Registration Dossier
Registration Dossier
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EC number: 201-245-8 | CAS number: 80-05-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
- NOAEL for systemic toxicity 5 mg/kg bw/d in rats and mice; LOAEL≥50 mg/kg bw/d.
- In rats no adverse effects on oestrus cycle, fertility, litter sizes, pre- and post-natal survival, growth and development at doses at doses up to ca. 200 mg/kg bw/d. With 500 mg/kg bw/d reduced litter size (at systemic toxic dose) in the Three Generation Reproduction Study (Tyl et al., 2002).
- In mice no adverse effects on fertility, litter sizes, pre- and post-natal survival at doses up to ca. 600 mg/kg bw/day. With 600 mg/kg bw/day delayed offspring development in the Two Generation Reproduction Study (Tyl et al., 2008).
- This is supported by the recently published Clarity Core Study, in which no clear adverse treatment-induced effects related to Fertility were observed.
Comprehensive guideline studies, which are described in this chapter, show that Bisphenol A is not a selective reproductive toxicant. Effects on fertility in rats and mice occur only at high doses of Bisphenol A and are a consequence of systemic toxicity.
Link to relevant study records
- Endpoint:
- two-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Conducted under OECD guidelines, OECD good laboratory practices
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study) TG 416 Enhanced
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: (P) 6 wks; (F1) 3 wks (at beginning of direct dosing)
- Housing: Animals were individually housed upon arrival, during acclimatisation, and upon the initiation of treatment periods; by mating pairs (1 male: 1 female) during the mating period; individually as plug-positive females from gestational day 0 to birth of litters; as individual dams with litters throughout the lactational period until weaning on PND 21; and individually as selected or extra retained F1 postweanlings. Housing was in solid-bottom polypropylene cages (5" x 11.5" x 7") with stainless-steel wire bar lids (Laboratory Products, Rochelle Park, NJ). Cage bedding was Sani-Chip (PJ Murphy Forest Products, Inc. Montville, NJ).
- Diet: Ad libitum. Purina Certified Ground Rodent Chow (No. 5002, PMI Feeds, Inc., St. Louis, MO) in glass mouse feeding jars with stainless steel snap-on or screw-on caps and stainless-steel wire-mesh inserts. The supplier provided contaminant levels; these were below certified levels. Each of the 3 lots of feed used were analysed for the phytoestrogens genistein (mean +- SEM: 192+-18.6 ppm), daidzein (177+-4.0 ppm), and glycitein (45+-8.9 ppm). Feed was stored at 60-70 degrees F, with the period of use not more than 6 months from the milling date.
- Water: Water was available ad libitum in glass water bottles with Teflon-lined, plastic screw caps and stainless-steel sipper tubes. Contaminant levels of the Durham City water were measured at regular intervals by the supplier and by Balazs Analytical Services, Inc. Contaminant levels were below the maximal levels established for potable water.
- Acclimation period: Animals quarantined 1 week upon arrival.
ENVIRONMENTAL CONDITIONS
- Temperature: 66-77 degrees F (19-25 degrees C)
- Humidity: 30-70%
- Photoperiod: 12 hours dark/12 hours light - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- Feed consumption measurements were recorded for all P and F1 parental animals at least every 7 days, every time the feed was changed, throughout the prebreed exposure period. Feed consumption collection periods corresponded with the collection of the animals' weekly body weight data and were employed to calculate intake of BPA on a mg of test article/kg body weight ratio. Feed consumption during pregnancy was recorded for GD 0-7, 7-14, and 14-17; during lactation maternal feed consumption was measured for PND 0-4, 4-7, 7-14, and 14-21. Maternal feed consumption after PND 14 was confounded by contribution from the pups. Feed was changed at least weekly.
- Details on mating procedure:
- One male and one female were selected randomly within a dose group and cohabited for 14 days, or until a copulation plug was observed in the vaginal tract of the female mouse. After observation of the vaginal plug, the mating pair were separated and individually housed.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Homogeneity and stability of BPA and E2 (positive control) in the dose feed were assessed and confirmed at room temperature for at least 9 days and at least 50 days when frozen at -20 degrees C.
- Duration of treatment / exposure:
- P generation animals were dosed from approximately 6 weeks of age. Dosing occurred for 8 weeks prior to mating. Selected F1 animals were administered dosed feed ad libitum 7 days/week for at least 8 weeks prior to mating. Dosing continued until sacrifice.
- Frequency of treatment:
- Animals ate Bisphenol A-containing food ad libitum.
- Details on study schedule:
- - F1 parental animals were mated at approximately 11-13 weeks of age.
- F1 females were selected for mating on PND 18; F1 males were selected on PND 21.
- Age at mating of the mated animals in the study: 14 weeks (P); 11-13 weeks (F1) - Remarks:
- Doses / Concentrations:
0, 0.018, 0.18, 1.8, 30, 300, and 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, 600 mg/kg body weight)
Basis:
nominal in diet - No. of animals per sex per dose:
- 28
- Control animals:
- yes, plain diet
- other: exposed to E2, positive control
- Details on study design:
- At weaning on PND 21, 28 F1 male pups/group were selected for mating, while 1 male pup/litter was randomly selected to be retained, with exposures continuing for 3 months, and necropsied when F1 parental males were necropsied.
At weaning (PND 21), up to 3 F1 and F2 pups/sex/litter were randomly selected and subjected to a gross necropsy with organs weighed and retained in fixative.
P and F1 parental males were sacrificed after completion of the gestation of their F1 and F2 litters, respectively. Sacrifice of P and parental F1 females occurred on the same day as weaning of the F1 and F2 litters. - Positive control:
- Positive control animals were fed E2 at 0.5 ppm.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, morning and evening
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Recorded initally and weekly throughout mating. For P females, weights taken during gestation on GD 0, 7, 14, and 17. Dams producing litters were weighed on PND 0, 4, 7, 14, and 21.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consumption periods corresponded with collection of animals' weekly body weight data and were employed to calculate intake of BPA on a mg BPA/kg body weight ratio. During pregnancy, feed consumption was measured on GD 0-7, 7-14, and 14-17; during lactation, feed consumption was measued on PND 0-4, 4-7, 7-14, and 14-21. There was no measurement of feed consumption during the period of cohabitation. - Oestrous cyclicity (parental animals):
- Evaluated by daily vaginal smears during the last 3 weeks of the prebreed exposure periods for P and F1 females. Also, a smear was taken after euthanasia of P and F1 females to determine stage of estrus at demise.
- Sperm parameters (parental animals):
- Parameters examined in P and F1 males: enumeration of testicular homogenisation-resistant spermatid heads and calculation of daily sperm production (DSP) and efficiency of DSP, sperm motility, sperm morphology.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum on 10 pups. Excess pups were killed and discarded.
GROSS EXAMINATION OF DEAD PUPS
Yes, for external and internal abnormalities; possible cause of death was determined, when possible, for pups born or found dead.
PARAMETERS EXAMINED
The following parameters were examined in [F1/F2 ] offspring: number and sex of pups, body weights, anogenital distance, physical abnormalities.
GROSS EXAMINATION OF DEAD PUPS
Pups that were stillborn, dead, or euthanised moribund during lactation were necropsied to determine cause of death and any malformations or variations. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals (paternal + 1 retained male/litter): All surviving animals sacrified after completion of gestation period of their litters.
- Maternal animals: All surviving animals sacrificed on the same day as weaning of their litters.
GROSS NECROPSY
- Gross necropsy performed with the addition of an examination of the uterus of parental females.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed and histopathology was performed: adrenal gland (paired), brain, epididymides (males), kidneys (weighed individually), liver, ovaries with oviducts (paired, females), pituitary, spleen, prostate (ventra and dorsolateral lobes, males), seminal vesicles with coagulating glands and their fluids (paired, males), testes (paired, males), thyroid, uterus+cervix+vagina (females), mammary glands (paired, axillary- no weights taken, just histopathology, females)
-Full histopathology was performed on the organs listed above for 10 animals/sex of the P/F1 parental generations in all dose groups. - Postmortem examinations (offspring):
- SACRIFICE
- On PND 21, F1 pups remaining after selection and all F2 pups were euthanised by CO2 inhalation.
- Retained F1 males were sacrificed at the same time as the F1 parental males (at approximately 14 weeks of age).
GROSS NECROPSY
- Gross necropsy performed on all animals.
-The status of the F1 weanling testes was evaluated at necropsy after euthanasia by abdominal incision and localisation of the testes low in the abdominal cavity at the inguinal ring (undescended) or in the scrotal sacs (descended). Gross lesions also were retained in fixative (BNF or Bouin’s for testes).
HISTOPATHOLOGY / ORGAN WEIGHTS
- For F1 and F2 pups, the following organs were weighed and histopathology was performed: brain, epididymides (males), kidneys (weighed individually), liver, ovaries with oviducts (paired, females), spleen, seminal vesicles with coagulating glands and their fluids (paired, males), testes (paired, males), thymus, uterus+cervix+vagina (females). For the first randomly selected pup/sex/litter, histopathological examination was performed on the reproductive organs. For the second pup/sex/litter, histopathological examination was performed on all retained tissues (systemic and reproductive) and identified target organs (if any). All retained tissues were placed in fixative (NBF or Bouin’s for testes), and all retained tissues examined histopathologically were embedded in paraffin.
- Full histopathology was performed on the following organ listed above for 10 F1 retained male animals per dose group: adrenal gland (paired), brain, epididymides (paired), kidneys (weighed individually), liver, pituitary, spleen, prostate (ventra and dorsolateral lobes, males), seminal vesicles with coagulating glands and their fluids (paired, males), testes (paired), thyroid. - Statistics:
- Statistical unit: the P and F1 parental male, the P and F1 parental female, the pregnant P and F1 female, the retained F1 male, or the F1 and F2 litter.
Quantitative continuous data compared using either parametric ANOVA under the standard assumptions or robust regression methods.
Frequency data, such as reproductive indices (e.g., mating and fertility indices), were not transformed. All indices were analyzed by Chi-Square test for Independence for differences among treatment groups (Snedecor and Cochran, 1967).
The criterion for statistical significance was p < 0.05. If the overall ANOVA or Chi-Square p value was significant, then the appropriate pairwise comparisons were made, and those pairwise comparisons that were statistically significant are presented in the summary tables. If the overall ANOVA or Chi-Square p value was not significant, then pairwise comparisons were not made.
Acquisition of developmental landmarks (e.g., VP and PPS), as well as AGD, was also analysed by Analysis of Covariance (ANCOVA; in addition to ANOVA analysis or robust regression analysis) using body weight at acquisition or measurement as the covariate. In addition, age at acquisition of puberty (VP, PPS) was also analyzed, with the individual body weights on PND 21 for females and on PND 30 for males as the covariate.
Correlated data (e.g., body and organ weights at necropsy of pups on PND 21, with more than 1 pup/sex/litter) were analysed using GEE techniques (Zeger and Liang, 1986) in the SUDAAN software package (RTI, 2001).
A test for statistical outliers (SAS Institute, Inc., 1999) was performed on parental body weights, feed consumption (in g/day), and F0 and F1 adult, F1 and F2 weanling, and F1 retained male organ weights. When examination of pertinent study data did not provide a plausible, biologically-sound reason for inclusion of the data flagged as “outlier,” the data were excluded from summarisation and analysis and were designated as outliers. - Reproductive indices:
- Mating, fertility, pregnancy, gestational indices, precoital intervals, epididymal sperm concentration, percent motile sperm, percent abnormal sperm, testicular homogenization-resistant spermatid head counts, daily sperm production, estrus cycle.
- Offspring viability indices:
- Number of live litters on PND 0, live birth index, number of total/live/dead pups, sex ratio (% males) per litter on PND 0.
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, treatment-related
- Dose descriptor:
- NOEL
- Remarks:
- Systemic
- Effect level:
- 30 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Based on centrilobular hypertrophy in adult males at 300 ppm
- Remarks on result:
- other: Generation: F0 and F1 (migrated information)
- Dose descriptor:
- NOAEL
- Remarks:
- Systemic
- Effect level:
- 300 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: The EU RAR 2003 concluded that the NOAEL is 300 ppm based on reduced body weight and effects on liver and kidney weight and histopathology at 3500 ppm
- Remarks on result:
- other: Generation: F0 and F1 (migrated information)
- Dose descriptor:
- NOEL
- Remarks:
- fertility
- Effect level:
- 3 500 ppm (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: No effects on fertility, reproductive organ weights and histopathology or sperm production were observed at any dose tested
- Remarks on result:
- other: Generation: F0 and F1 (migrated information)
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 300 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- organ weights and organ / body weight ratios
- Dose descriptor:
- NOAEL
- Generation:
- other: F2 Generation
- Effect level:
- 300 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- organ weights and organ / body weight ratios
- Reproductive effects observed:
- not specified
- Conclusions:
- The authors concluded that BPA is not a selective reproductive or developmental toxicant in mice.
- Executive summary:
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 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 non-monotonic dose response curves for any parameter. The systemic NOEL was 30 ppm BPA (approximately 5 mg/kg-day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg-day).
- Endpoint:
- three-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study designed and conducted using an internationally accepted reproductive toxicity protocol under Good Laboratory Practice (GLP) regulations (U.S. EPA, 1989).
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 837.3800, 1998
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Housing: During the acclimation period and upon initiation of treatment, animals were housed individually in stainless-steel hanging cages. Mating pairs and sperm/plug positive females (from gestation day 0 to weaning of litters on postnatal day 21) were housed in solid-bottom polypropylene cages (Laboratory Products, Rochelle Park, NJ) with Sani-Chip cage bedding (PJ Murphy Forest Products, Inc., Montville, NJ)
- Diet (e.g. ad libitum): ad libitum Purina Certified Ground Rodent Chow (No. 5002, PMI Feeds, Inc., St. Louis, MO)
- Water (e.g. ad libitum): ad libitum by automatic water system or by glass water bottles
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
-
DIET PREPARATION
- Appropriate amounts were mixed with Purina Certified Rodent Chow (No. 5002, PMI Feeds): BPA was dissoved in a fixed volume of acetone and added to a premix feed aliquot. Stability of formulations at 15 ppb and 7500 and 10,000 ppm was confirmed at -20 degrees C for 50 days and at room temperature in open containers to simulate cageside conditions for at least 9 days. Homogeneity was confirmed by assaying one sample each at 15 ppb and 7500 and 10,000 ppm in triplicate from 3 locations within the blender.
- Storage temperature of food: -20 degrees C. - Details on mating procedure:
- - Male/female ratio per cage: 1 male/1 female per cage
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm positive
- After successful mating, each dam was caged in a solid-bottom polypropylene cage with Sani-Chip cage bedding until weaning of litters on postnatal day 21. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Aliquots from all dosed feed preparations were analysed for BPA concentration and diet was only used if within +/- 15% of the nominal. Feed analyses were performed using negative ion chemical ionisation gas chromatography-mass spectrometry.
