Steven G. Hentges

Two-Generation Reproductive Toxicity Study of Dietary Bisphenol A in CD-1 (Swiss) Mice

Dietary bisphenol A (BPA) was evaluated in a mouse two-generation study at 0, 0.018, 0.18, 1.8, 30, 300, or 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, or 600 mg BPA/kg/day, 28 per sex per group). A concurrent positive control group of dietary 17beta-estradiol (0.5 ppm; 28 per sex) confirmed the sensitivity of CD-1 mice to an endogenous estrogen. There were no BPA-related effects on adult mating, fertility or gestational indices, ovarian primordial follicle counts, estrous cyclicity, precoital interval, offspring sex ratios or postnatal survival, sperm parameters or reproductive organ weights or histopathology (including the testes and prostate). Adult systemic effects: at 300 ppm, only centrilobular hepatocyte hypertrophy; at 3500 ppm, reduced body weight, increased kidney and liver weights, centrilobular hepatocyte hypertrophy, and renal nephropathy in males. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights (with seminiferous tubule hypoplasia), slightly delayed preputial separation (PPS), and apparently increased the incidence of treatment-related, undescended testes only in weanlings, which did not result in adverse effects on adult reproductive structures or functions; this last finding is considered a developmental delay in the normal process of testes descent. It is likely that these transient effects were secondary to (and caused by) systemic toxicity. Gestational length was increased by 0.3 days in F1/F2 generations; the toxicological significance, if any, of this marginal difference is unknown. At lower doses (0.018-30 ppm), there were no treatment-related effects and no evidence of nonmonotonic dose-response curves for any parameter. The systemic no observable effect level (NOEL) was 30 ppm BPA (approximately 5 mg/kg/day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg/day). Therefore, BPA is not considered a selective reproductive or developmental toxicant in mice.

Developmental Neurotoxicity Study of Dietary Bisphenol A in Sprague-Dawley Rats

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F1 offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F1 offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F1 offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).

The chemistry of metal carbonyl anions. VII. Reduction of cyclopentadienylmetal carbonyls in hexamethylphosphoramide

The alkali metal reductions of a series of monomeric and dimeric cyclopentadienyl-substituted metal carbonyls in hexamethylphosphoramide provide principally C5H5V(CO)32- from C5H5V(CO)4, Mn(CO)43- from C5H5Mn(CO)3, Co(CO)4- from C5H5Co(CO)2, and C5H5M(CO)y- from corresponding [C5H5M(CO)y]2, where M = Mo, Fe and Ni for y = 3, 2 and 1, respectively. The spectral and chemical properties of many of these reduced species are described.

Relevance of drinking water as a source of human exposure to bisphenol A

A comprehensive search of studies describing bisphenol A (BPA) concentrations in drinking water and source waters (i.e., surface water and groundwater) was conducted to evaluate the relevance of drinking water as a source of human exposure and risk. Data from 65 papers were evaluated from North America (31), Europe (17), and Asia (17). The fraction of drinking water measurements reported as less than the detection limit is high; 95%, 48%, and 41%, for North America, Europe, and Asia, respectively. The maximum quantified (in excess of the detection limit) BPA concentrations from North America, Europe, and Asia are 0.099 μg/l, 0.014 μg/l, and 0.317 μg/l. The highest quantified median and 95th percentile concentrations of BPA in Asian drinking water are 0.026 μg/l and 0.19 μg/l, while high detection limits restricted the determination of representative median and 95th percentile concentrations in North America and Europe. BPA in drinking water represents a minor component of overall human exposure, and compared with the lowest available oral toxicity benchmark of 16 μg/kg-bw/day (includes an uncertainty factor of 300) gives margins of safety >1100. Human biomonitoring data indicate that ingestion of drinking water represents <2.8% of the total intake of BPA.

The chemistry of metal carbonyl anions. II. Synthesis and reactions of the pentacarbonylmetallate dianions of chromium, molybdenum, and tungsten with metallic monohalides

