In vitro percutaneous absorption and metabolism of Bisphenol A (BPA) through fresh human skin.

Bisphenol A (BPA) is a high production volume compound. It is mainly used as a monomer to make polymers for various applications including food-contact materials. The primary route of exposure to BPA in the general population is through oral intake (EFSA 2015) however, other potential sources of exposure have also been identified, such as dermal contact. In the present study, the percutaneous absorption through human skin has been investigated in an in vitro study according to OECD TG 428 (Skin Absorption: In Vitro Method). In order to investigate potential dermal BPA metabolism during absorption, radiolabelled BPA was applied to fresh, metabolically competent, human skin samples (ring labelled 14C BPA concentrations tested were 2.4, 12, 60 and 300mg/L). Measured as total radioactivity the mean absorbed dose (receptor compartment) ranged from 1.7-3.6% of the applied doses and the dermal delivery (epidermis+dermis+receptor compartment), sometimes also named bioavailable dose was 16-20% of the applied doses, with the majority of the radioactivity associated with epidermis compared to dermis and receptor fluid. No metabolism was observed in any of the epidermis samples; however some metabolism was observed in dermis and receptor fluid samples with formation of BPA-glucuronide and BPA-sulfate, and some polar metabolites.

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.

Development of a method for the determination of bisphenol A at trace concentrations in human blood and urine and elucidation of factors influencing method accuracy and sensitivity.

Bisphenol A (BPA) is an industrial chemical used to make polymers including some used in food contact applications. Virtually complete presystemic clearance of orally administered BPA occurs in humans by metabolism to BPA-glucuronide (BPA-G), but some biomonitoring studies report low concentrations of free (parent) BPA in human blood and urine. Trace contamination of BPA from exogenous sources or hydrolysis of BPA-G to free BPA, either during or after biomonitoring specimen collection, may have contributed to the reported concentrations of free BPA. An analytical method for the determination of free BPA in human blood and urine was developed and validated in two independent laboratories, using the latest generation of high-performance liquid chromatography-tandem mass spectrometry instrumentation to ensure the desired high sensitivity and selectivity. The method was designed to account for and/or eliminate background contamination from all sources and demonstrated that contamination could occur from devices used for specimen collection or storage, as well as other sources. The method employed an internal standard (BPA-d(8)) and demonstrated accuracy and reproducibility in both matrices fortified with BPA or a surrogate analyte ((13)C-BPA) at a low quantitation limit (0.1-0.2 ng/mL). For validation, five replicate samples were analyzed to evaluate reproducibility. Importantly, it was demonstrated that the conditions of the method did not result in the hydrolysis of BPA-G to free BPA, another possible source of error in BPA analysis. Application of the principles defined by this method will be critical to assure valid analytical results in any future biomonitoring studies.

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 F(1) 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. F(1) 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 F(1) 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).

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.

One-generation reproductive toxicity study of dietary 17beta-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).

Two-generation reproductive toxicity evaluation of dietary 17beta-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.

Induction of micronuclei by phenol in the mouse bone marrow: I. Association with chemically induced hypothermia.

Doses of xenobiotics at or near LD50 may result in substantial hypothermia in mice. Hypothermia has previously been associated with an increase in micronuclei (MN) formation. The present series of investigations examined the potential for phenol to induce hypothermia in mice and its correlation to previously reported MN induction. In order to examine the potential etiology of phenol-induced MN, evaluation of kinetochore status of MN was also carried out. Phenol-induced hypothermia was assessed in CD1 mice following a single ip dose of phenol ranging from 0-500 mg/kg. Phenol at 300 mg/kg or above caused significant and prolonged hypothermia in male and female mice (up to 7 degrees C decrease). In the micronucleus test, single ip doses of phenol to CD1 mice at 0, 30, 100, or 300 mg/kg produced a significant and prolonged hypothermia and a significant increase in MN only at 300 mg/kg; no marked effect on either body temperature or MN was observed at lower doses. A statistically significant increase in kinetochore-positive MN was observed at the 300-mg/kg dose; however, the response was considerably less than that observed for a known spindle poison. Hence, the induction of MN by phenol occurred only at a dose that produced substantial and prolonged physiologic hypothermia, but interruption of the cell spindle apparatus appeared to play only a minor role in MN formation. These data are suggestive of a threshold mechanism for the induction of MN by phenol treatment in mice.

Acetone in drinking water does not modulate humoral immunity in mice as measured by the antibody, plaque-forming cell assay.

It has been reported that the repeated topical, nonoccluded application of acetone may modulate antibody production in mice, thus producing humoral immunosuppression. However, the evaporative loss expected following nonoccluded dermal application of acetone makes the systemic effect seem unlikely. This study was designed to investigate the immunotoxicity potential of acetone in mice following a more direct systemic route of dosing via drinking water for 28 days. CD-1 male mice consumed average daily acetone doses of 121, 621 or 1144 mg/kg/day. The antibody, plaque-forming cell (AFC) assay was performed to measure the T cell-dependent, anti-sheep red blood cell immunoglobulin M (IgM) response, and hematology and thymus weights were evaluated to provide additional insight into the potential effects to the immune system. Body weights, white blood cell (WBC), numbers, red blood cell (RBC) counts, and hemoglobin and hematocrit levels showed no treatment-related effects at any dose of acetone. Eosinophil percentages were variable but also showed no dose-related trends. Spleen and thymus weights were not statistically different from controls and there were no effects on spleen cellularity or AFC response as a result of acetone administration. The AFC responses ranged from 1088 to 1401 AFCs/10(6) splenocytes and were not statistically different from controls (1277 AFCs/10(6) cells). Mice treated with cyclophosphamide (20 mg/kg) on days 25 to 28 demonstrated a 94% reduction in AFC/10(6) cells. Thus, the direct systemic administration of acetone did not produce evidence for immunotoxicity in CD-1 mice and the no observed adverse effect level (NOAEL) in this study was determined to be 1144 mg/kg/day.

Evaluation of oral and intravenous route pharmacokinetics, plasma protein binding, and uterine tissue dose metrics of bisphenol A: a physiologically based pharmacokinetic approach.

Bisphenol A (BPA) is a weakly estrogenic monomer used in the production of polycarbonate plastic and epoxy resins, both of which are used in food contact and other applications. A physiologically based pharmacokinetic (PBPK) model of BPA pharmacokinetics in rats and humans was developed to provide a physiological context in which the processes controlling BPA pharmacokinetics (e.g., plasma protein binding, enterohepatic recirculation of the glucuronide [BPAG]) could be incorporated. A uterine tissue compartment was included to allow the correlation of simulated estrogen receptor (ER) binding of BPA with increases in uterine wet weight (UWW) in rats. Intravenous- and oral-route blood kinetics of BPA in rats and oral-route plasma and urinary elimination kinetics in humans were well described by the model. Simulations of rat oral-route BPAG pharmacokinetics were less exact, most likely the result of oversimplification of the GI tract compartment. Comparison of metabolic clearance rates derived from fitting rat i.v. and oral-route data implied that intestinal glucuronidation of BPA is significant. In rats, but not humans, terminal elimination rates were strongly influenced by enterohepatic recirculation. In the absence of BPA binding to plasma proteins, simulations showed high ER occupancy at doses without uterine effects. Restricting free BPA to the measured unbound amount demonstrated the importance of including plasma binding in BPA kinetic models: the modeled relationship between ER occupancy and UWW increases was consistent with expectations for a receptor-mediated response with low ER occupancy at doses with no response and increasing occupancy with larger increases in UWW.