- Duration of treatment / exposure:
- P: Animals were exposed for 10 weeks before mating, for 2 weeks during mating, and during 3 weeks of gestation; dams were also exposed during 3 weeks of lactation.
F1/F2: Animals were exposed in utero and during lactation. Direct dietary exposure began after weaning and continued for 10 weeks, then animals were dosed during 2 weeks of mating, 3 weeks of gestataion, and dams for 3 weeks of lactation.
F3: Animals were exposed in utero and during lactation. Direct dietary exposure began after weaning and continued for 10 weeks. - Frequency of treatment:
- Bisphenol A in feed was available ad libitum to animals.
- Details on study schedule:
- - F1 parental animals were not mated until 10 weeks after selection from the F1 litters.
- Selection of animals to mate from the F1 generation was made when F1 pups were 21 days of age.
- Age at mating of all animals in the study: 13 weeks. - Remarks:
- Doses / Concentrations:
0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm (0, 0.001, 0.02, 0.3, 5, 50, 500 mg/kg body weight)
Basis:
nominal in diet - No. of animals per sex per dose:
- 30 males/30 females per dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected to encompass the ranges of low oral BPA doses at which male mouse offspring prostate weights were reported to be significantly increased, doses at which testis weight and efficiency of daily sperm production (DSP) were reported to be significantly reduced in rat offspring, and doses that provide a maximum tolerated dose (MTD) expected to exceed metabolic capability of the adult liver and to produce reductions in body weight or other indications of systemic toxicity. Target dietary concentrations were based on the chosen BPA intakes in mg/kg-day for the female rats.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Oestrous cyclicity (parental animals):
- Estrous cyclicity was evaluated for the last three weeks of the pre-breed period for the P, F1, and F2 parental animals, and in the 3 week post-weaning period for F3 retained females. Stage of estrus was also determined at necropsy for P, F1, and F2 parental and F3 retained females.
- Sperm parameters (parental animals):
- For all parental generations, parameters examined included: epididymal sperm number, epididymal sperm motility, epididymal sperm morphology, testicular homogenization-resistant spermatid head counts, daily sperm production, efficiency of daily sperm production.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 10 pups/litter (equivalent number per sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1/F2/F3 offspring: number of live pups per litter, day 4 survival index, lactational index, anogenital distance, body weight/litter, age at vaginal patency, body weight at vaginal patency, number of nipples/areolae per male pup, age at preputial separation, body weight at preputial separation
- Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: Parental males were sacrificed after delivery of their litters.
- Maternal animals: Maternal animals were sacrificed after weaning of their pups.
HISTOPATHOLOGY / ORGAN WEIGHTS
- For adult males, histopathology was performed on the following organs: adrenal glands, coagulating glands, epididymis, kidneys, liver, pitutiary gland, preputial gland, prostate, seminal vesicles, spleen, testis, and urinary bladder (one animal in the P generation only). Organ weights were measured for the following organs: liver, paired kidneys, paired testes, paired epididymides, prostate, preputial gland, and seminal vesicles.
- For adult females (all generations), histopathology was done on the following organs: adrenal glands, cervix, kidneys, liver, ovary, oviduct, pituitary, spleen, urinary bladder (one animal in the P generation only), uterus, and vagina. Enumeration of ovarian primordial follicles was performed for high-dose females. Organ weights were measured for the following organs: liver, paired kidneys, paired ovaries, and uterus. - Postmortem examinations (offspring):
- SACRIFICE
- The F1/F2 offspring not selected as parental animals and all F3 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Up to 3 pups/sex/litter were randomly selected, necropsied, and selected organs were weighed.
ORGAN WEIGHTS
Organ weights were measured for weanling animals, but no details are given in the text as to which organs were weighed. - Statistics:
- The unit of comparison was the individual animal or the litter, as appropriate. Data from the cohorts were combined for summarisation and statistical analyses.
Quantitative continuous data were compared among the 6 treatment groups and the vehicle control group. For the litter-derived percentage data, ANOVA was weighted according to litter size. GLM analysis was used to determine the significance of the dose-response relationship and to determine whether significant dosage effects had occurred for selected measures. A one-tailed test was used for all pairwise comparisons to the vehicle control group, except two-tailed tests were used for parental and pup body and organ weights, feed consumption, percent males per litter, and AGD per sex per litter.
Nonparametric tests for continuous data were used to determine if significant differences were present among the groups or to identify significant dose-response trends. Frequency data were analyzed for differences among treatment groups and for pairwise comparisons.
For acquisition of developmental landmarks and AGD, ANOVA and ANCOVA, with body weight (at birth, PND 0, for AGD; at acquisition of puberty and on study day [SD] 7 for females [VP] and SD 14 for males [PPS]) as the covariate, were used for pairwise comparisons. For correlated data, SUDAAN software was used for analysis of overall significance, presence of trend, and pairwise comparisons to the control group values. For all statistical tests, the significance limit of 0.05 (one- or two-tailed) was used as the criterion for significance.
A test for statistical outliers was performed on parental body weights and feed consumption (in g/day) and parental and weanling offspring organ weights at necropsy. If examination of pertinent study data did not provide a plausible biologically sound reason for inclusion of the data flagged as “outlier,” the data were excluded from summarisation and analysis and were designated as outliers. - Reproductive indices:
- Estrous cycle length, precoital interval, gestational length, number of implantation sites/dam, number of pups/litter, percent postimplantation loss/litter, paired ovarian primordial follicle counts, epididymal sperm concentration
- Offspring viability indices:
- Number of live pups/litter, day 4 survival index, number surviving on day 21.
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Reproductive performance:
- effects observed, treatment-related
- Dose descriptor:
- NOEL
- Remarks:
- systemic
- Effect level:
- 75 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Based on body weight reduction in adult females at 750 ppm
- Remarks on result:
- other: Generation: F0, F1, F2 (migrated information)
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 750 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: The EU RAR 2003 concluded that the NOAEL for systemic toxicity in this study is 50 mg/kg/day, based on consistent and significant reductions in body weight gain in both sexes and renal tubule degeneration in females only at 7500 ppm
- Remarks on result:
- other: Generation: F0, F1 and F2 (migrated information)
- Dose descriptor:
- NOAEL
- Remarks:
- reproductive
- Effect level:
- 750 ppm
- Sex:
- male/female
- Basis for effect level:
- other: Total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD)
- Remarks on result:
- other: Generation: F1 and F2 (migrated information)
- Clinical signs:
- not specified
- Mortality / viability:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- not specified
- Histopathological findings:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 750 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- organ weights and organ / body weight ratios
- Dose descriptor:
- NOAEL
- Generation:
- other: F2 generation
- Effect level:
- 750 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- organ weights and organ / body weight ratios
- Dose descriptor:
- NOAEL
- Generation:
- other: F3 generation
- Effect level:
- 750 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- organ weights and organ / body weight ratios
- Reproductive effects observed:
- not specified
- Conclusions:
- The authors concluded that based on the results of this study, BPA should not be considered a selective reproductive toxicant.
- Executive summary:
Bisphenol A (BPA) was administered 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) in the diet ad libitum to 30 CD 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.
- Endpoint:
- two-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted in compliance with regulatory guidelines and in accordance with the principles for Good Laboratory Practice (GLP).
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan, Inc. (Yokohama, Japan)
- Age at study initiation: 5 weeks (males); 10 weeks (females)
- Housing: Animals were housed individually (except during the acclimation, mating and nursing periods) in suspended stainless steel cages. From gestation day 17 to the day of weaning, individual dams and litters were reared using wood chips as bedding (White Flake, Charles River Japan, Inc.)
- Diet: Ad libitum gamma-irradiated basal diet (CRF-1, Oriental Yeast Col, Ltd., Tokyo, Japan). Contaminant levels of BPA were below the detection limit.
- Water: Ad libitum filtered tap water. Contaminant levels of BPA were below the detection limit.
- Acclimation period: 8 days (males); 15 days (females)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 50-60
- Photoperiod: 12 hrs dark / 12 hrs light) - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Rats were dosed once daily by gastric intubation with BPA dissolved in distilled water for 10 weeks (males) or 2 weeks (females) prior to the mating period. The volume of each dose was adjusted to 2 ml/kg body weight based on the latest body weight measurement. Control rats received distilled water only. The stability of test formulations in a dark, cool place was confirmed for up to 21 days.
- Details on mating procedure:
- The vaginal smears of each female rat were recorded daily for 2 weeks, and only rats showing repeated 4- or 5-day estrous cycles were used in the experiment. Each female was mated with a male rat of the same dosage group. The mating periods for P and F1 animals were 2 and 3 weeks, respectively. The presence of sperm in the vaginal smear and/or a vaginal plug was considered evidence of successful mating. At weaning on PND 22, F1 weanlings (25 per sex per group) were randomly selected to serve as parent animals for the F2 generation. For each group, one or two male and female F1 weanlings were selected from each P dam. F1 animals that did not mate during the 3-week mating period wer reassigned to an untreated partner and cohabited. For F1 matings, cohabitation of siblings was avoided.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- During use, formulations of BPA were maintained in a dark, cool place for no more than 21 days, and were 90-105.3% of the target concentration.
- Duration of treatment / exposure:
- P: Rats were exposed for 10 weeks (males) or 2 weeks (females) prior to the mating period. Each female was mated with a male rat of the same dosage group, with exposure continuing throughout the mating period. Exposure was also continued throughout gestation and lactation.
F1: F1 animals were exposed in utero and during lactation, then direct exposure began on PND 23 and continued for 10 weeks prior to their mating period, as well as during mating, gestation, and lactation periods.
F2: F2 animals were exposed in utero and during lactation, then direct exposure began on PND 22 for 4 weeks (males) or 11 weeks (females). - Frequency of treatment:
- Once daily during treatment periods
- Details on study schedule:
- - F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: 13 weeks - Remarks:
- Doses / Concentrations:
0, 0.2, 2, 20, and 200 µg/kg
Basis:
actual ingested - No. of animals per sex per dose:
- 25
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were determined based on previous studies in which significant changes in reproductive parameters of offspring were found after maternal feeding of BPA at 2 and 20 ug/kg on days 11 to 17 of pregnancy in mice.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes - Oestrous cyclicity (parental animals):
- Daily vaginal lavage samples of each P and F1 female were evaluated for estrous cyclicity throughout the 2-week pre-cohabitation period and during cohabitation until evidence of copulation was detected. In F2 females, vaginal smears were also evaluated for 2 weeks at 9 to 11 weeks of age.
- Sperm parameters (parental animals):
- Parameters examined in all P and F1 male parental generations on the day of scheduled terminal sacrifice: epididymis weight, sperm count in epididymides, sperm motility, and sperm morphology.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight, weight gain, and developmental landmarks (pinna detachment, incisor eruption, eye opening, testes descent, surface righting, negative geotaxis reflex, mid-air righting reflex, day of preputial separation or vaginal opening, and anogenital distance). - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals after the mating period.
- Maternal animals: All surviving animals after weaning of pups.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.
HISTOPATHOLOGY / ORGAN WEIGHTS
- Brain, thyroid, thymus, liver, spleen, heart, lung, uterus, prostate, testes, kidneys, adrenals, ovaries, and epididymes weights were recorded. Weights of the pituitary and seminal vesicle were measured after fixation.
- Ovary, uterus, vagina, testis, epididymis, seminal vesicle, coagulating gland, prostate, hypothalamus, pituitary, liver, adrenal, thyroid, and mammary gland from P and F1 adults were prepared for microscopic examination. Histopathologic evaluations were also conducted on the hearts of all male P generation adults, on kidneys and lungs of control and highest dose group F1 adult males, and on the seminal vesicles and coagulating glands of male F2 adults in all groups. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 22 days of age.
- The F2 offspring not chosen for further study were sacrificed at 22 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.
HISTOPATHOLOGY / ORGAN WEIGHTS
- Brain, thyroid, thymus, liver, spleen, heart, lung, uterus, prostate, testes, kidneys, adrenals, ovaries, and epididymes weights were recorded in one male and one female F1 and F2 animal selected from each dam. In these F2 male weanlings, histopathologic examinations were performed on the seminal vesicle and coagulating gland in all groups. - Statistics:
- Statistical analysis of offspring before weaning was carried out using the litter as the experimental unit.
The incidence of females with normal estrous cycles, copulation and fertility, gestation indices, and the neonatal sex ratio were analysed by Chi-square test or Fisher's exact test.
All other parameters were analysed for statistical significance as follows: Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent variances at the 5% level of significance. If the variances were equivalent, the groups were compared by one-way ANOVA. If significant differences were found, Dunnett's multiple comparison test was performed. If the groups did not have equivalent variances, the Kruskal-Wallis test was used to assess the overall effects. Whenever significant differences were noted, pair-wise comparisons were made by Mann-Whitney U-test.
The 0.05 level of probability was used as the criterion for significance. - Reproductive indices:
- Estrous cycle length, precoital interval, copulation index, fertility index, gestation index, gestation length, number of implantation sites/dam, delivery index, epididymal sperm counts.
- Offspring viability indices:
- Number of pups delivered, viability index during lactation.
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 0.2 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicologically significant effects were observed at any dose studied.
- Remarks on result:
- other: Generation: P, F1, F2 (migrated information)
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 0.2 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicologically significant effects were observed at any dose studied.
- Dose descriptor:
- NOAEL
- Generation:
- other: F2 generation
- Effect level:
- 0.2 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicologically significant effects were observed at any dose studied.
- Reproductive effects observed:
- not specified
- Conclusions:
- The authors concluded that, under the conditions of this study, no adverse effects of BPA on reproduction or development were detected.
- Executive summary:
In this rat two-generation reproductive study, groups of 25 male and 25 female rats were given BPA at 0.2, 2, 20, or 200 ug/kg-day by gastric intubation throughout the study beginning at the onset of a 10- and 2 -week premating period, in P generation males and females, respectively, and continuing through the mating, gestation, and lactation periods. No BPA-related clinical signs or effects on body weight or food consumption were observed in any generation. There were no BPA-related changes in surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna detachment, incisor eruption, eye opening, testes descent, preputial separation, or vaginal opening in F1 and F2 generations, or behaviour in the open field or water-filled multiple T-maze in the F1 generation. No BPA-related changes in estrous cyclicity, copulation index, fertility index, number of implantations, gestation length, litter size, pup weight, pup sex ratio, pup viability, or other functional reproductive measures were noted in any generation. A few statistically significant changes in the AGD per cube root of body weight ratio were found at 0.2 and 20 ug/kg in F1 males, at 2, 20, and 200 ug/kg in F1 females, and at 20 and 200 ug/kg in F2 females; however, the changes in AGD were consistently small (within 5% of control values), and no continuous changes in the AGD or AGD per cube root of body weight were detected. There were no BPA-related changes in epididymal sperm counts or motility in P and F1 males. No BPA-related necropsy findings or effects on organ weight including the reproductive organs were found in any generation. Histopathologic examinations revealed no evidence of BPA-related changes in any organs including the reproductive organs of both sexes. The data indicate that oral doses of BPA between 0.2 and 200 ug/kg over two generations did not cause significant compound-related changes in reproductive or developmental parameters in rats.