Alkali metal amalgam reductions of M(CO)6 in tetrahydrofuran (THF) have provided salts of the pentacarbonylmetallate dianions. Cesium amalgam reacts with M(CO)6 at room temperature to yield Cs2M(CO)5, while sodium or potassium amalgams provide only (Na or K)2Cr(CO)5 in good yield in addition to a substantial amount of (Na or K)2Cr2(CO)10. Molybdenum and tungsten carbonyl give primarily (Na or K)2M2(CO)10 in THF even after 24 h of reflux; however, infrared evidence indicates the presence of monomeric dianions in these solutions. Reaction of the monomeric dianions with Ph3MCl (M = Ge, Sn, Pb) provides air- and moisture-stable substituted carbonyl anions of the type Ph3MM′(CO)5− (M′ = Cr, Mo, W) which have been isolated as tetraethylammonium salts. Pure Na2Cr(CO)5, prepared from the interaction of Cr(CO)6 with sodium metal in liquid ammonia or HMPA, has also been used to provide similar substituted carbonyl anions of the type ECr(CO)5− where E = Me3Sn and C5H5Fe(CO)2. The triphenyltin derivatives have also been prepared independently by the interaction of Ph3SnLi with M′(CO)6 in THF at room temperature as well as from the reaction of Ph3SnCl with M′2(CO)102− in THF.

Estimating potential risks to terrestrial invertebrates and plants exposed to bisphenol A in soil amended with activated sludge biosolids.

Bisphenol A (BPA) is a high production volume substance primarily used to produce polycarbonate plastic and epoxy resins. During manufacture and use, BPA may enter wastewater treatment plants. During treatment, BPA may become adsorbed to activated sludge biosolids, which may expose soil organisms to BPA if added to soil as an amendment. To evaluate potential risks to organisms that make up the base of the terrestrial food web (i.e., invertebrates and plants) in accordance with international regulatory practice, toxicity tests were conducted with potworms (Enchytraeids) and springtails (Collembolans) in artificial soil, and six plant types using natural soil. No-observed-effect concentrations (NOEC) for potworms and springtails were equal to or greater than 100 and equal to or greater than 500 mg/kg (dry wt), respectively. The lowest organic matter-normalized NOEC among all tests (dry shoot weight of tomatoes) was 37 mg/kg-dry weight. Dividing by an assessment factor of 10, a predicted-no-effect concentration in soil (PNEC(soil)) of 3.7 mg/kg-dry weight was calculated. Following international regulatory guidance, BPA concentrations in soil hypothetically amended with biosolids were calculated using published BPA concentrations in biosolids. The upper 95th percentile BPA biosolids concentration in North America is 14.2 mg/kg-dry weight, and in Europe is 95 mg/kg-dry weight. Based on recommended biosolids application rates, predicted BPA concentrations in soil (PEC(soil)) would be 0.021 mg/kg-dry weight for North America and 0.14 mg/kg-dry weight for Europe. Hazard quotients (ratio of PEC(soil) and PNEC(soil)) for BPA were all equal to or less than 0.04. This indicates that risks to representative invertebrates and plants at the base of the terrestrial food web are low if exposed to BPA in soil amended with activated sludge biosolids.

Two-Generation Reproductive Toxicity Evaluation of Dietary 17β-Estradiol (E2; CAS No. 50-28-2) in CD-1 (Swiss) Mice

No information exists on reproductive/developmental effects in mice exposed to dietary 17beta-estradiol (E2) over multiple generations. Therefore, under OECD Test Guideline 416 with enhancements, CD-1 mice (F0 generation, 25 mice/sex/group) were exposed to dietary E2 at 0, 0.001, 0.005, 0.05, 0.15, or 0.5 ppm ( approximately 0, 0.2, 1, 10, 30, or 100 mug E2/kg body weight/day) for 8 weeks prebreed, 2 weeks mating, approximately 3 weeks gestation, and 3 weeks lactation. At weaning, selected F1 offspring (F1 parents; 25/sex/group) and extra retained F1 males (one per litter) were exposed to the same dietary concentrations and durations as the F0 generation; study termination occurred at F2 weaning; F1/F2 weanlings (up to three per sex per litter) were necropsied with organs weighed. At 0.5 ppm, effects were increased F1/F2 perinatal loss, prolonged F0/F1 gestational length, reduced numbers of F2 (but not F1) litters/group, reduced F1/F2 litter sizes, accelerated vaginal patency (VP) and delayed preputial separation (PPS), increased uterus + cervix + vagina weights (UCVW) in F0/F1 adults and F1/F2 weanlings, and decreased testes and epididymides weights (TEW) in F1/F2 weanlings. At 0.15 ppm, effects were increased UCVW in F0/F1 adults and F1/F2 weanlings, accelerated VP, delayed PPS, and reduced TEW in F1/F2 weanlings. At 0.05 ppm, UCVW were increased in F1/F2 weanlings, and PPS was delayed only in extra retained F1 males. There were no biologically significant or treatment-related effects on F0/F1 parental body weights, feed consumption, or clinical observations, or on F0/F1 estrous cyclicity, F0/F1 andrology, or F1/F2 anogenital distance at any dose. The no observable effect level was 0.005 ppm E2 ( approximately 1 mug/kg/day). Therefore, the mouse model is sensitive to E2 by oral administration, with effects on reproductive development at doses of 10- 100 mug/kg/day.

