm count, daily sperm production, or testes histopathology) of male offspring from dams exposed to BPA during gestation and lactation. DES administered in the drinking water at 0. 1 ppm resulted in decreased body weight, body weight change, and food consumption in adult females. In addition, an increase in the duration of gestation and a decrease in the number of pups delivered and number of live pups were also observed in animals exposed to DES. In conclusion, these results do not confirm the previous findings of Sharpe et al. (1996) and show that low doses of BPA had no effects on male sexual development in the rat.

Normal reproductive organ development in CF-1 mice following prenatal exposure to bisphenol A.

Bisphenol A (BPA) is a monomer used in the manufacture of a multitude of chemical products, including epoxy resins and polycarbonate. The objective of this study was to evaluate the effects of BPA on male sexual development. This study, performed in CF-1 mice, was limited to the measurement of sex-organ weights, daily sperm production (DSP), epididymal sperm count, and testis histopathology in the offspring of female mice exposed to low doses of BPA (0, 0.2, 2, 20, or 200 microg/kg/day), by deposition in the mouth on gestation days 11-17. Male sexual development determinations were made in offspring at 90 days-of-age. Since this study was conducted to investigate and clarify low-dose effects reported by S. C. Nagel et al., 1997, Environ. Health Perspect. 105, 70-76, and F. S. vom Saal et al., 1998, Toxicol. Indust. Health 14, 239-260, our study protocol purposely duplicated the referenced studies for all factors indicated as critical by those investigators. An additional group was dosed orally with 0.2 microg/kg/day of diethylstilbestrol (DES), which was selected based on the maternal dose reported to have maximum effect on the prostate of developing offspring, by F. S. vom Saal (1996, personal communication), vom Saal et al. (1997, Proc. Natl. Acad. Sci. U S A 94, 2056-2061). Tocopherol-stripped corn oil was used as the vehicle for BPA and DES, and was administered alone to control animals. No treatment-related effects on clinical observations, body weight, or food consumption were observed in adult females administered any dose of BPA or DES. Similarly, no treatment-related effects on growth or survival of offspring from dams treated with BPA or DES were observed. The total number of pups born per litter was slightly lower in the 200-microg/kg/day BPA group when compared to controls, but this change was not considered treatment-related since the litter size was within the normal range of historical controls. There were no treatment-related effects of BPA or DES on testes histopathology, daily sperm production, or sperm count, or on prostate, preputial gland, seminal vesicle, or epididymis weights at doses previously reported to affect these organs or at doses an order of magnitude higher or lower. In conclusion, under the conditions of this study, the effects of low doses of BPA reported by S. C. Nagel et al., 1997 (see above) and F. S. vom Saal et al., 1998 (see above), or of DES reported by F. S. vom Saal et al., 1997 (see above) were not observed. The absence of adverse findings in the offspring of dams treated orally with DES challenges the "low-dose hypothesis" of a special susceptibility of mammals exposed perinatally to ultra-low doses of even potent estrogenic chemicals. Based on the data in the present study and the considerable body of literature on effects of BPA at similar and much higher doses, BPA should not be considered as a selective reproductive or developmental toxicant.

In vitro metabolism of 4-vinylcyclohexene in rat and mouse liver, lung, and ovary.