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP; exceeding guideline requirements; Research Conducted at FDA Laboratory in Jefferson, AR
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Bisphenol A was administered by oral gavage from gestation day 6 through the start of labor and then directly to pups from postnatal day (PND) 1 (day of birth = PND 0) until termination at PND 90 ± 5 to Sprague-Dawley rats from the NCTR breeding colony (Sprague-Dawley/CD23/NCTR BR). Bisphenol A doses were 2.5, 8, 25, 80, 260, 840, 2,700, 100,000, and 300,000 μg/kg body weight (bw)/day.
Vehicle (0.3% carboxymethylcellulose) and naïve control groups were included to assess any effects of the gavage procedure on the endpoints measured. Two doses (0.5 and 5.0 μg/kg bw/day) of the synthetic estrogenic substance ethinyl estradiol (EE2) were also included.
The litter was the unit of statistical analysis and the target litter number was 20 per dose group (actual n = 18 – 23). Rats were fed soy- and alfalfa-free diet and caged in polysulfone cages with hardwood chip bedding, glass water bottles with food-grade silicone stoppers. Prior to mating female rats were randomized to treatment groups stratified by body weight to give approximately equivalent mean body weights in each group. During mating females were examined daily for presence of an in situ vaginal plug or sperm-positive smear.
Data collected included body weights, weekly feed consumption, litter parameters, anogenital distances at PND 1 and PND 90, measures of sexual development (vaginal opening and time to first estrus for females; nipple retention, testicular descent, and preputial separation for males), vaginal cytology, clinical chemistry, organ weights, and histopathology. Vaginal cytologies were collected from PND 69 until termination in animals utilized for histopathology; the cytology data were used to attempt to terminate the females in estrus. A subset of females was also removed from dosing at PND 90 ± 5 and assessed for cyclicity from PND 150 to PND 170. For clinical chemistry, blood was taken from subsets of pups at PND 15, PND 80 and at terminal necropsy on PND 90 ± 5. Sperm motility, testicular spermatid head count, caudal epididymal sperm count, and sperm morphology were assessed at terminal necropsy. Organ weights collected at terminal necropsy included adrenals, brain, dorsolateral and ventral prostate, epididymides, heart, kidneys, liver, ovaries, pituitary (after fixation), seminal vesicles with coagulating gland, spleen, testes, thymus, thyroid (after fixation), uterus, epididymal, ovarian and parametrial (combined), and retroperitoneal fat pads. Organs evaluated microscopically included adrenals, aorta (thoracic), bone marrow (femur), brain, right epididymis, heart, kidneys, liver, 5th left mammary gland (inguinal) from both sexes, ovaries, oviduct, pancreas, pituitary, prostate (dorsolateral and ventral), seminal vesicles with coagulating gland, spleen, right testis, thymus, thyroid, uterus, and vagina. - GLP compliance:
- yes
- Species:
- rat
- Strain:
- other: Sprague-Dawley/CD23/NCTR BR
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Details on mating procedure:
- Males were placed in wire-bottomed breeding cages for acclimation approximately 48 h prior to introduction of the female. The breeding pairs were housed together and the females were examined daily until evidence of mating had occurred or for 14 days, whichever occurred first. Either the presence of an in situ copulation plug or sperm in the vaginal smear was considered evidence of mating [gestation day (GD) 0] and triggered separation of the breeding pair. The males were removed from the study and euthanized after a successful mating and the female was placed in a separate solid-bottomed polysulfone cage with hardwood chip bedding.
- Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- Bisphenol A was administered by oral gavage from gestation day 6 through the start of labor and then directly to pups from postnatal day (PND) 1 (day of birth = PND 0) until termination at PND 90 ± 5
- Frequency of treatment:
- once a day
- Remarks:
- Doses / Concentrations:
2.5, 8, 25, 80, 260, 840, 2,700 µg/kg/day
Basis:
analytical conc. - Remarks:
- Doses / Concentrations:
100,000, and 300,000 µg/kg/day
Basis:
analytical conc. - No. of animals per sex per dose:
- The litter was the unit of statistical analysis and the target litter number was 20 per dose group (actual n = 18 – 23).
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- Statistics:
- Corrigendum published in TOXICOLOGICAL SCIENCES, 153(1), 2016, 212: Although details of the statistical analyses performed are included in the Supplementary Materials provided with this article (Toxicological Sciences 139 (1), 174–197, 2014), it should have been noted that the pairwise comparisons of dose groups to the vehicle control were not corrected for multiple comparisons for the data reported in Tables 4 and 7–9. By convention, histopathologydata are not corrected in National Toxicology Program studies.
- Dose descriptor:
- NOAEL
- Effect level:
- 2.7 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: Generation: all genererations (migrated information)
- Dose descriptor:
- LOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- female
- Basis for effect level:
- other: see 'Remark'
- Remarks on result:
- other: Generation: P and F1 (migrated information)
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 2.7 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: next higher dose 100 mg/kg/dya; see results for more details
- Reproductive effects observed:
- not specified
- Executive summary:
Under the conditions of this study, BPA had clear adverse effects at doses of 100,000 and 300,000 µg/kg bw/day, with the majority of these effects observed in females. In the study defined “low BPA” dose range of 2.5–2700 g/kg bw/day, which was the primary focus of this study, potential effects could not be clearly linked to treatment as they were observed sporadically across the dose groups and did not occur in a consistent grouping across organs as did effects of EE2 (0.5 and 5.0 µg/kg bw/day) or “high BPA” (100,000 and 300,000 µg/kg bw/day).
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at EPA Laboratory in Research Triangle Park, NC
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- Endpoints investigated: Body weight at PND2, AGD at PND2, Areolae/nipple retention at PND13, Body weight in adulthood, Nipple retention in adulthood, Glans penis weight, Ventral prostate weight, Seminal vesicle weight, Paired testes weight, Paired epididymides weight, LABC muscle weight, Cowper’s gland weight,
Liver weight, Kidney weight, Paired adrenal gland weight, Spleen weight, Heart weight, Paired lung weight, Brain weight, Pituitary weight, Testosterone, serum levels, LH, serum levels, PRL, serum levels, Estradiol, serum levels, Corticosterone serum levels, Thyroxine levels serum levels, Sperm number per epididymis, Reproductive tract malformations, Histopathology of reproductive tissues. - GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Long-Evans
- Sex:
- male
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on mating procedure:
- Adult female LE rats (Charles River Laboratories, Raleigh, NC) of approximately 90 days of age were mated by the supplier and shipped on GD2. Mating was confirmed by sperm presence in vaginal smears (day of sperm plug positive = GD1).
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- GD7 - PND18
- Frequency of treatment:
- Daily
- Details on study schedule:
- Male offspring were euthanized beginning at PND150.
- Remarks:
- Doses / Concentrations:
2, 20, and 200 μg/kg/day
Basis:
actual ingested - No. of animals per sex per dose:
- The total number of individual males and litters in Block 1a by treatment were as follows: vehicle control—20, 8; BPA (2 lg/kg/day—18, 7; 20 lg/kg/day—18, 7; 200 lg/kg/day—20, 9); and EE (0.05 lg/kg/day—16, 6; 0.5 lg/kg/day—20, 8; 5 lg/kg/day—20, 7; and 50 lg/kg/day—20, 7). The total number of individual males and litters, respectively, necropsied in Block 1b by treatment were as follows: vehicle control—11, 6; and EE (5 lg/kg/day—9, 7; and 50 lg/kg/day—3, 2). Finally, the total number of individual males and litters, respectively, necropsied in Block 2 by treatment were as follows: vehicle control—35, 10; BPA (20 lg/kg/day—16, 6; 200 lg/kg/day—34, 9; and EE (0.05 lg/kg/day—17, 6; 0.15 lg/ kg/day—18, 6; 0.5 lg/kg/day—26, 7; 1.5 lg/kg/day—28, 9; 5 lg/kg/day—41, 8; 15 lg/kg/day—23, 6; and 50 lg/kg/day—1, 1).
- Control animals:
- yes, concurrent vehicle
- other: Positive control groups: 0.05–50 μg/kg/day EE2
- Dose descriptor:
- NOAEL
- Effect level:
- 0.2 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no adverse effect reported
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 0.2 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Treatment with 2, 20, or 200 μg Bisphenol A/kg/day or EE at 0.05–1.5 μg/kg/day did not significantly affect any male endpoint in the current study.
- Reproductive effects observed:
- not specified
- Executive summary:
Many chemicals released into the environment are capable of disrupting normal sex steroid balance, including the oral contraceptive ethinyl estradiol (EE) and the plastic monomer bisphenol A (BPA). EE and BPA are reported to impair reproductive organ development in laboratory animals; however, effects of lower doses of these chemicals have been debated. The goal of the current study was to determine whether relatively low oral doses of EE or BPA would alter male reproductive morphology and associated hormone levels of Long Evans hooded rat. Dams were gavaged with corn oil vehicle, EE (0.05–50 μg/kg/day) or BPA (2, 20, and 200 μg/kg/day) during pregnancy through lactation from gestational day 7 to postnatal day (PND) 18. Anogenital distance was measured at PND2 and nipple retention was measured at PND14 in male pups. Male offspring were euthanized beginning at PND150, and sera and organs were collected for analyses. Adult body weight was significantly decreased in males exposed to 50 μg EE/kg/day. Developmental EE exposure reduced androgen-dependent tissue weights in a dose-dependent fashion; for example, seminal vesicle and paired testes weights were reduced with ≥ 5 μg EE/kg/day. Epididymal sperm counts were also significantly decreased with 50 μg EE/kg/day. In contrast, treatment with 2, 20, or 200 μg BPA/kg/day or EE at 0.05–1.5 μg/kg/day did not significantly affect any male endpoint in the current study. These results demonstrate that developmental exposure to oral micromolar doses of EE can permanently disrupt the reproductive tract of the male rat.
- Endpoint:
- reproductive toxicity, other
- Remarks:
- A study to characterize and evaluate the toxicologic potential of BPA following perinatal only or chronic exposure (see chapters 7.5.1 and 7.7) in rats under the conditions of a chronic, extended-dose response design.
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- according to guideline
- Guideline:
- other: guideline-compliant research standard conducted at the FDA designed by NCTR and NIEHS scientists
- Principles of method if other than guideline:
- Mating was conducted prior to the start of dosing on GD 6, and thus breeding success was not an endpoint for analysis in this study. Because pups within litter and sex were assigned at weaning to different dosing arms and sacrifice times, litter correlation was not a consideration for endpoints evaluated after weaning in this study.Therefore the in utero exposure is described in details in chapter 7.8.2 and the perinatal and chronic exposure in chapters 7.5.1 and 7.7.
- GLP compliance:
- yes
- Limit test:
- no
- Justification for study design:
- The core GLP study was designed to characterize and evaluate the toxicologic potential of BPA following perinatal only (stop-dose study arm) or chronic exposure (continuous-arm study; see also chapters 7.7 and 7.5.1) in rats under the conditions of a chronic, extended-dose response design. Sacrifice was conducted at one year of age (PND 365 ± 20) and the terminal sacrifice was conducted at two years of age (PND 730 ± 20). The interim (one-year) sacrifice group was included to allow evaluation of long-term exposure effects with less confounding due to background lesions of aging than would be expected at two years. Organ weights and clinical pathology (clinical chemistry and hematology) data were also collected from interim, but not terminal, sacrifice animals given the expected increased moribund removal later in the study and the level of variability in older animals due to age-related disease. Because the sensitivity of the animal model to exogenous estrogen was considered important, EE2 was in cluded for this purpose rather than necessarily to compare effects of the two agents. PND 1 pups (in utero exposure is described in chapter 7.8.2) were weighed and daily dosed until weaning at PND 21. After weaning the pubs were assigned either to the chronic study (continuousdose
arm and stop-dose study arm) or to the hypothesis-driven studies of academic investigators. In the chronic study postweaning animals were dosed daily throughout the study (termination at 1 or 2 years) = continuous-dose arm) or were held without further treatment until termination (1 or 2 years). This stop-dose study arm was included to assess any effects that were due to early exposure only. - Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The two high BPA dosing suspensions were mixed by directly adding BPA solid to the vehicle with sonication, and the suspensions were stirred constantly. The three low BPA and the two EE2 dosing
solutions were mixed by serial dilutions of stock solutions. - Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- CD23/NctrBR
- Details on species / strain selection:
- The animal model used in these studies was the Sprague-Dawley rat maintained at NCTR. This colony had its origins in the late 1970s from Charles River Sprague-Dawley founders and has been used in toxicology studies with hormonally active agents for over a decade at NCTR13-20.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- see chapter 7.5.1 and 7.8.2
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Details on exposure:
- with modified Hamilton Microlab® ML511C programmable 115 V pumps. Dosing containers were constantly stirred and dose volume calculation and dispensing were automate. For animals younger than PND 5, the gavage needle was inserted to the opening of the esophagus, but did not enter the esophagus.
