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.

Quantitative analysis of unconjugated and total bisphenol A in human urine using solid-phase extraction and UPLC-MS/MS: method implementation, method qualification and troubleshooting.

The aim of the presented investigation was to document challenges encountered during implementation and qualification of a method for bisphenol A (BPA) analysis and to develop and discuss precautions taken to avoid and to monitor contamination with BPA during sample handling and analysis. Previously developed and published HPLC-MS/MS methods for the determination of unconjugated BPA (Markham et al. Journal of Analytical Toxicology, 34 (2010) 293-303) [17] and total BPA (Markham et al. Journal of Analytical Toxicology, 38 (2014) 194-203) [20] in human urine were combined and transferred into another laboratory. The initial method for unconjugated BPA was developed and evaluated in two independent laboratories simultaneously. The second method for total BPA was developed and evaluated in one of these laboratories to conserve resources. Accurate analysis of BPA at sub-ppb levels is a challenging task as BPA is a widely used material and is ubiquitous in the environment at trace concentrations. Propensity for contamination of biological samples with BPA is reported in the literature during sample collection, storage, and/or analysis. Contamination by trace levels of BPA is so pervasive that even with extraordinary care, it is difficult to completely exclude the introduction of BPA into biological samples and, consequently, contamination might have an impact on BPA biomonitoring data. The applied UPLC-MS/MS method was calibrated from 0.05 to 25ng/ml. The limit of quantification was 0.1ng/ml for unconjugated BPA and 0.2ng/ml for total BPA, respectively, in human urine. Finally, the method was applied to urine samples derived from 20 volunteers. Overall, BPA can be analyzed in human urine with acceptable recovery and repeatability if sufficient measures are taken to avoid contamination throughout the procedure from sample collection until UPLC-MS/MS analysis.

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 total bisphenol a at trace levels in human blood and urine and elucidation of factors influencing method accuracy and sensitivity.

This publication describes a method for the determination of total bisphenol A (BPA and conjugated BPA) following enzyme hydrolysis and is intended as a companion to our previously developed analytical method for the determination of free BPA (the aglycone) in human blood and urine using high-performance liquid chromatography-tandem mass spectrometry ( 1). That free BPA method provided a means to account for and/or eliminate background contamination and demonstrated accuracy and reproducibility in both matrices fortified with BPA or a surrogate analyte ((13)C BPA) at a low method quantitation limit (MQL) of 0.1-0.2 ng/mL. In contrast to the free BPA method results and based on stringent accuracy, precision and confirmation criteria set for the MQLs of the method developed for total BPA, the MQL achieved in blood was 1.020-2.550 and 0.510-1.020 ng/mL in urine. These data showed higher MQLs than the desired MQLs of 0.5 ng/mL (blood) and 0.2 ng/mL (urine) with increased variability between analyses which demonstrates the importance of generating method validation data with each analysis. In contrast, the MQL achieved for (13)C BPA-G (monoglucuronide as a surrogate analyte in blood was 0.2-0.5 and 0.2 ng/mL in urine illustrating that the method is capable of meeting lower MQL requirements if the contribution from exogenous BPA can be well controlled. This method for the determination total BPA in human blood and urine is intended to be used in conjunction with the free BPA method ( 1) to obtain accurate and complete BPA biomonitoring data to support human exposure assessments.

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).

Acute and chronic toxicity testing of bisphenol A with aquatic invertebrates and plants.

Bisphenol A (BPA, 4,4'-isopropylidine diphenol) is a commercially important chemical used primarily as an intermediate in the production of polycarbonate plastic and epoxy resins. Extensive effect data are currently available, including long-term studies with BPA on fish, amphibians, crustaceans, and mollusks. The aim of this study was to perform additional tests with a number of aquatic invertebrates and an aquatic plant. These studies include acute tests with the midge (Chironomus tentans) and the snail (Marisa cornuarietis), and chronic studies with rotifers (Brachionus calyciflorus), amphipods (Hyalella azteca), and plants (Lemna gibba). The effect data on different aquatic invertebrate and plant species presented in this paper correspond well with the effect and no-effect concentrations (NOECs) available from invertebrate studies in the published literature and are within the range found for other aquatic species tested with BPA.

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.

Two generation reproduction study of ethylbenzene by inhalation in Crl-CD rats.

BACKGROUND: This study was conducted to evaluate the potential adverse effects of ethylbenzene (EB) on reproductive capability from whole-body inhalation exposure of F0 and F1 parental animals. METHODS: Four groups of Crl:CD(SD)IGS BR rats (30/sex/group for F0 and 25/sex/group for F1) were exposed to 0, 25, 100, and 500 ppm EB for 6 hr/day for at least 70 consecutive days before mating. Inhalation exposure for the F0 and F1 females continued throughout mating, gestation through gestation day (GD) 20, and lactation days (LD) 5-21. On LD 1-4, females received EB in corn oil via oral gavage at dose levels of 26, 90, and 342 mg/kg/day (divided into three equal doses, approximately 2 hr apart), as calculated from a physiologically-based pharmacokinetic (PBPK) model to provide similar maternal blood area-under-concentration (AUC) as provided by inhalation. Pups were weaned on postnatal day (PND) 21 and exposure of the F1 generation started on PND 22. Estimates of internal exposure were determined by measuring EB concentrations in blood collected from F1 dams (4/group) and their culled pups 1 hr after the last gavage dose on PND 4. On PND 22, blood was collected from these same F1 dams and their weanlings for EB analysis 1 hr after a 6-hr inhalation exposure. The remainder of the F2 generation was not directly exposed. RESULTS: EB exposure did not affect survival or clinical observations. Male rats in the 500 ppm group in both generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, ovarian follicle counts, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, pup weights, developmental landmarks, and postnatal survival were unaffected. No adverse exposure-related macroscopic pathology was noted at any level. CONCLUSIONS: Increased liver weights were found in the animals exposed to 500 ppm. F1 maternal whole blood EB concentrations of 0.49, 3.51, or 18.28 mg/L were found 1 hr after administration of a composite oral dose of 26, 90, or 342 mg/kg/day, respectively, but no detectable EB was found in blood samples of their F2 PND 4 culled pups. F1 maternal mean whole blood EB levels 1 hr after a 6-hr inhalation exposure on postpartum day (PPD) 22 was 0.11 mg/L (25 ppm), 0.56 mg/L (100 ppm), and 11 mg/L (500 ppm). For the offspring exposed with their dams on PND 22, F2 pup blood EB concentrations ranged from 0.017-0.039 mg/L (25 ppm), 0.165-0.465 mg/L (100 ppm), and 8.82-15.74 mg/L (500 ppm). Because decreased weight gain in the 500 ppm males was transient and no histopathological changes were associated with the increased liver weights in the 500 ppm male and female groups, these changes were not considered adverse. Therefore, for parental systemic toxicity, 100 ppm was considered a NOEL and 500 ppm a NOAEL in this study. The 500 ppm exposure concentration was considered a NOAEL for F0 and F1 reproductive toxicity and offspring developmental endpoints.