4-Vinylcyclohexene (4-VCH), the dimer of 1,3-butadiene, is an ovarian toxicant in mice due to the formation of a diepoxide metabolite, but the tissue-specific site of formation of the metabolites is unknown. Microsomal preparations from liver, lung, and ovaries obtained from female Crl:CD BR rats and female B6C3F1 mice were tested for their ability to metabolize the following reactions: 4-VCH to 4-VCH-1,2-epoxide and 4-VCH-7,8-epoxide; 4-VCH-1,2-epoxide to 4-VCH diepoxide and 4-VCH-1,2-diol; 4-VCH-7,8-epoxide to 4-VCH diepoxide and 4-VCH-7,8-diol; and hydrolysis of 4-VCH diepoxide. Microsomes were incubated with the test chemical and the reaction products were analyzed by gas chromatography. Rat liver and lung microsomes and mouse liver and lung microsomes metabolized 4-VCH to 4-VCH-1,2-epoxide at detectable rates. Mouse liver had a Vmax for the reaction that was 56-fold higher than that for rat liver (11.1 and 0.20 nmol/min/mg protein, respectively). The Vmax for mouse lung was 2-fold higher than that for rat lung. 4-VCH-1,2-epoxide formation was not detected in ovarian microsomes from rats or mice. Metabolism of 4-VCH to 4-VCH-7,8-epoxide was detected in microsomes from rat liver and mouse liver and lung, at rates very low compared to those for metabolism to the 1,2-epoxide. Rat and mouse liver had very similar K(m) and Vmax values for metabolism of 4-VCH-1,2-epoxide to 4-VCH diepoxide. The Vmax for rat liver was 3.69 and for mouse liver was 5.35 nmol/min/mg protein. Rat and mouse ovaries did not have detectable capacity to metabolize 4-VCH-1,2-epoxide to the diepoxide. Rat and mouse liver and lung have very similar K(m) and Vmax values for metabolism of 4-VCH-7,8-epoxide to the diepoxide, while ovaries did not have detectable rates for this reaction. Hydrolysis of 4-VCH-1,2-epoxide to 4-VCH-1,2-diol was at similar rates in rat and mouse liver microsomes. Hydrolysis of 4-VCH-7,8-epoxide to 4-VCH-7,8-diol was detected only in rat liver microsomes. Hydrolysis of 4-VCH diepoxide was detected in rat and mouse liver and lung, and in rat ovary microsomes. The Vmax for rat liver was 9-fold greater than that for mouse liver (5.51 and 0.63 nmol/min/mg protein, respectively), and lung and ovary tissues were not as active as rat liver. The balance of activation versus detoxication reactions in rats and mice suggests that the mouse may be more susceptible to 4-VCH toxicity because of generation of high levels of epoxide metabolites. In general, the mouse is more efficient at metabolism of 4-VCH to epoxides than is the rat. In contrast, the rat may be more efficient at hydrolysis of epoxides. Thus, the rat would tend to produce a lower concentration of epoxide metabolites than the mouse, at equal doses of 4-VCH.

Estrogen modulation: tiered testing for human hazard evaluation. American Industrial Health Council, Reproductive and Developmental Effects Subcommittee.

Recent concerns about the potential of certain chemicals to modulate estrogen-regulated processes have led to questions as to how chemicals should be tested for such effects. Therefore, AIHC has developed a comprehensive, resource-efficient, and flexible tiered strategy for estrogen modulation (EM) testing. Levels of evaluation include Tier 0, in which exposure, along with alerts based on structure-activity, persistence, bioaccumulation, and other data, are assessed to prioritize chemicals for preliminary testing. In Tier I, short term in vitro, ex vivo, and/or in vivo assays are used to obtain a preliminary indication of EM potential. Among these, an in vivo response assay is considered the most reliable at this time. However, none of these tests are intended for risk assessment, but rather to aid in choosing chemicals for further testing and in guiding the extent of that testing. Tier II is aimed at risk assessment and involves whole animal tests that contain EM-sensitive end points (e.g., two-generation reproduction study). Tier III consists of hypothesis-driven research reserved for situations where targeted research can reduce levels of uncertainty. This tiered approach provides a framework for the strategic and effective application of EM test methods to address specific information needs on a case by case basis.

Review of risk assessments on 1,3-butadiene (1985-1991).

During the past decade, several assessments have estimated the carcinogenic risk of inhaled 1,3-butadiene (BD) to the general population as well as for occupational exposures. Although most of the risk assessments have been based on inhalation bioassays in mice, some of the assessments have included the 2-year inhalation bioassay in rats. Because of the marked species differences in the carcinogenic response to butadiene, estimates of risk vary over nine orders of magnitude going from the most sensitive target organ in female mice to less sensitive male rats. An important tool in determining which estimate is most relevant for extrapolation to humans is to ascertain consistency with human experience. Estimated workplace cancer risks which are based on the assumption that humans are as responsive as the mouse suggest added risks of 200 or more out of 1000 workers (1 in 5) exposed to 2 ppm butadiene (assume 40 years of exposure). This estimate is clearly inconsistent with what has been seen and this would not have been missed in epidemiology studies. Risk assessments based on the growing understanding of the mechanisms of toxicity and the importance of various tissues ability to produce and maintain reactive epoxide metabolites demonstrate why rats are much less susceptible than the mouse. By developing a mechanistic understanding of humans, it is possible to refine the traditional approach to risk assessment, whereby better risk assessments can be made. This paper presents a summary of eight risk assessments that have been developed to date for butadiene.