- Details on mating procedure:
- not applicable (s. 7.8.2)
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- At the start of the study and approximately every 8–10 weeks over the course of the study, all dose level preparations were assayed by the Chemistry Support Group prior to delivery to the animal rooms and certified to be within 10% of the target concentration with a % CV of ≤10%. In addition, at intervals spaced 4-7 months apart, BPA and EE2 dosing preparations from the animal rooms were
assayed at the end of their use to verify the dose concentrations. - Duration of treatment / exposure:
- F1 in utero exposure: P0 dams on GD 6 (GD 0 = sperm- or plug-positive day) and continued until initiation of parturition (GD 21)
F1 from PND1 to PND21 (stop-dose arm study)
F1 from PND 1 to 1 year or 2 years (continuous-dose arm study) - Frequency of treatment:
- once a day
- Details on study schedule:
- see 7.5.1 and 7.8.2
- Dose / conc.:
- 2.5 other: µg/kg bw/day
- Dose / conc.:
- 25 other: µg/kg bw/day
- Dose / conc.:
- 250 other: µg/kg bw/day
- Dose / conc.:
- 2 500 other: µg/kg bw/day
- Dose / conc.:
- 25 000 other: µg/kg bw/day
- No. of animals per sex per dose:
- 70-78 dams/dose from which data were collected
At weaning, up to a maximum of three pups/sex/litter were assigned to the chronic study. Same-sex littermates were not assigned to the same combination of study dose arm and time of sacrifice so that litter of origin was not a factor to be considered in the statistical analysis of endpoints collected after weaning. 20 to 26 pups/sex/BPA dose group were assigned to the one-year interim continuous-dose a ssessment
19 to 22 pups/sex/BPA dose group were assigned to the one-year interim stop-dose assessment 26 to 50 pups/sex/BPA dose group/dose arm were assigned to the two year continuous-dose assessment
46 to 50 pups/sex/BPA dose group/dose arm were assigned to the two year study stop-dose assessment
26 pups/sex/EE2 dose group were assignedto the one-year interim continuous-dose assessment
26 pups/sex/EE2 dose group were assigned to the two-year study continuous-dose assessment
The remaining pups from those litters with more than three same-sex pups were assigned to thehypothesis-driven studies of academic investigators. The reason that stop-dose EE2 groups were not included in the study was solely an animal facility space and resource consideration, given the number of animals that needed to be provided and housed for both this study and the NIEHS-funded academic CLARITY-BPA grantee studies. - Control animals:
- yes, concurrent vehicle
- Details on study design:
- see chapters 7.5.1 and 7.8.2
- Positive control:
- ethinyl estradiol (EE2) (0.05 and 0.5 μg/kg bw/day)
- Parental animals: Observations and examinations:
- see chapter 7.8.2
- Oestrous cyclicity (parental animals):
- not examined
- Sperm parameters (parental animals):
- not examined
- Litter observations:
- see chapter 7.8.2
- Postmortem examinations (parental animals):
- see chapter 7.8.2
- Postmortem examinations (offspring):
- see chapter 7.5.1 and 7.7
- Statistics:
- The full statistical analysis reports for all protocol-specified endpoints, including detailed methods and results for each analysis, including the omnibus tests, are found in Supplemental Appendices of the study report. The pairwise comparisons to the vehicle control and trend tests are the comparisons of interest that are presented in the tables in study report. The statistical methodology for each endpoint is summarized in the study report. Statistical comparisons were conducted within sex and, for data collected after weaning, within dosing arm (continuous-dose or stop-dose). For pairwise comparisons, the five BPA dose groups were compared to the vehicle control group. Similarly, the two EE2 reference estrogen dose groups were compared to the vehicle control. Tests were conducted at the 0.05 significance level and, in most cases, were two-sided. Exceptions were one-sided tests for the pairwise comparisons of histopathology lesion incidence and severity to vehicle controls and trend tests for abnormal estrous cycles. Although a p-value of <0.05 was used to flag a result as significant, the actual p-values are included in some of the tables in the study report and in all the statistical report appendices (Supplemental Appendices of the study report) to aid in the further evaluation of the statistical and biological significance of each result. Trend tests for treatment effect (either increased or decreased relative to vehicle control) with increasing dose were conducted only for vehicle control and BPA treatment groups, except for non-neoplastic and neoplastic lesions, where trend tests were also conducted within the vehicle control and EE2 groups. Because pups within litter and sex were assigned at weaning to different dosing arms and sacrifice times, litter correlation was not a consideration for endpoints evaluated after weaning in this study.
- Reproductive indices:
- not applicable
- Offspring viability indices:
- not applicable
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- 25 000 other: µg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- Critical effects observed:
- not specified
- Clinical signs:
- not specified
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- for details see chapter 7.5.1
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- for details see chapter 7.5.1
- Food consumption and compound intake (if feeding study):
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- for details see chapter 7.5.1
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- for details see chapter 7.5.1
- Urinalysis findings:
- not examined
- Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- for details see chapter 7.5.1
- Gross pathological findings:
- not specified
- Histopathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- for details see chapter 7.5.1
- Description (incidence and severity):
- - Vaginal opening from PND 22:
There were no treatment-related effects on these endpoints for any dose of either compound. No treatment effects were observed in the stop-dose BPA groups. Body weight at vaginal opening could not be analyzed for the stop-dose animals because weight at vaginal opening was not recorded for many of the animals due to a technical error. While no formal analysis was conducted comparing
vehicle controls in the continuous- and stop-dose arms, the mean vaginal opening date appears to be later regardless of BPA treatment in the stop-dose groups.
- Vaginal Cytology Estrous (Cycle Analysis at approx. 16 weeks of Age):
Continuous-Dose Arm
There were no significant differences from the vehicle control among the BPA dose groups. The high EE2 dose had a highly significant effect on the estrous cycle, with 96% of the animals showing extended estrus as compared to 12% of the vehicle controls. When all types of abnormal cycles were considered, 100% of the high EE2 dose group animals showed abnormal cycles compared to 27% of the vehicle controls.
Stop-Dose Arm
There were no BPA treatment-related effects on the estrous cycle in the stop-dose animals.
- Onset of Aberrant Estrous cycles in Aging Animals
Continuous-Dose Arm
There was no treatment effect of BPA. None of the dose groups differed significantly in the median onset time of 56.8 weeks in the vehicle control group. As expected based on the previously mentioned analysis of estrous cycle data at 16 weeks, the onset of aberrant cycles occurred significantly earlier in the 0.5 μg EE2/kg bw/day dose group.
Stop-Dose Arm
The sole significant effect was a delay in the mediantime of onset in the 2,500 μg BPA/kg bw/day dose group (57 weeks versus 42 weeks in vehicle controls). While no formal analysis was conducted to compare the continuous-dose vehicle control group with the stop-dose vehicle control group, the estimated median time of onset of aberrant cycling appeared shorter in the stop-dose vehicle control group.
- Sperm analysis (Interim sacrifice, PND 365 ± 20):
There were no significant treatment effects observed for either compound and Dose Arm group. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- LOAEL
- Generation:
- F1
- Effect level:
- 25 000 other: µg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Several observations may be treatment related, including the effects mentioned above in the f emale reproductive tract (ovary, uterus, and vagina) and in the male pituitary.
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 2 500 other: µg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: other: see information on LOAEL
- Reproductive effects observed:
- not specified
- Executive summary:
The core GLP study was designed to characterize and evaluate the toxicologic potential of BPA following perinatal only (stop-dose study arm) or chronic exposure (continuous-arm study) in
Sprague-Dawley rats from the NCTR breeding colony (Sprague-Dawley/CD23/NctrBR) under the conditions of a chronic, extended-dose response design.
For that purpose first of all dams (P0) were daily dosed by gavage with 2.5, 25, 250, 2,500, and 25,000 μg BPA/kg body weight (bw)/day. Also reference estrogen groups (0.05 and 0.5 µg ethinyl estradiol/kg bw/day) and vehicle control group ( 0.3% CMC) were included. In utero exposure began on GD 6 and continued until the initiation of parturition.
Then the pups (F1) were daily dosed by gavage from postnatal day (PND) 1 (day of birth = PND 0) until termination at one year or two years was examined . BPA doses were unchanged 2.5, 25, 250, 2,500, and 25,000 μg/kg body weight (bw)/day. A vehicle (0.3% carboxymethylcellulose (CMC)) control group was also included. In addition to animals that were dosed daily throughout the study, a stop-dose study arm was included with animals dosed daily until PND 21 and then held without further treatment until termination at one year or two years. Two dose groups which received orally the active estrogen ethinyl estradiol (EE2; (0.05 and 0.5 μg/kg bw/day) were also included in the continuous-dose arm but not in the stop dose study arm of the study. Data collected included body weights, age at vaginal opening, vaginal cytology, clinical pathology (interim sacrifice only), sperm parameters (interim sacrifice only), organ weights (interim sacrifice only), and histopathology (both interim and terminal sacrifices).
For body weight, clinical pathology endpoints and organ weights, some statistically significant effects of continuous- or stop-dose BPA treatments were observed. These effects were of questionable relevance
to BPA toxicity given that they were seen only in single-dose groups, in several cases differed from the vehicle control by less than 10%, and, in the case of organ weights, were not significant when adjusted
for body weight. No significant effects on sperm parameters or testicular histopathology were noted in the BPA dose groups. An increase in exfoliated germ cells and lymphocyte cellular infiltration in the
epididymis at one year in the continuous-dose study arm and hyperplasia in the pars distalis of the pituitary at two years in both continuous- and stop-dose study arms at 25,000 μg BPA/kg bw/day were
notable effects in males. Hyperplasia of the pituitary pars distalis was also observed in 0.5 μg EE2/kg bw/day terminal sacrifice males. For the epididymal lesions, there were no potentially related lesions
noted in the testes to increase confidence in this observation as an effect of toxicological significance. Other non-neoplastic lesions in males showed variable increases in some dose groups without a pattern
in dose response. BPA treatment did not have adverse effects on the estrous cycle. The increased vaginal epithelial hyperplasia observed at 25,000 μg BPA/kg bw/day may be a plausible estrogenic effect when considered together with the observations mentioned below on the uterus. Lesions observed in the uterus (cystic endometrial hyperplasia, squamous metaplasia, apoptosis of the endometrial luminal epithelial cells), particularly at the interim sacrifice, suggest that the 25,000 μg BPA/kg bw/day dose may exert weak estrogen-like effects on the uterus. In the ovary the magnitude of the increase in follicular cysts at the high BPA dose and the trend evident in the two highest BPA groups suggests that this is a treatment-related effect, although the lack of effect on this endpoint in the continuous-dose animals cannot be readily explained.
An increase in exfoliated germ cells and lymphocyte cellular infiltration in the epididymis at one year in the continuous-dose study arm and hyperplasia in the pars distalis of the pituitary at two years in both continuous- and stop-dose study arms at 25,000 μg BPA/kg bw/day were notable effects in males. Hyperplasia of the pituitary pars distalis was also observed in 0.5 μg EE2/kg bw/day terminal sacrifice males. For the epididymal lesions, there were no potentially related lesions noted in the testes to increase confidence in this observation as an effect of toxicological significance. Other non-neoplastic lesions in males showed variable increases in some dose groups without a pattern in dose response.
In conclusion, in the core study, differences between BPA treatment groups, particularly below 25,000 μg BPA/kg bw/day, and the vehicle control group detected by the low-stringency statistical tests applied to
histopathology lesions, were not dose responsive, sometimes occurring in only one low or intermediate dose group, and did not demonstrate a clear pattern of consistent responses within or across organs
within the stop- and continuous-dose arms and the interim and terminal sacrifices. In contrast, the high EE2 dose elicited several strong effects in females in a clearly interpretable and biologically plausible
as estrogenic effects.
At 25,000 μg BPA/kg bw/day, several observations may be treatment related, including the effects mentioned above in the female reproductive tract (ovary, uterus, and vagina) and in the male pituitary.
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at EPA Laboratory in Research Triangle Park, NC
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- Parameters investigated: On PND2, the sex, body weight, and anogenital distance (AGD) of each female pup were recorded; On PND14, the female rats were sexed and weighed and the number of areolae/nipples on the ventral surface of each female was determined. After weaning at PND23, female rats were weighed and checked for VO every other day until completion. At weaning, a subset of the developmentally exposed female and male rats from the second block of the study were paired with an age-matched, nonsiblings for at least 4 months and monitored regularly for the presence of litters. Saccharin Preference. Figure-8 maze activity. lordosis behavior and uterine weight. Reproductive Organ Morphology.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Long-Evans
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on mating procedure:
- Adult female LE rats (Charles River Laboratories, Raleigh, NC) of approximately 90 days of age were mated by the supplier and shipped on GD2. Mating was confirmed by sperm presence in vaginal smears (day of sperm plug positive = GD1).
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Dams were dosed: GD7 - PND 21
- Frequency of treatment:
- daily
- Details on study schedule:
- Parameters investigated: On PND2, the sex, body weight, and anogenital distance (AGD) of each female pup were recorded; On PND14, the female rats were sexed and weighed and the number of areolae/nipples on the ventral surface of each female was determined. After weaning at PND23, female rats were weighed and checked for VO every other day until completion. At weaning, a subset of the developmentally exposed female and male rats from the second block of the study were paired with an age-matched, nonsiblings for at least 4 months and monitored regularly for the presence of litters. Saccharin Preference. Figure-8 maze activity. lordosis behavior and uterine weight. Reproductive Organ Morphology.
- Remarks:
- Doses / Concentrations:
2, 20 and 200 µg/kg/day
Basis:
actual ingested - No. of animals per sex per dose:
- This study was performed in two blocks. The first block involved 169 dams with 13–29 dams per treatment group: oil vehicle, EE2 (0.05, 0.5, 5, or 50 lg/
kg/day), or BPA (2, 20, or 200 lg/kg/day). The second block (block 2) was performed to expand the range of EE2 doses tested and involved 82 dams with
6–14 dams per treatment group: oil vehicle, EE2 (0.05, 0.15, 0.5, 1.5, 5, 15, or 50 lg/kg/day), or BPA (20 or 200 lg/kg/day). - Control animals:
- yes, concurrent vehicle
- other: Positive control groups: 0.05, 0.15, 0.5, 1.5, 5, 15, or 50 µg/kg/day
- Dose descriptor:
- NOAEL
- Effect level:
- 0.2 mg/kg diet
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: No adverse effect
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 0.2 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Sex:
- female
- Basis for effect level:
- other: see 'Remark'
- Reproductive effects observed:
- not specified
- Executive summary:
The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in uteroand during lactation would alter the expression of well-characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female Long-Evans rat offspring. Pregnant rats were gavaged with vehicle, EE2 (0.05–50 μg/kg/day), or BPA (2, 20, and 200 μg/kg/day) from day 7 of gestation to postnatal day (PND) 18, and the female offspring were studied. EE2 (50 μg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes, and induced malformations of the external genitalia (5 μg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 μg/kg) and absence of lordosis behavior (15 μg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat.
Referenceopen allclose all
REPRODUCTIVE PERFORMANCE: At 3500 ppm, gestational length was statistically significantly increased (authors noted that the toxicological significance of this is unknown).
ORGAN WEIGHTS (PARENTAL ANIMALS): Increased liver and kidney weights in males at 3500 ppm, increase in kidney weight at 300 ppm for males (the authors did not consider this to be toxicologically significant because of the very small increase in absolute kidney weight as compared to controls and the absence of histopathological findings). In adult females, there was a statistically significant increase in liver and kidney weights at 3500 ppm.
HISTOPATHOLOGY (PARENTAL ANIMALS): In males, increased incidence of liver centrilobular hepatocyte hypertrophy at 300 ppm (minimal severity) and 3500 ppm (minimal to mild severity). Increased incidence of renal nephropathy at 3500 ppm (minimal severity).
BODY WEIGHT (OFFSPRING): F1 pups (males and females) had reduced body weights from PND7-PND21 in the 3500 ppm group. Body weight on PND 30 was significantly reduced in F1 weanling males in the 3500 ppm group. Body weights on PND 21 and at acquisition of puberty were significantly reduced in F1 females in the 3500 ppm group.
