John M. Waechter

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).

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

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.

Factors Affecting the Accuracy of Bisphenol A and Bisphenol A-Monoglucuronide Estimates in Mammalian Tissues and Urine Samples

ABSTRACT Bisphenol A (BPA) (CAS Number 80–05-7; EINECS Number 201–245-8) is used in the production of plastics having food contact applications. Some biomonitoring studies have reported free BPA in blood or urine of humans. Since complete first-pass metabolism of orally administered BPA to BPA-monoglucuronide (BPA-G) occurs in humans, the presence of free BPA in human specimens raises questions as to the origin and/or possible sources of the free BPA. We hypothesized that BPA-G instability during specimen collection and analysis contributes to the presence of free BPA in the biological samples. Investigation of the in vitro hydrolysis of BPA-G in blood plasma, tissue homogenates, and diluted urine from laboratory rats and in aqueous/organic solutions commonly used for extraction in BPA analyses lent support to the hypothesis of BPA-G instability as a possible source of free BPA determinations in the biological specimens. Hydrolysis of BPA-G occurred at neutral pH and room temperature in diluted urine and in rat placental or fetal tissue homogenates at room temperature. Hydrolysis of BPA-G in aqueous/organic solutions began within minutes at pH 2 and 80°C. BPA-G was degraded to an unidentified compound in a urine/water mixture or when stored in a 25/75 mixture of urine/acetonitrile at pH 9 at either 22 or 80°C. Based upon these experiments, it was concluded that methods demonstrating BPA-G stability or accounting for its instability during analysis are warranted in studies designed to measure free BPA in biological specimens.

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).

Two-Generation Reproduction Study and Immunotoxicity Screen in Rats Dosed with Phenol via the Drinking Water

This study evaluated the potential reproductive toxicity of phenol in a rat two-generation reproduction study, which included additional study endpoints, such as sperm count and motility, developmental landmarks, histological evaluation of suspect target organs (liver, kidneys, spleen, and thymus), weanling reproductive organ weights, and an immunotoxicity screening plaque assay. Phenol was administered to 30 Sprague-Dawley rats/sex/group in the drinking water at concentrations of 0, 200, 1000, or 5000 ppm. Parental (P1) animals were treated for 10 weeks prior to mating, during mating, gestation, lactation, and until sacrifice. The F1 generation (P1 offspring) was treated using a similar regimen, while the F2 generation was not treated. After mating, 10 P1 males/group were evaluated using standard clinical pathology parameters and an immunotoxicity screening plaque assay. Significant reductions in water and food consumption were observed in the 5000-ppm group in both generations; corollary reductions in body weight/body weight gain were also observed. Mating performance and fertility in both generations were similar to controls, and no adverse effects on vaginal cytology or male reproductive function were observed. Vaginal opening and preputial separation were delayed in the 5000-ppm group, and were considered to be secondary to the reduction in F1 body weight. Litter survival of both generations was reduced in the 5000-ppm group. Absolute uterus and prostate weights were decreased in the F1 generation at all dose levels; however, no underlying pathology was observed and there was no functional deficit in reproductive performance. Therefore, these findings were not considered to be adverse. No evidence of immunotoxicity was noted in the 5000-ppm group. The effects noted at the high concentration were presumed to be associated with flavor aversion to phenol in the drinking water. Based on a comprehensive examination of all parameters, the no-observable-adverse-effect level (NOAEL) for reproductive toxicity of phenol administered in drinking water to rats is 1000 ppm. The corresponding daily intake of phenol for an adult rat at the NOAEL of 1000 ppm is equivalent to about 70 mg/kg/day for males and 93 mg/kg/day for females.

Two-Week (Ten-Day) Inhalation Toxicity and Two-Week Recovery Study of Phenol Vapor in the Rat

The toxicity of phenol vapor was evaluated in male and female Fischer 344 rats (20/sex/group) via flow-past nose-only inhalation exposure. The test animals were exposed to target concentrations of 0 (air control), 0.5, 5.0, or 25 parts per million (ppm) of phenol in air for 6 hours/day, 5 days/week, for 2 weeks. High pressure liquid chromatography (HPLC) measurement of phenol test atmospheres determined mean (± standard deviation) analytical concentrations of 0.0 ± 0.0, 0.52 ± 0.078, 4.9 ± 0.57, and 25 ± 2.2 ppm, respectively. After 2 weeks of exposure, 10 test animals/sex/group were sampled for clinical chemistry and hematology parameters, and then sacrificed. Histopathological examination included the nasopharyngeal tissues, larynx, trachea, lungs with mainstem bronchi, kidney, fiver, and spleen. The remaining 10 animals/sex/group were retained for a 2-week recovery period. Recovery groups of animals were evaluated as described previously and then sacrificed. No signs of toxicity in clinical observations (including overt neurological signs), body weights, food consumption, clinical pathology, organ weights, macroscopic pathology or microscopic pathology were seen during the exposures or at either sacrifice interval. In conclusion, 2-week inhalation exposures to phenol vapor at concentrations up to and including 25 ppm did not produce any adverse effects.

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/106 splenocytes and were not statistically different from controls (1277 AFCs/106 cells). Mice treated with cyclophosphamide (20 mg/kg) on days 25 to 28 demonstrated a 94% reduction in AFC/106 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.