A neurotoxicity assessment of high flash aromatic naphtha.

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide-boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 Aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent also known as High Flash Aromatic Naphtha (HFAN). A program was initiated to assess the toxicological properties of HFAN since there may be human exposure, especially in the workplace. The current study was conducted to assess the potential for neurotoxicity in the rat. Adult male Sprague-Dawley rats of approximately 300 grams body weight, in groups of twenty, were exposed by inhalation to HFAN for 90 days at concentrations of 0, 100, 500, and 1500 ppm. During this period the animals were tested monthly for motor activity and in a functional observation battery. After three months of exposure, for 6 hours/day, 5 days/week, 10 animals/group/sex were sacrificed and selected nervous system tissue was examined histopathologically. No signs of neurotoxicity were seen in any of the evaluated parameters, nor was there evidence of pathologic changes in any of the examined tissues.

Effect of vitamin E and other amelioratory agents on the fenvalerate-mediated skin sensation.

Previous investigations have demonstrated that dermal exposure to fenvalerate or other synthetic pyrethroid insecticides can produce a skin sensory response characterized by an itching/tingling sensation in humans and animals. The objective of this investigation performed in guinea pigs was to establish treatments which would be effective against pyrethroid-mediated skin sensation. Two classes of agents were tested. Barrier agents, which block penetration of substances through the skin, did not significantly reduce the fenvalerate-mediated skin sensations. Post-treatments with steroidal Dermolate, antihistamine Delamine or anti-inflammatory aspirin did not significantly reduce the pyrethroid-mediated skin sensation. However, Bicozene (a local anesthetic cream) and Tashan (a vitamin A, D, and E-containing cream) were effective in reducing the pyrethroid-mediated skin sensations. Prior (30 min and 5h) dermal application of vitamin E was found to be effective in significantly reducing the fenvalerate-mediated skin sensation; even when applied 29 h prior to fenvalerate exposure, there appeared to be a reduced skin response. Piperonyl butoxide (PBO), a pesticide synergist, reduced the fenvalerate skin sensations when applied either directly to the skin or in conjunction with the pyrethroid.

Pyrethroid-mediated skin sensory stimulation characterized by a new behavioral paradigm.

Occupational exposures to pyrethroids have been associated with skin sensory effects characterized by transient itching/tingling sensations. This effect had not been detected in "Draize" skin irritation tests and exists in the absence of visible skin irritation. The objective of this investigation was to develop an animal model to study this phenomenon. Guinea pigs were treated with pyrethroid solutions on one side of their shaved back and control substances on the other side. The animals responded by licking, rubbing, scratching, or biting the test sites and activity was quantified by counting the number of times the animals responded to pyrethroid or control treated sites. Animals responded to pyrethroid more than to control substances and this behavioral activity was apparently maximum during the first hour and essentially over by the fourth hour after treatment. The sensory response did not directly correlate with overt visible signs of skin irritation. However, a chemical irritant (oil of mustard) was able to restimulate the behavioral activity when applied within 24 hr after pyrethroid application. Skin sensory stimulation produced by cyano-containing synthetic pyrethroids was significantly greater than that produced by a non-cyano-containing pyrethroid. This behavioral model provides a quantitative means to evaluate pyrethroid nonerythematous skin sensory stimulation.

Evaluation of hepatic storage of sulfobromophthalein in rats and dogs.