SEXUAL MATURATION (OFFSPRING): Absolute age at acquisition of puberty was delayed for F1 weanling males at 3500 ppm. When adjusted for PND 30 body weight, there was no significant delay. For females, when adjusted for body weight on PND 21, there was an acceleration in acquisition of puberty, but when adjusted for body weight at the time of acquisition, there was no acceleration.
ORGAN WEIGHTS (OFFSPRING): F1 weanling males had significantly increased thymus weights in the 300 ppm dose group. F1 adult males had increased liver and kidney weights at 3500 ppm. F1 parental males had statistically significant increases in kidney weights at 1.8, 30, and 300 ppm (authors concluded that these were not treatment-related due to lack of dose response, lack of correlating histopathological findings and no effect in F1 retained males). F1 and F2 (male and female) weanlings had reduced spleen weights at 3500 ppm. F1 and F2 weanling males had reduced testes weights in the 3500 ppm group. F2 weanling males had significantly decreased seminal vesicle with coagulating gland weights at 3500 ppm. F1 parental males had a slight, but significant, decrease in absolute paired epidydimal weights at 3500 ppm (the authors did not consider this to be treatment related because this did not occur in the F0 or F1 retained males).
HISTOPATHOLOGY (OFFSPRING): F1 adult females had an increased incidence of centrilobular hepatocyte hypertrophy. In F1 adult males, there was an increased incidence of liver centrilobular hepatocyte hypertrophy at 300 ppm (minimal severity) and 3500 ppm (minimal to mild severity) and an inncreased incidence of renal nephropathy at 3500 ppm (minimal severity). F1 and F2 weanling males in the 3500 ppm group had an increased incidence of minimal to mild hypoplasia of the seminiferous tubules. F1 weanling males had an increase in the incidence of centrilobular hepatocellular cytoplasmic alteration in the liver at 300 and 3500 ppm.
OTHER: F1 males had statistically significantly reduced absolute anogenital distance on PND 21 at 3500 ppm and reduced anogenital distance adjusted for terminal body weight at 300 and 3500 ppm. The authors did not consider these findings to be treatment-related owing to the absence of effects on PND 0 for F1/F2 males and on PND 21 for F2 males.
REPRODUCTIVE PERFORMANCE: F1 females had a statistically significant increase in gestational length at 3500 ppm BPA. The authors noted that the toxicological significance of this minor difference is unknown.
GROSS PATHOLOGY: Increased incidence of undescended testes in F1 weanling males at 3500 ppm. Authors noted that these effects were likely secondary to and caused by systemic toxicity.
REPRODUCTIVE: At 3500 ppm, gestational length was statistically significantly increased for F1 females (authors noted that the toxicological significance of this is unknown).
Reproductive function: sperm measures: At 7500 ppm, epididymal sperm production was significantly reduced in the F1 generation and daily sperm production was significantly reduced in the F3 generation.
Reproductive performance: At 7500 ppm, there was a significant reduction in number of implants and number of total pups per litter in all generations. At 0.3 ppm, the number of implant sites per dam was significantly reduced in the F2 generation and the number of total pups per litter was significantly reduced in the F3 generation.
Non-reproductive organ weights (males): At 7500 ppm, absolute organ weights at necropsy were significantly reduced for liver, kidneys, adrenal glands, spleen, pituitary and brain in P, F1, and F2 parental and F3 retained adult animals. Relative weights of kidneys were increased in all generations at this dose level. At 750 ppm, P/F1/F2/F3 males had significant reductions in liver weights; relative liver weights were significantly reduced for P, F2 and F3 animals. Relative kidney weights were significantly increased for males in the F2 generation dosed at 750 ppm. Relative paired kidney weights were significantly reduced at 4.5 ppm for F1 males.
Non-reproductive organ weights (females): At 7500 ppm, absolute liver weights were significantly reduced for F3 females, relative liver weights were significantly increased in P and F2 females, absolute kidney weights were significantly reduced for all generations, and relative kidney weights were significantly increased for F0 and F2 females. Absolute and relative liver weights were significantly increased in F2 females at 0.015 ppm.
For organ weights, the authors noted that relative organ weights were affected by reduced body weights, and other changes in absolute and relative organ weights (other than liver, kidneys, adrenal glands, spleen, pituitary and brain) were not consistent across generations and did not exhibit a dose-response pattern.
Reproductive organ weights (males): Absolute seminal vesicle weights and absolute prostate weights were significantly reduced at 7500 ppm for all generations, relative seminal vesicle weights were significantly increased in F1 males only. Absolute testes weights and absolute epididymides weights were significantly reduced for F1, F2 and F3 males at 7500 ppm, while relative testes weights and relative epididymides weights were significantly increased for all generations at this dose level. For the preputial gland, there was a significant decrease in weight at 7500 ppm, but only for the F1 generation.
At 750 ppm, absolute testes weights were significantly decreased in F2 and F3 males, absolute epididymides weights were significantly decreased for F3 males, relative epididymides weights were significantly increased for F2 males, and relative seminal vesicle weights were significantly increased for F2 males. Absolute testes weights were significantly reduced in the F2 generation at 0.3 ppm and in the F3 generation at 0.015 and 0.3 ppm. Absolute seminal vesicle weights were significantly increased in P generation males at 4.5 ppm. The authors noted that there were no dose- or treatment-related effects on the absolute and relative weights of the testes, epididymides, prostate, or seminal vesicles plus coagulating glands.
Reproductive organ weights (females): At 7500 ppm, absolute ovary weights were significantly reduced for all generations, relative ovary weights were significantly reduced in the P, F1 and F2 generations, and absolute uterus weights were significantly reduced in P, F1 and F2 generations. Absolute and relative ovary weights were significantly reduced at 0.015, 4.5 and 75 ppm for F2 females, and absolute uterus weights were significantly reduced in P generation females at 0.015 ppm.
Histopathology: Slight to mild renal tubular degeneration and chronic hepatic inflammation were observed at a higher incidence in P, F1, and F2 females at 7500 ppm.
Body weight: Per litter pup body weights were reduced at 7500 ppm for F1, F2, and F3 offspring on postnatal days 7, 14 and 21. F1 litters also had significantly reduced pup body weights per litter on postnatal day 4 when all pups were analysed together, but not when the sexes were analysed separately.
Anogenital distance: Anogenital distance (AGD) was significantly increased in F2 females at all dose levels except 75 and 7500 ppm.
Sexual maturation: There was a significant delay in age at vaginal patency (VP) for F1, F2 and F3 females at 7500 ppm and for F2 females at 75 ppm. When adjusted for body weight at acquisition, VP was only delayed at 7500 ppm for all three generations. Male offspring had a significant delay in age at preputial separation (PPS) in the F1 generation at 750 and 7500 ppm, in the F2 generation at 0.3, 75, 750 and 7500 ppm, and in the F3 generation at 7500 ppm. When adjusted for body weight at acquisition, PPS was delayed in the F1 generation at 750 and 7500 ppm and in F2 and F3 generations at 7500 ppm.
Organ weights: Data was not shown, but according to the text, absolute organ weights were decreased at 7500 ppm for F1, F2, and F3 weanling males and females sacrificed on postnatal day 21. The authors noted that there were reductions in organ weights at lower doses, but they were not consistently affected in F1, F2, and F3 weanlings or reproducible in specific dose groups. Relative organ weights at 7500 ppm were increased, which the authors noted was caused by reduced body weights at this dietary dose.
There were no clinical signs of toxicity and no significant mortality in any generation.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
An increase in body weight was observed in P generation males at 0.2 ug/kg BPA during the first week of dosing. Food consumption was decreased in P females at 20 ug/kg during weeks 2 and 3; in F1 males at 20 and 200 ug/kg during weeks 3 and 4; in F1 females at 200 ug/kg during week 7; and in F2 females at 20 ug/kg during weeks 4 and 5. These changes were unaccompanied by changes in body weight or body weight gain. The changes in body weight and food consumption were occasional and inconsistent and seem unlikely to be toxicologically significant.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There was a decrease in the incidence of females having normal estrous cycles in F1 animals at 20 ug/kg BPA. This decrease was not dose-dependent or consistent across generations, and BPA did not affect reproductive outcome, so it was concluded that BPA did not adversely affect the estrous cycle.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Gestation length was decreased in F1 animals at 20 ug/kg BPA. This effect was also not dose-dependent or consistent across generations, and did not affect reproductive outcome, so it was concluded that BPA did not adversely affect reproductive performance.
ORGAN WEIGHTS (PARENTAL ANIMALS)
P generation males had decreased heart weight at 200 ug/kg. F1 adult males had decreased lung weight at 0.2 and 200 ug/kg, decreased kidney weight at 200 ug/kg, and decreased testes weight at 20 ug/kg. F1 adult females had decreased ovarian weight at 0.2 ug/kg. F2 adult males had decreased seminal vesicle weight at 200 ug/kg. Relative organ weights were not affected in P and F1 animals at any dose, and it was concluded that BPA did not affect organ weights.
OTHER FINDINGS (PARENTAL ANIMALS)
P generation females had decreased serum LH concentrations at 0.2, 2, and 20 ug/kg and decreased serum T3 concentrations at 200 ug/kg; however, these changes were not dose-dependent and were inconsistent across sexes and generations, so it was concluded that these changes are not biologically significant.
Male F1 pups had decreased body weight at 20 ug/kg on PND 14 and 21. Because this change was inconsistent and occasional, it is unlikely to be toxicologically significant.
SEXUAL MATURATION (OFFSPRING)
Testes descent was delayed in F2 pups at 20 and 200 ug/kg, but this was believed to be a biologic variation, as the time to descent in treated F2 males was equivalent to the control value for F1 males. The AGD per cube root of body weight ratio in F1 males was decreased at 0.2 ug/kg on PND 57 and at 20 ug/kg on PND 106, 113, and the day of sacrifice. This ratio was also decreased in F1 females at 200 ug/kg on PND2 and increased in F1 females at 2 and 20 ug/kg on PND7. In F2 females, this ratio was decreased at 20 ug/kg on PND 64, 71, 85, and 92 and on the day of sacrifice, and at 200 on PND 57, 64, and the day of sacrifice. These changes were occasional and inconsistent across sexes and generations and are not BPA-related or toxicologically significant.
ORGAN WEIGHTS (OFFSPRING)
F1 male weanlings had decreased absolute lung weight at 20 and 200 ug/kg and decreased absolute kidney weight 20 ug/kg. In F2 male weanlings, lung weight was decreased at 20 ug/kg and relative heart weight was decreased at 200 ug/kg. At 2 ug/kg in F2 males, increased relative liver weight, decreased absolute and relative weights of the seminal vesicle, and decreased weight of the thyroid glands were observed. These changes were occasional and inconsistent, and it was concluded that BPA did not affect organ weights.
Clear adverse effects of BPA, including depressed gestational and postnatal body weight gain, effects on the ovary (increased cystic follicles, depleted corpora lutea, and antral follicles), and serum hormones (increased serum estradiol and prolactin and decreased progesterone), were observed only at the two high doses of BPA. For more details see document attached to the study summary.
Many chemicals released into the environment are capable of disrupting normal sex steroid balance, including the oral contraceptive ethinyl estradiol (EE) and the plastic monomer bisphenol A (BPA). EE and BPA are reported to impair reproductive organ development in laboratory animals; however, effects of lower doses of these chemicals have been debated. The goal of the current study was to determine whether relatively low oral doses of EE or BPA would alter male reproductive morphology and associated hormone levels of Long Evans hooded rat. Dams were gavaged with corn oil vehicle, EE (0.05–50 μg/kg/day) or BPA (2, 20, and 200 μg/kg/day) during pregnancy through lactation from gestational day 7 to postnatal day (PND) 18. Anogenital distance was measured at PND2 and nipple retention was measured at PND14 in male pups. Male offspring were euthanized beginning at PND150, and sera and organs were collected for analyses. Adult body weight was significantly decreased in males exposed to 50 μg EE/kg/day. Developmental EE exposure reduced androgen-dependent tissue weights in a dose-dependent fashion; for example, seminal vesicle and paired testes weights were reduced with ≥ 5 μg EE/kg/day. Epididymal sperm counts were also significantly decreased with 50 μg EE/kg/day. In contrast, treatment with 2, 20, or 200 μg BPA/kg/day or EE at 0.05–1.5 μg/kg/day did not significantly affect any male endpoint in the current study. These results demonstrate that developmental exposure to oral micromolar doses of EE can permanently disrupt the reproductive tract of the male rat.
TABLE 1: End Points Evaluated in F1 Female LE Rats and F0 Dams Treated Orally with BPA or EE2 during Gestation and Lactation End points.
| BPA | EE2 | |
| AGD PND2 | No effect | Increased at 50 |
| Female F1 pup body weight (g) PND2 | No effect | Reduced at 50 |
| Age at vaginal opening | No effect | Accelerated at 5 |
| Weight at vaginal opening | No effect | Reduced at 5 |
| % Displaying cleft phallus at weaning | No effect | Increased at 5 |
| External genitalia UVD (mm) at necropsy | No effect | Reduced at 5 |
| Breeding data: fecundity | No effect | Reduced at 5 |
| Saccharin preference | No effect | Male-like at 5 |
| LQ: activated with 50 mg adult EE2 | No effect | Reduced at 15 |
| Locomotor activity in Figure-8 maze (photocell counts): ovariectomized | No effect | No effect |
| Locomotor activity in Figure-8 maze (photocell counts): ovariectomized plus oral EE2 | No effect | No effect |
| F1 weaning weight (g) | No effect | Reduced at 50 |
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 50 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Multiple comprehensive guideline studies, including multi-generation and lifelong exposure studies are available.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
EFSA Opinion 2015 concluded on reproductive and developmental toxicity:
Overall, the better powered, better conducted studies in animals found few consistent effects of in-utero exposure to Bisphenol A on reproductive development at dose levels at or below 3.6 mg Bisphenol A/kg/day HED. On balance, the evidence remains contradictory and highly variable between studies. The CEF Panel noted that there is some evidence for effects of Bisphenol A exposure on several parameters indicative for changes in the reproductive system in adult male animals at dose levels below 3.6 mg/kg bw per day, although these effects were modest. It is not possible to conclude that these changes are reflective of changes in reproductive performance, since the studies rarely included a forced/continuous breeding phase in adulthood to establish reduced fertility. However, in several multigenerational studies no effects were observed at dose levels as low as 3mg/kg bw per day up to at least 50 mg/kg bw per day.