One-generation reproductive toxicity study of dietary 17β-estradiol (E2; CAS No. 50-28-2) in CD-1® (Swiss) mice

There is no information on reproductive/developmental effects in mice from dietary estrogen. Therefore, 10 adult CD-1 mice/sex/group were administered dietary 17beta-estradiol (E2) at 0, 0.005, 0.05, 0.5, 2.5, 5, 10, and 50 ppm for 2-week prebreed, mating, gestation, lactation. F1 weanlings (3/sex/litter) were necropsied and 2/sex/litter were retained, with exposure, until vaginal patency (VP) or preputial separation (PPS) and then necropsied. Results included complete infertility at 2.5-50 ppm with normal mating indices. At 0.5 ppm (and above), F0 adult female uterus plus cervix plus vagina weights (UCVW) were increased. At 0.5 ppm: prolonged gestational length; increased F1 stillbirth index; reduced live birth index and litter size; decreased testes and epididymides weights at weaning; unaffected AGD on pnd 0 and 21; delayed PPS; increased undescended testes; unaffected prostate weight; accelerated VP; enlarged vaginas; fluid-filled uteri. At 0.05 ppm: no F0 reproductive effects, increased F1 weanling UCVW; delayed PPS. The NOEL was 0.005 ppm ( approximately 1 microg/kg/day).

Adult fathead minnow, Pimephales promelas, partial life-cycle reproductive and gonadal histopathology study with bisphenol A.

Bisphenol A (BPA) is an intermediate used to produce epoxy resins and polycarbonate plastics. Although BPA degrades rapidly in the environment with aquatic half-lives from 0.5 to 6 d, it can be found in aquatic systems because of widespread use. To evaluate potential effects from chronic exposure, fathead minnows were exposed for 164 d to nominal concentrations of 1, 16, 64, 160, and 640 µg/L BPA. Population-level endpoints of survival, growth, and reproduction were assessed with supplemental endpoints (e.g., vitellogenin, gonad histology), including gonad cell type assessment and quantification. No statistically significant changes in growth, gonad weight, gonadosomatic index, or reproduction variables (e.g., number of eggs and spawns, hatchability) were observed; however, there was a significant impact on male survival at 640 µg/L. Vitellogenin increased in both sexes at 64 µg/L or higher. Gonad cell type frequencies were significantly different from controls at 160 µg/L or higher in males with a slight decrease in spermatocytes compared with less mature cell types, and at 640 µg/L in females with a slight decrease in early vitellogenic cells compared with less mature cells. The decrease in spermatocytes did not correspond to a decrease in the most mature sex cell type (spermatozoa) and did not impair male fertility, as hatchability was not impacted. Overall, marginal shifts in gametogenic cell maturation were not associated with any statistically significant effects on population-relevant reproductive endpoints (growth, fecundity, and hatchability) at any concentration tested.

Extensive metabolism and route-dependent pharmacokinetics of bisphenol A (BPA) in neonatal mice following oral or subcutaneous administration.

Orally administered bisphenol A (BPA) undergoes efficient first-pass metabolism to produce the inactive conjugates BPA-glucuronide (BPA-G) and BPA-sulfate (BPA-S). This study was conducted to evaluate the pharmacokinetics of BPA, BPA-G and BPA-S in neonatal mice following the administration of a single oral or subcutaneous (SC) dose. This study consisted of 3 phases: (1) mass-balance phase in which effective dose delivery procedures for oral or SC administration of (3)H-BPA to postnatal day three (PND3) mice were developed; (2) pharmacokinetic phase during which systemic exposure to total (3)H-BPA-derived radioactivity in female PND3 mice was established; and (3) metabolite profiling phase in which 50 female PND3 pups received either a single oral or SC dose of (3)H-BPA. Blood was collected from 5 pups/route/time-point at various times post-dosing, the blood plasma samples were pooled by group, and time-point and samples were profiled by HPLC with fraction collection. Fractions were analyzed for total radioactivity and data used to reconstruct radiochromatograms and to integrate individual peaks. The identity of the BPA, BPA-G, and BPA-S peaks was confirmed using authentic standards and LC-MS/MS analysis. The result of this study revealed that female PND3 mice have the capacity to metabolize BPA to BPA-G, BPA-S and other metabolites after both routes of administration. Systemic exposure to free BPA is route-dependent as the plasma concentrations were lower following oral administration compared to SC injection.