Sulfobromophthalein (BSP) is stored in the liver during its passage from plasma to bile. The soluble hepatic anion-binding protein, ligandin, may be responsible for BSP storage since BSP binds to this protein. Studies were undertaken in rats and dogs to determine whether the amount of ligandin in the liver is a major determinant of storage. Ligandin content in dog liver was found to be 0.252 mg BSP bound/kg body wt which was about one-half that in rat liver (0.504 mg BSP bound/kg body wt). Relative to plasma, BSP accumulation in liver tissue (or storage of BSP) following single bolus injection of BSP was about 5 times greater in rats than in dogs. Lack of correlation between BSP accumulation and ligandin content suggests that intracellular storage of this anion is not solely determined by its binding to ligandin. Differences in the carrier-mediated transport processes on either/or both sinusoidal and canalicular sides of the hepatocyte may be responsible for this affect.

Carbon tetrachloride-induced hepatotoxicity: studies in developing rats and protection by zinc.

This investigation was designed to evaluate carbon tetrachlorid (CCl4)-induced hepatotoxicity in developing rats and in adult rats pretreated with zinc. Hepatotoxicity of CCl4 in rats as young as 4 days of age was similar to that in adults. However, CCl4 metabolism, measured by in vitro binding of 14CCl4 to hepatic microsomal protein and lipid, was significantly lower in 4- and 14-day old rats than in adults. The sensitivity of young animals to CCl4 toxicity may be due to metabolic ketosis since blood concentrations of acetoacetate were 3-5 times higher in young rats than in adult animals. It has previously been shown that adult rats are more sensitive to CCl4 toxicity when they are in the ketonic state. The protection from CCl4 toxicity that was afforded adult rats pretreated with zinc was determined to be independent of the effect of zinc on CCl4 metabolism. Since treatment with zinc results in a large increase in hepatic concentration of metallothionein and that some product of 14CCl4 appeared to bind to zinc-induced metallothionein, it was suggested that metallothionein may protect against CCl4-induced liver damage by sequestering reactive metabolites of CCl4. These studies represent two examples of how the toxicity of a chemical whose toxicity is mediated via a metabolite can be modified by factors independent of metabolic activation.

Characteristics of hepatic excretory function during development.

The purpose of this investigation was to characterize the development of hepatic excretory function in rats. Cumulative (40 min) intestinal ouabain content was lower and plasma ouabain concentrations were higher in 15-day-old rats than in 21-, 25-, 35- and 45-day-old animals and reached adult levels when rats were 35 days old. Impaired transport of ouabain in rat neonates correlated to low initial ouabain concentration in liver, which suggested that the inability of young rats to accumulate ouabain in liver may be the most important determinant for functional insufficiency. Bile duct ligation and bile salt infusion, treatments that primarily depress and enhance (respectively) excretion of sulfobromophthalein from liver into bile, markedly altered the disappearance of sulfobromophthalein from plasma of adult rats but did not appreciably affect sulfobromophthalein disappearance from blood of 15-day-old rats. The effect of bile duct ligation on ouabain transport in 15-day-old rats was also not as dramatic as the effect produced in adult rats. Hepatic uptake is rapid in adult rats and overall excretion is limited by a slower rate of transport from liver into bile. The lower rate of uptake in 15-day-old rats may limit overall transport function in young animals.

Effect of polybrominated biphenyls on the development of hepatic excretory function.

Polybrominated biphenyls (PBBs) stimulate hepatic drug metabolism in adult and developing rats. The purpose of this investigation was to determine the influence of PBBs on the development of the liver as an organ for chemical excretion. Exposure of developing rats to polybrominated biphenyls (PBBs) by feeding 50 ppm of PBBs to pregnant or lactating mothers and rat weanlings did not produce overt toxicity when compared to controls over a 49-day postnatal period. However, prenatal and postnatal dietary exposure to PBBs resulted in elevated liver weight. In 15-day-old rats, increased liver weight after PBBs was correlated with enhanced ouabain transport from plasma into bile. Liver weight was also elevated in 21-, 35- and 49-day-old rats treated with PBBs, but this effect was not associated with stimulation of ouabain transport in these animals. Stimulation of ouabain transport after PBBs in 15-day-old rats was associated with increased hepatic uptake of ouabain. Stimulation in 15-day-old rats and not older rats may be attributed to the relative importance of uptake for ouabain transport in 15-day-old rats.