Using a WoE approach, the CEF Panel assigned a likelihood level of “as likely as not” to reproductive and developmental effects of Bisphenol A at low doses (below the HED of 3.6 mg/kg bw per day). Since the likelihood level for this endpoint is less than "likely" (see Appendix A), this endpoint was not taken forward for assessing the toxicological reference point, but was taken into account in the evaluation of uncertainty for hazard characterisation and risk characterisation (Section 4.3)."
EFSA Opinion 2015 concluded concerning potential Non-Monotonic-Dose-Responses (NMDR):
“The CEF Panel developed criteria for nonmonotonic dose-responses (NMDRs) and reviewed studies reporting a NMDR for Bisphenol A. None of the studies fulfil these criteria. Overall the CEF Panel concluded that the available data do not provide evidence that Bisphenol A exhibits a NMDR for the endpoints considered (reproductive/developmental toxicity, neurotoxicity/behavioural effects, metabolic effects, proliferative changes in mammary gland). “
SCOEL Recommendation 2014 concluded on reproductive and developmental toxicity:
"Overall, in standard reproductive and developmental studies in rodents, effects on reproduction have been seen only at high doses showing also other toxic effects. Even though several non-guideline studies suggest effects on reproductive and developmental parameters at lower dose levels (< 5 mg/kg bw), the data are contradictory and are not supported by the recent FDA/NTCR study with a wide-dose range (Delclos et al 2014). In humans, based on Chinese epidemiological studies, there is some concern for impaired sperm quality but, for example, the effect of other concurrent exposures cannot be excluded. In addition, there are some concerns on the potential developmental neurotoxicity of Bisphenol A based on animal studies suggesting effects on memory and learning and anxiety-like behaviour. However, since the data are very inconsistent it is difficult to conclude on the relevance of these findings."
SCOEL Recommendation 2014 concluded concerning potential low-dose effects:
“Even though there are some concerns related to the long-term effects of Bisphenol A at exposure levels lower than 5 mg/kg bw after exposure during the foetal and early postnatal period, the results of these studies are controversial and there is no clear support for these effects at low dose levels from good quality animal studies (including the recent study by Delcloset al2014). Therefore, at present SCOEL did not consider them relevant for deriving the recommended OEL.”
Short description of key information: SCOEL Recommendation 2014 concluded concerning potential low-dose effects: “Even though there are some concerns related to the long-term effects of Bisphenol A at exposure levels lower than 5 mg/kg bw after exposure during the foetal and early postnatal period, the results of these studies are controversial and there is no clear support for these effects at low dose levels from good quality animal studies (including the recent study by Delcloset al2014). Therefore, at present SCOEL did not consider them relevant for deriving the recommended OEL.” EFSA Opinion 2015 concluded concerning potential Non-Monotonic-Dose-Resopnses (NMDR): “The CEF Panel developed criteria for nonmonotonic dose-responses (NMDRs) and reviewed studies reporting a NMDR for Bisphenol A. None of the studies fulfil these criteria. Overall the CEF Panel concluded that the available data do not provide evidence that Bisphenol A exhibits a NMDR for the endpoints considered (reproductive/developmental toxicity, neurotoxicity/behavioural effects, metabolic effects, proliferative changes in mammary gland).“
The 2008 updated EU RAR concluded:
"A new two-generation study in mice by Tyl et al. (published in 2008) provides a comprehensive and definitive investigation on the effects of Bisphenol A on reproduction at exposure levels spanning the low (ug/kg/day) to high (mg/kg/day) ranges. This study showed that Bisphenol A causes adverse effects on pregnancy and the offspring at 600 mg/kg/day, an exposure level that also caused mild parental toxicity. Fertility was not affected by Bisphenol A exposure. A NOAEL for reproductive toxicity of 50 mg/kg/day was identified and should be used in the risk assessment."
The 2003 EU RAR concluded:
"No human data on reproductive toxicity of Bisphenol A are available. Bisphenol A has been shown to have endocrine modulating activity in a number of screening assays, with a potency that generally ranged from 3 to 5 orders of magnitude less than that of oestradiol. The effects of Bisphenol A on fertility and reproductive performance have been investigated in two-generation and multi-generation studies in the rat and a continuous breeding study in mice. Effects were seen in both species at approximately the same dose level and it is considered that the NOAEL of 50 mg/kg/day identified in the rat multi-generation study is also likely to produce no adverse effects in mice for which there is only a LOAEL of 300 mg/kg/day for a small decrease in epididymal weight in F1 males. The NOAEL of 50 mg/kg/day from the multi-generation study will be used for risk characterisation purposes, in relation to effects on fertility."
Effects on developmental toxicity
Description of key information
- NOAEL for systemic toxicity 5 mg/kg bw/d in rats and mice; LOAEL >= 50 mg/kg bw/d
- In rats no adverse effects on oestrous cycle, fertiliy, litter sizes, pre- and post-natal survival, growth, and development at doses up to ca. 200 mg/kg bw/d. With 500 mg/kg bw/d reduced litter size (at systemic toxic dose) in a Three Generation Reproduction Study (Tyl et al., 2002)
- In mice no adverse effects on fertility, litter sizes, pre- and post-natal survival at doses up to 600 mg/kg bw/d. With 600 mg/kg bw/d delayed offspring development in a Two Generation Reproduction Study (Tyl et al., 2008).
- This is supported by the recently published Clarity Core study, in which no treatment effects were observed for the number of implantation sites, litter size, sex ratio, litter weight by sex and mean pup weight at birth by sex up to the highest tested dose group (25,000 µg/kg bw/d)
Comprehensive guideline studies, which are described in this chapter, show that Bisphenol A is not a selective reproductive toxicant:
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Conducted under OECD guidelines, OECD good laboratory practices
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study) TG 416 Enhanced
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: (P) 6 wks; (F1) 3 wks (at beginning of direct dosing)
- Housing: Animals were individually housed upon arrival, during acclimatisation, and upon the initiation of treatment periods; by mating pairs (1 male: 1 female) during the mating period; individually as plug-positive females from gestational day 0 to birth of litters; as individual dams with litters throughout the lactational period until weaning on PND 21; and individually as selected or extra retained F1 postweanlings. Housing was in solid-bottom polypropylene cages (5" x 11.5" x 7") with stainless-steel wire bar lids (Laboratory Products, Rochelle Park, NJ). Cage bedding was Sani-Chip (PJ Murphy Forest Products, Inc. Montville, NJ).
- Diet: Ad libitum. Purina Certified Ground Rodent Chow (No. 5002, PMI Feeds, Inc., St. Louis, MO) in glass mouse feeding jars with stainless steel snap-on or screw-on caps and stainless-steel wire-mesh inserts. The supplier provided contaminant levels; these were below certified levels. Each of the 3 lots of feed used were analysed for the phytoestrogens genistein (mean +- SEM: 192+-18.6 ppm), daidzein (177+-4.0 ppm), and glycitein (45+-8.9 ppm). Feed was stored at 60-70 degrees F, with the period of use not more than 6 months from the milling date.
- Water: Water was available ad libitum in glass water bottles with Teflon-lined, plastic screw caps and stainless-steel sipper tubes. Contaminant levels of the Durham City water were measured at regular intervals by the supplier and by Balazs Analytical Services, Inc. Contaminant levels were below the maximal levels established for potable water.
- Acclimation period: Animals quarantined 1 week upon arrival.
ENVIRONMENTAL CONDITIONS
- Temperature: 66-77 degrees F (19-25 degrees C)
- Humidity: 30-70%
- Photoperiod: 12 hours dark/12 hours light - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- Feed consumption measurements were recorded for all P and F1 parental animals at least every 7 days, every time the feed was changed, throughout the prebreed exposure period. Feed consumption collection periods corresponded with the collection of the animals' weekly body weight data and were employed to calculate intake of BPA on a mg of test article/kg body weight ratio. Feed consumption during pregnancy was recorded for GD 0-7, 7-14, and 14-17; during lactation maternal feed consumption was measured for PND 0-4, 4-7, 7-14, and 14-21. Maternal feed consumption after PND 14 was confounded by contribution from the pups. Feed was changed at least weekly.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Homogeneity and stability of BPA and E2 (positive control) in the dose feed were assessed and confirmed at room temperature for at least 9 days and at least 50 days when frozen at -20 degrees C.
- Details on mating procedure:
- One male and one female were selected randomly within a dose group and cohabited for 14 days, or until a copulation plug was observed in the vaginal tract of the female mouse. After observation of the vaginal plug, the mating pair were separated and individually housed.
- F1 parental animals were mated at approximately 11-13 weeks of age.
- F1 females were selected for mating on PND 18; F1 males were selected on PND 21.
- Age at mating of the mated animals in the study: 14 weeks (P); 11-13 weeks (F1) - Duration of treatment / exposure:
- P generation animals were dosed from approximately 6 weeks of age. Dosing occurred for 8 weeks prior to mating. Selected F1 animals were administered dosed feed ad libitum 7 days/week for at least 8 weeks prior to mating. Dosing continued until sacrifice.
- Frequency of treatment:
- Animals ate Bisphenol A-containing food ad libitum.
- Duration of test:
- Two-generation
- No. of animals per sex per dose:
- 28
- Control animals:
- yes, plain diet
- other: exposed to 0.5 ppm E2, positive control
- Details on study design:
- At weaning on PND 21, 28 F1 male pups/group were selected for mating, while 1 male pup/litter was randomly selected to be retained, with exposures continuing for 3 months, and necropsied when F1 parental males were necropsied.
At weaning (PND 21), up to 3 F1 and F2 pups/sex/litter were randomly selected and subjected to a gross necropsy with organs weighed and retained in fixative.
P and F1 parental males were sacrificed after completion of the gestation of their F1 and F2 litters, respectively. Sacrifice of P and parental F1 females occurred on the same day as weaning of the F1 and F2 litters. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, morning and evening
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Recorded initally and weekly throughout mating. For P females, weights taken during gestation on GD 0, 7, 14, and 17. Dams producing litters were weighed on PND 0, 4, 7, 14, and 21.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consumption periods corresponded with collection of animals' weekly body weight data and were employed to calculate intake of BPA on a mg BPA/kg body weight ratio. During pregnancy, feed consumption was measured on GD 0-7, 7-14, and 14-17; during lactation, feed consumption was measured on PND 0-4, 4-7, 7-14, and 14-21. There was no measurement of feed consumption during the period of cohabitation.
SACRIFICE
- Maternal animals: All surviving animals sacrificed on the same day as weaning of their litters.
GROSS NECROPSY
- Gross necropsy performed with the addition of an examination of the uterus of parental females.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed and histopathology was performed: adrenal gland (paired), brain, epididymides, kidneys (weighed individually), liver, ovaries with oviducts (paired), pituitary, spleen, thyroid, uterus+cervix+vagina, mammary glands (paired, axillary- no weights taken, just histopathology)
-Full histopathology was performed on the organs listed above for 10 animals/sex of the P/F1 parental generations in all dose groups. - Ovaries and uterine content:
- Estrous cyclicity was evaluated by daily vaginal smears during the last 3 weeks of the prebreed exposure periods for P and F1 females. Also, a smear was taken after euthanasia of P and F1 females to determine stage of estrous at demise.
- Fetal examinations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum on 10 pups. Excess pups were killed and discarded.
GROSS EXAMINATION OF DEAD PUPS
Yes, for external and internal abnormalities; possible cause of death was determined, when possible, for pups born or found dead.
PARAMETERS EXAMINED
The following parameters were examined in [F1/F2 ] offspring: number and sex of pups, body weights, anogenital distance, physical abnormalities.
GROSS EXAMINATION OF DEAD PUPS
Pups that were stillborn, dead, or euthanised moribund during lactation were necropsied to determine cause of death and any malformations or variations.
SACRIFICE
- On PND 21, F1 pups remaining after selection and all F2 pups were euthanised by CO2 inhalation.
- Retained F1 males were sacrificed at the same time as the F1 parental males (at approximately 14 weeks of age).
GROSS NECROPSY
- Gross necropsy performed on all animals.
-The status of the F1 weanling testes was evaluated at necropsy after euthanasia by abdominal incision and localisation of the testes low in the abdominal cavity at the inguinal ring (undescended) or in the scrotal sacs (descended). Gross lesions also were retained in fixative (BNF or Bouin’s for testes).
HISTOPATHOLOGY / ORGAN WEIGHTS
- For F1 and F2 pups, the following organs were weighed and histopathology was performed: brain, epididymides (males), kidneys (weighed individually), liver, ovaries with oviducts (paired, females), spleen, seminal vesicles with coagulating glands and their fluids (paired, males), testes (paired, males), thymus, uterus+cervix+vagina (females). For the first randomly selected pup/sex/litter, histopathological examination was performed on the reproductive organs. For the second pup/sex/litter, histopathological examination was performed on all retained tissues (systemic and reproductive) and identified target organs (if any). All retained tissues were placed in fixative (NBF or Bouin’s for testes), and all retained tissues examined histopathologically were embedded in paraffin.
- Full histopathology was performed on the following organ listed above for 10 F1 retained male animals per dose group: adrenal gland (paired), brain, epididymides (paired), kidneys (weighed individually), liver, pituitary, spleen, prostate (ventra and dorsolateral lobes, males), seminal vesicles with coagulating glands and their fluids (paired, males), testes (paired), thyroid. - Statistics:
- Statistical unit: the P and F1 parental male, the P and F1 parental female, the pregnant P and F1 female, the retained F1 male, or the F1 and F2 litter.
Quantitative continuous data compared using either parametric ANOVA under the standard assumptions or robust regression methods.
Frequency data, such as reproductive indices (e.g., mating and fertility indices), were not transformed. All indices were analyzed by Chi-Square test for Independence for differences among treatment groups (Snedecor and Cochran, 1967).
The criterion for statistical significance was p < 0.05. If the overall ANOVA or Chi-Square p value was significant, then the appropriate pairwise comparisons were made, and those pairwise comparisons that were statistically significant are presented in the summary tables. If the overall ANOVA or Chi-Square p value was not significant, then pairwise comparisons were not made.
Acquisition of developmental landmarks (e.g., VP and PPS), as well as AGD, was also analysed by Analysis of Covariance (ANCOVA; in addition to ANOVA analysis or robust regression analysis) using body weight at acquisition or measurement as the covariate. In addition, age at acquisition of puberty (VP, PPS) was also analyzed, with the individual body weights on PND 21 for females and on PND 30 for males as the covariate.
Correlated data (e.g., body and organ weights at necropsy of pups on PND 21, with more than 1 pup/sex/litter) were analysed using GEE techniques (Zeger and Liang, 1986) in the SUDAAN software package (RTI, 2001).
A test for statistical outliers (SAS Institute, Inc., 1999) was performed on parental body weights, feed consumption (in g/day), and F0 and F1 adult, F1 and F2 weanling, and F1 retained male organ weights. When examination of pertinent study data did not provide a plausible, biologically-sound reason for inclusion of the data flagged as “outlier,” the data were excluded from summarisation and analysis and were designated as outliers. - Indices:
- - Reproductive indices: mating, fertility, pregnancy, gestational indices, precoital intervals, epididymal sperm concentration, percent motile sperm, percent abnormal sperm, testicular homogenization-resistant spermatid head counts, daily sperm production, estrus cycle.
- Offspring viability indices: number of live litters on PND 0, live birth index, number of total/live/dead pups, sex ratio (% males) per litter on PND 0. - Historical control data:
- Not reported.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
ORGAN WEIGHTS: There was a statistically significant increase in liver and kidney weights at 3500 ppm. - Dose descriptor:
- NOEL
- Effect level:
- 30 ppm
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Remarks:
- Systemic
- Effect level:
- 300 ppm
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Effect level:
- 300 ppm
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
SURVIVAL: There was a significant reduction in the F2 survival index at 14-21 days of age in the 300 ppm dose group.
BODY WEIGHT (OFFSPRING): F1 pups (males and females) had reduced body weights from PND7-PND21 in the 3500 ppm group. Body weight on PND 30 was significantly reduced in F1 weanling males in the 3500 ppm group. Body weights on PND 21 and at acquisition of puberty were significantly reduced in F1 females in the 3500 ppm group.
SEXUAL MATURATION (OFFSPRING): Absolute age at acquisition of puberty was delayed for F1 weanling males at 3500 ppm. When adjusted for PND 30 body weight, there was no significant delay. For females, when adjusted for body weight on PND 21, there was an acceleration in acquisition of puberty, but when adjusted for body weight at the time of acquisition, there was no acceleration.
ORGAN WEIGHTS (OFFSPRING): F1 weanling males had significantly increased thymus weights in the 300 ppm dose group. F1 adult males had increased liver and kidney weights at 3500 ppm. F1 parental males had statistically significant increases in kidney weights at 1.8, 30, and 300 ppm (authors concluded that these were not treatment-related due to lack of dose response, lack of correlating histopathological findings and no effect in F1 retained males). F1 and F2 (male and female) weanlings had reduced spleen weights at 3500 ppm. F1 and F2 weanling males had reduced testes weights in the 3500 ppm group. F2 weanling males had significantly decreased seminal vesicle with coagulating gland weights at 3500 ppm. F1 parental males had a slight, but significant, decrease in absolute paired epidydimal weights at 3500 ppm (the authors did not consider this to be treatment related because this did not occur in the F0 or F1 retained males).
HISTOPATHOLOGY (OFFSPRING): F1 adult females had an increased incidence of centrilobular hepatocyte hypertrophy. In F1 adult males, there was an increased incidence of liver centrilobular hepatocyte hypertrophy at 300 ppm (minimal severity) and 3500 ppm (minimal to mild severity) and an inncreased incidence of renal nephropathy at 3500 ppm (minimal severity). F1 and F2 weanling males in the 3500 ppm group had an increased incidence of minimal to mild hypoplasia of the seminiferous tubules. F1 weanling males had an increase in the incidence of centrilobular hepatocellular cytoplasmic alteration in the liver at 300 and 3500 ppm.
OTHER: F1 males had statistically significantly reduced absolute anogenital distance on PND 21 at 3500 ppm and reduced anogenital distance adjusted for terminal body weight at 300 and 3500 ppm. The authors did not consider these findings to be treatment-related owing to the absence of effects on PND 0 for F1/F2 males and on PND 21 for F2 males.
REPRODUCTIVE PERFORMANCE: F1 females had a statistically significant increase in gestational length at 3500 ppm BPA. The authors noted that the toxicological significance of this minor difference is unknown.
GROSS PATHOLOGY: Increased incidence of undescended testes in F1 weanling males at 3500 ppm. Authors noted that these effects were likely secondary to and caused by systemic toxicity.
REPRODUCTIVE: At 3500 ppm, gestational length was statistically significantly increased for F1 females (authors noted that the toxicological significance of this is unknown). - Dose descriptor:
- NOAEL
- Effect level:
- 300 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: See remarks
- Remarks on result:
- other:
- Remarks:
- At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights, slightly delayed 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.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The authors concluded that BPA is not a selective developmental toxicant in mice.
- Executive summary:
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, or reproductive organ weights or histopathology. Maternal systemic effects were increased liver and kidney weight at 3500 ppm. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights, slightly delayed 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 non-monotonic dose-response curves for any parameter. The systemic NOEL and developmental NOEL were 300 ppm BPA (approximately 50 mg/kg-day).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study designed and conducted using an internationally accepted reproductive toxicity protocol under Good Laboratory Practice (GLP) regulations (U.S. EPA, 1989).
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 837.3800, 1998
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Housing: During the acclimation period and upon initiation of treatment, animals were housed individually in stainless-steel hanging cages. Mating pairs and sperm/plug positive females (from gestation day 0 to weaning of litters on postnatal day 21) were housed in solid-bottom polypropylene cages (Laboratory Products, Rochelle Park, NJ) with Sani-Chip cage bedding (PJ Murphy Forest Products, Inc., Montville, NJ)
- Diet (e.g. ad libitum): ad libitum Purina Certified Ground Rodent Chow (No. 5002, PMI Feeds, Inc., St. Louis, MO)
- Water (e.g. ad libitum): ad libitum by automatic water system or by glass water bottles
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: feed
- Vehicle:
- acetone
- Details on exposure:
- DIET PREPARATION
- Appropriate amounts were mixed with Purina Certified Rodent Chow (No. 5002, PMI Feeds): BPA was dissoved in a fixed volume of acetone and added to a premix feed aliquot. Stability of formulations at 15 ppb and 7500 and 10,000 ppm was confirmed at -20 degrees C for 50 days and at room temperature in open containers to simulate cageside conditions for at least 9 days. Homogeneity was confirmed by assaying one sample each at 15 ppb and 7500 and 10,000 ppm in triplicate from 3 locations within the blender.
- Storage temperature of food: -20 degrees C. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Aliquots from all dosed feed preparations were analysed for BPA concentration and diet was only used if within +/- 15% of the nominal. Feed analyses were performed using negative ion chemical ionisation gas chromatography-mass spectrometry.
- Details on mating procedure:
- - Male/female ratio per cage: 1 male/1 female per cage
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm positive
- After successful mating, each dam was caged in a solid-bottom polypropylene cage with Sani-Chip cage bedding until weaning of litters on postnatal day 21. - Duration of treatment / exposure:
- P: Animals were exposed for 10 weeks before mating, for 2 weeks during mating, and during 3 weeks of gestation; dams were also exposed during 3 weeks of lactation.
F1/F2: Animals were exposed in utero and during lactation. Direct dietary exposure began after weaning and continued for 10 weeks, then animals were dosed during 2 weeks of mating, 3 weeks of gestataion, and dams for 3 weeks of lactation.
F3: Animals were exposed in utero and during lactation. Direct dietary exposure began after weaning and continued for 10 weeks. - Frequency of treatment:
- Bisphenol A in feed was available ad libitum.
- Duration of test:
- Three-generation study
- No. of animals per sex per dose:
- 30 males/30 females per dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - F1 parental animals were not mated until 10 weeks after selection from the F1 litters.
- Selection of animals to mate from the F1 generation was made when F1 pups were 21 days of age.
- Age at mating of all animals in the study: 13 weeks.
- Dose selection rationale: Doses were selected to encompass the ranges of low oral BPA doses at which male mouse offspring prostate weights were reported to be significantly increased, doses at which testis weight and efficiency of daily sperm production (DSP) were reported to be significantly reduced in rat offspring, and doses that provide a maximum tolerated dose (MTD) expected to exceed metabolic capability of the adult liver and to produce reductions in body weight or other indications of systemic toxicity. Target dietary concentrations were based on the chosen BPA intakes in mg/kg-day for the female rats. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
ESTROUS CYCLICITY: Yes
- Estrous cyclicity was evaluated for the last three weeks of the pre-breed period for the P, F1, and F2 parental animals, and in the 3 week post-weaning period for F3 retained females. Stage of estrus was also determined at necropsy for P, F1, and F2 parental and F3 retained females.
SACRIFICE
- Maternal animals: Maternal animals were sacrificed after weaning of their pups.
HISTOPATHOLOGY / ORGAN WEIGHTS
- For adult females (all generations), histopathology was done on the following organs: adrenal glands, cervix, kidneys, liver, ovary, oviduct, pituitary, spleen, urinary bladder (one animal in the P generation only), uterus, and vagina. Enumeration of ovarian primordial follicles was performed for high-dose females. Organ weights were measured for the following organs: liver, paired kidneys, paired ovaries, and uterus. - Fetal examinations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 10 pups/litter (equivalent number per sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1/F2/F3 offspring: number of live pups per litter, day 4 survival index, lactational index, anogenital distance, body weight/litter, age at vaginal patency, body weight at vaginal patency, number of nipples/areolae per male pup, age at preputial separation, body weight at preputial separation.
SACRIFICE
- The F1/F2 offspring not selected as parental animals and all F3 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Up to 3 pups/sex/litter were randomly selected, necropsied, and selected organs were weighed.
ORGAN WEIGHTS
Organ weights were measured for weanling animals, but no details are given in the text as to which organs were weighed. - Statistics:
- The unit of comparison was the individual animal or the litter, as appropriate. Data from the cohorts were combined for summarisation and statistical analyses.
Quantitative continuous data were compared among the 6 treatment groups and the vehicle control group. For the litter-derived percentage data, ANOVA was weighted according to litter size. GLM analysis was used to determine the significance of the dose-response relationship and to determine whether significant dosage effects had occurred for selected measures. A one-tailed test was used for all pairwise comparisons to the vehicle control group, except two-tailed tests were used for parental and pup body and organ weights, feed consumption, percent males per litter, and AGD per sex per litter.
Nonparametric tests for continuous data were used to determine if significant differences were present among the groups or to identify significant dose-response trends. Frequency data were analyzed for differences among treatment groups and for pairwise comparisons.
For acquisition of developmental landmarks and AGD, ANOVA and ANCOVA, with body weight (at birth, PND 0, for AGD; at acquisition of puberty and on study day [SD] 7 for females [VP] and SD 14 for males [PPS]) as the covariate, were used for pairwise comparisons. For correlated data, SUDAAN software was used for analysis of overall significance, presence of trend, and pairwise comparisons to the control group values. For all statistical tests, the significance limit of 0.05 (one- or two-tailed) was used as the criterion for significance.
A test for statistical outliers was performed on parental body weights and feed consumption (in g/day) and parental and weanling offspring organ weights at necropsy. If examination of pertinent study data did not provide a plausible biologically sound reason for inclusion of the data flagged as “outlier,” the data were excluded from summarisation and analysis and were designated as outliers. - Indices:
- REPRODUCTIVE INDICES
- Estrous cycle length, precoital interval, gestational length, number of implantation sites/dam, number of pups/litter, percent postimplantation loss/litter, paired ovarian primordial follicle counts, epididymal sperm concentration
OFFSPRING VIABILITY INDICES
- Number of live pups/litter, day 4 survival index, number surviving on day 21. - Historical control data:
- Not reported.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Body weight: Body weights during the pre-breed and mating period were significantly reduced for F1 females at 7500 ppm. Body weights were significantly reduced during gestation/lactation for P, F1, and F2 parental females at 7500 ppm, during gestation and lactation at 750 ppm for P and F2 females and during lactation for the 750 ppm F1 parental females. At terminal sacrifice, body weights were reduced for all generations at 7500 ppm, and in F1 females and F1/F2 males at 750 ppm.
Reproductive performance: At 7500 ppm, there was a significant reduction in number of implants and number of total pups per litter in all generations. At 0.3 ppm, the number of implant sites per dam was significantly reduced in the F2 generation and the number of total pups per litter was significantly reduced in the F3 generation.
Non-reproductive organ weights: At 7500 ppm, absolute liver weights were significantly reduced for F3 females, relative liver weights were significantly increased in P and F2 females, absolute kidney weights were significantly reduced for all generations, and relative kidney weights were significantly increased for F0 and F2 females. Absolute and relative liver weights were significantly increased in F2 females at 0.015 ppm.
For organ weights, the authors noted that relative organ weights were affected by reduced body weights, and other changes in absolute and relative organ weights (other than liver, kidneys, adrenal glands, spleen, pituitary and brain) were not consistent across generations and did not exhibit a dose-response pattern.
Reproductive organ weights: At 7500 ppm, absolute ovary weights were significantly reduced for all generations, relative ovary weights were significantly reduced in the P, F1 and F2 generations, and absolute uterus weights were significantly reduced in P, F1 and F2 generations. Absolute and relative ovary weights were significantly reduced at 0.015, 4.5 and 75 ppm for F2 females, and absolute uterus weights were significantly reduced in P generation females at 0.015 ppm.
Histopathology: Slight to mild renal tubular degeneration and chronic hepatic inflammation were observed at a higher incidence in P, F1, and F2 females at 7500 ppm. - Dose descriptor:
- NOEL
- Remarks:
- Systemic
- Effect level:
- 75 ppm
- Based on:
- test mat.
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- Based on body weight reduction in adult females at 750 ppm
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 750 ppm
- Based on:
- test mat.
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- The EU RAR 2003 concluded that the NOAEL for systemic toxicity in this study is 50 mg/kg/day, based on consistent and significant reductions in body weight gain in both sexes and renal tubule degeneration in females only at 7500 ppm
- Dose descriptor:
- NOAEL
- Effect level:
- 750 ppm
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Viability: The number of live pups per litter (on postnatal day 0) was significantly reduced for the F1, F2, and F3 pups at 7500 ppm.
Body weight: Per litter pup body weights were reduced at 7500 ppm for F1, F2, and F3 offspring on postnatal days 7, 14 and 21. F1 litters also had significantly reduced pup body weights per litter on postnatal day 4 when all pups were analysed together, but not when the sexes were analysed separately.
Anogenital distance: Anogenital distance (AGD) was significantly increased in F2 females at all dose levels except 75 and 7500 ppm.
Sexual maturation: There was a significant delay in age at vaginal patency (VP) for F1, F2 and F3 females at 7500 ppm and for F2 females at 75 ppm. When adjusted for body weight at acquisition, VP was only delayed at 7500 ppm for all three generations. Male offspring had a significant delay in age at preputial separation (PPS) in the F1 generation at 750 and 7500 ppm, in the F2 generation at 0.3, 75, 750 and 7500 ppm, and in the F3 generation at 7500 ppm. When adjusted for body weight at acquisition, PPS was delayed in the F1 generation at 750 and 7500 ppm and in F2 and F3 generations at 7500 ppm.
Organ weights: Data was not shown, but according to the text, absolute organ weights were decreased at 7500 ppm for F1, F2, and F3 weanling males and females sacrificed on postnatal day 21. The authors noted that there were reductions in organ weights at lower doses, but they were not consistently affected in F1, F2, and F3 weanlings or reproducible in specific dose groups. Relative organ weights at 7500 ppm were increased, which the authors noted was caused by reduced body weights at this dietary dose. - Dose descriptor:
- NOAEL
- Effect level:
- 7 500 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: See remarks
- Remarks on result:
- other:
- Remarks:
- 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).
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The authors concluded that based on the results of this study, BPA should not be considered a selective reproductive toxicant.
- Executive summary:
Bisphenol A (BPA) was administered 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) in the diet ad libitum to 30 CD 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.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted in compliance with regulatory guidelines and in accordance with the principles for Good Laboratory Practice (GLP).
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 416 (Two-Generation Reproductive Toxicity Study)
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan, Inc. (Yokohama, Japan)
- Age at study initiation: 5 weeks (males); 10 weeks (females)
- Housing: Animals were housed individually (except during the acclimation, mating and nursing periods) in suspended stainless steel cages. From gestation day 17 to the day of weaning, individual dams and litters were reared using wood chips as bedding (White Flake, Charles River Japan, Inc.)
- Diet: Ad libitum gamma-irradiated basal diet (CRF-1, Oriental Yeast Col, Ltd., Tokyo, Japan). Contaminant levels of BPA were below the detection limit.
- Water: Ad libitum filtered tap water. Contaminant levels of BPA were below the detection limit.
- Acclimation period: 8 days (males); 15 days (females)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 50-60
- Photoperiod: 12 hrs dark / 12 hrs light) - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Rats were dosed once daily by gastric intubation with BPA dissolved in distilled water for 10 weeks (males) or 2 weeks (females) prior to the mating period.. The volume of each dose was adjusted to 2 ml/kg body weight based on the latest body weight measurement. Control rats received distilled water only. The stability of test formulations in a dark, cool place was confirmed for up to 21 days.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- During use, formulations of BPA were maintained in a dark, cool place for no more than 21 days, and were 90-105.3% of the target concentration.
- Details on mating procedure:
- The vaginal smears of each female rat were recorded daily for 2 weeks, and only rats showing repeated 4- or 5-day estrous cycles were used in the experiment. Each female was mated with a male rat of the same dosage group. The mating periods for P and F1 animals were 2 and 3 weeks, respectively. The presence of sperm in the vaginal smear and/or a vaginal plug was considered evidence of successful mating. At weaning on PND 22, F1 weanlings (25 per sex per group) were randomly selected to serve as parent animals for the F2 generation. For each group, one or two male and female F1 weanlings were selected from each P dam. F1 animals that did not mate during the 3-week mating period wer reassigned to an untreated partner and cohabited. For F1 matings, cohabitation of siblings was avoided.
- Duration of treatment / exposure:
- P: Rats were exposed for 10 weeks (males) or 2 weeks (females) prior to the mating period. Each female was mated with a male rat of the same dosage group, with exposure continuing throughout the mating period. Exposure was also continued throughout gestation and lactation.
F1: F1 animals were exposed in utero and during lactation, then direct exposure began on PND 23 and continued for 10 weeks prior to their mating period, as well as during mating, gestation, and lactation periods.
F2: F2 animals were exposed in utero and during lactation, then direct exposure began on PND 22 for 4 weeks (males) or 11 weeks (females). - Frequency of treatment:
- Once daily during treatment periods
- Duration of test:
- Two-generation
- No. of animals per sex per dose:
- 25
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Details on study schedule:
- Dose selection rationale: Doses were determined based on previous studies in which significant changes in reproductive parameters of offspring were found after maternal feeding of BPA at 2 and 20 ug/kg on days 11 to 17 of pregnancy in mice. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes
POST-MORTEM EXAMINATIONS: Yes
- Maternal animals: All surviving animals sacrificed after weaning of pups.
- Gross necropsy consisted of external and internal examinations.
- Weights of brain, thyroid, thymus, liver, spleen, heart, lung, uterus, kidneys, adrenals, and ovaries were recorded. Weight of the pituitary was measured after fixation.
- Ovary, uterus, vagina, hypothalamus, pituitary, liver, adrenal, thyroid, and mammary gland from P and F1 adults were prepared for microscopic examination.
SERUM HORMONE LEVELS:
Levels of testosterone, thyroxine (T4), triiodothyronine (T3), estradiol, prolactin (PRL), luteinising hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH) were measured in serum collected from 6 proestrous females randomly selected from the P and F1 animals on the day of scheduled terminal sacrifice.
OTHER:
- Estrous cyclicity: Daily vaginal lavage samples of each P and F1 female were evaluated for estrous cyclicity throughout the 2-week pre-cohabiation period and during cohabitation until evidence of copulation was detected. In F2 females, vaginal smears were also evaluated for 2 weeks at 9 to 11 weeks of age. - Fetal examinations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight, weight gain, and developmental landmarks (pinna detachment, incisor eruption, eye opening, testes descent, surface righting, negative geotaxis reflex, mid-air righting reflex, day of preputial separation or vaginal opening, and anogenital distance).
POST-MORTEM EXAMINATIONS: Yes
- The F1 offspring not selected as parental animals were sacrificed at 22 days of age.
- The F2 offspring not chosen for further study were sacrificed at 22 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination).
- Gross necropsy consisted of external and internal examinations.
- Brain, thyroid, thymus, liver, spleen, heart, lung, uterus, prostate, testes, kidneys, adrenals, ovaries, and epididymes weights were recorded in one male and one female F1 and F2 animal selected from each dam. In these F2 male weanlings, histopathologic examinations were performed on the seminal vesicle and coagulating gland in all groups.
BEHAVIOURAL TESTING:
A behavioural test in an open field was conducted on three successive days in all F1 rats at 5 to 6 weeks of age. A test in a water-filled multiple T-maze was conducted in 6 F1 rats per sex per group at 6 to 7 weeks of age.
SERUM HORMONE LEVELS:
Levels of testosterone, thyroxine (T4), triiodothyronine (T3), estradiol, prolactin (PRL), luteinising hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH) were measured in serum collected from 6 males and 6 proestrous females randomly selected from the F1 animals on the day of scheduled terminal sacrifice. - Statistics:
- Statistical analysis of offspring before weaning was carried out using the litter as the experimental unit.
The incidence of females with normal estrous cycles, copulation and fertility, gestation indices, and the neonatal sex ratio were analysed by Chi-square test or Fisher's exact test.
All other parameters were analysed for statistical significance as follows: Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent variances at the 5% level of significance. If the variances were equivalent, the groups were compared by one-way ANOVA. If significant differences were found, Dunnett's multiple comparison test was performed. If the groups did not have equivalent variances, the Kruskal-Wallis test was used to assess the overall effects. Whenever significant differences were noted, pair-wise comparisons were made by Mann-Whitney U-test.
The 0.05 level of probability was used as the criterion for significance. - Indices:
- REPRODUCTIVE INDICES:
Estrous cycle length, precoital interval, copulation index, fertility index, gestation index, gestation length, number of implantation sites/dam, delivery index, epididymal sperm counts.
OFFSPRING VIABILITY INDICES:
Number of pups delivered, viability index during lactation. - Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs of toxicity and no significant mortality in any generation.
BODY WEIGHT AND FOOD CONSUMPTION
Food consumption was decreased in P females at 20 ug/kg during weeks 2 and 3; in F1 females at 200 ug/kg during week 7; and in F2 females at 20 ug/kg during weeks 4 and 5. These changes were unaccompanied by changes in body weight or body weight gain. These changes were also occasional and inconsistent and seem unlikely to be toxicologically significant.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE
There was a decrease in the incidence of females having normal estrous cycles in F1 animals at 20 ug/kg BPA.This decrease was not dose-dependent or consistent across generations, and BPA did not affect reproductive outcome, so it was concluded that BPA did not adversely affect the estrous cycle.
REPRODUCTIVE PERFORMANCE
Gestation length was decreased in F1 animals at 20 ug/kg BPA. This effect was also not dose-dependent or consistent across generations, and did not affect reproductive outcome, so it was concluded that BPA did not adversely affect reproductive performance.
ORGAN WEIGHTS
F1 adult females had decreased ovarian weight at 0.2 ug/kg, but relative organ weight was not affected at any dose, so it was concluded that BPA did not affect organ weights.
OTHER FINDINGS
P generation females had decreased serum LH concentrations at 0.2, 2, and 20 ug/kg and decreased serum T3 concentrations at 200 ug/kg; however, these changes were not dose-dependent and were inconsistent across sexes and generations, so it was concluded that these changes are not biologically significant. - Dose descriptor:
- NOAEL
- Effect level:
- 0.2 mg/kg bw/day
- Basis for effect level:
- other: other:
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
BODY WEIGHT
Male F1 pups had decreased body weight at 20 ug/kg on PND 14 and 21. Because this change was inconsistent and occasional, it is unlikely to be toxicologically significant.
SEXUAL MATURATION
Testes descent was delayed in F2 male pups at 20 and 200 ug/kg. The AGD per cube root of body weight ratio in F1 males was decreased at 0.2 ug/kg on PND 57 and at 20 ug/kg on PND 106, 113, and the day of sacrifice. This ratio was also decreased in F1 females at 200 ug/kg on PND2 and increased in F1 females at 2 and 20 ug/kg on PND7. In F2 females, this ratio was decreased at 20 ug/kg on PND 64, 71, 85, and 92 and on the day of sacrifice, and at 200 on PND 57, 64, and the day of sacrifice. These changes were occasional and inconsistent across sexes and generations and are not BPA-related or toxicologically significant.
ORGAN WEIGHTS
F1 male weanlings had decreased absolute lung weight at 20 and 200 ug/kg and decreased absolute kidney weight 20 ug/kg. In F2 male weanlings, lung weight was decreased at 20 ug/kg and relative heart weight was decreased at 200 ug/kg. At 2 ug/kg in F2 males, increased relative liver weight, decreased absolute and relative weights of the seminal vesicle, and decreased weight of the thyroid glands were observed. These changes were occasional and inconsistent, and it was concluded that BPA did not affect organ weights. - Dose descriptor:
- NOAEL
- Effect level:
- 0.2 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicologically significant effects were observed at any dose studied.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The authors concluded that, under the conditions of this study, no adverse effects of BPA on reproduction or development were detected.
- Executive summary:
In this rat two-generation reproductive study, groups of 25 male and 25 female rats were given BPA at 0.2, 2, 20, or 200 ug/kg-day by gastric intubation throughout the study beginning at the onset of a 10- and 2 -week premating period, in P generation males and females, respectively, and continuing through the mating, gestation, and lactation periods. No BPA-related clinical signs or effects on body weight or food consumption were observed in any generation. There were no BPA-related changes in surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna detachment, incisor eruption, eye opening, testes descent, preputial separation, or vaginal opening in F1 and F2 generations, or behaviour in the open field or water-filled multiple T-maze in the F1 generation. No BPA-related changes in estrous cyclicity, copulation index, fertility index, number of implantations, gestation length, litter size, pup weight, pup sex ratio, pup viability, or other functional reproductive measures were noted in any generation. A few statistically significant changes in the AGD per cube root of body weight ratio were found at 0.2 and 20 ug/kg in F1 males, at 2, 20, and 200 ug/kg in F1 females, and at 20 and 200 ug/kg in F2 females; however, the changes in AGD were consistently small (within 5% of control values), and no continuous changes in the AGD or AGD per cube root of body weight were detected. There were no BPA-related changes in epididymal sperm counts or motility in P and F1 males. No BPA-related necropsy findings or effects on organ weight including the reproductive organs were found in any generation. Histopathologic examinations revealed no evidence of BPA-related changes in any organs including the reproductive organs of both sexes. The data indicate that oral doses of BPA between 0.2 and 200 ug/kg over two generations did not cause significant compound-related changes in reproductive or developmental parameters in rats.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- according to guideline
- Guideline:
- other: guideline-compliant research standard conducted at the FDA designed by NCTR and NIEHS scientists
- Principles of method if other than guideline:
- Rats were obtained as weanlings from the NCTR breeding colony and placed under study conditions (soy- and alfalfa-free diet (5K96, LabDiet, Purina Mills), polysulfone cages, hardwood chip bedding, glass water bottles, and food-grade silicone stoppers) until mating. Study materials were monitored for background BPA levels; the only material with detectable levels of BPA was the diet, which had less than 3 ppb BPA. Prior to mating to males that were not siblings or first cousins, female rats were stratified by body weight and were randomized to treatment groups to give approximately equivalent mean starting body weights in each group. Each morning after pairing, females were examined for evidence of mating (presence of an in situ vaginal plug or sperm-positive vaginal smear). Upon evidence of mating, the females were separated from the males and individually housed; this day was considered GD 0. Daily gavage dosing of the dams began on GD 6 and continued until the initiation of parturition.
Pups without evident malformations were randomly culled to a maximum of five males and five females on PND 1. Direct gavage dosing of these pups started on PND 1. This post-natal study arms (= Core study) are described in details in chapters 7.5.1 and 7.7 because no mating of the animals was part of the Core study.
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The two high BPA dosing suspensions were mixed by directly adding BPA solid to the vehicle with sonication, and the suspensions were stirred constantly. The three low BPA and the two EE2 dosing solutions were mixed by serial dilutions of stock solutions. - Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- CD23/NctrBR
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: National Center for Toxicological Research (NCTR) breeding colony (Jefferson, AR)
- Age at study initiation: at weaning on PND 21; 10–15 weeks of age at mating
- Weight at study initiation:
- Fasting period before study:
- Housing: housed two to three per cage in polysulfone cages with hardwood chip bedding, and given water from glass water bottles with silicone stoppers.The rats were held under these conditions until they were individually housed prior to mating.
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: one to two weeks for females, or for 48 hours for males
ENVIRONMENTAL CONDITIONS
Temperature: 23°± 3°C
Relative humidity: 50% ± 20%
Room fluorescent light: 12 hours/day (on 6 AM, off 6 PM)
Room air changes: at least 10/hour
- Route of administration:
- other: gavage with modified Hamilton Microlab® ML511C programmable 115 V pumps
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- used as a 0.3% (w/w) aqueous solution
- Details on exposure:
- - Concentration in vehicle:
The target concentrations of the dose preparations were 0.5, 5, 50, 500, and 5000 μg/mL 0.3% CMC for the 2.5, 25, 250, 2,500, and 25,000 μg/kg bw/day dose groups, respectively. For EE2, the target concentrations were 0.01 and 0.1 μg/mL 0.3% CMC for the 0.05 and 0.5 μg/kg bw/day dose groups, respectively.
- Amount of vehicle (if gavage):
5 mL/kg bw/day
- Diet Assessment: Contaminants, Including Background BPA:
Pelleted rodent chow, verified casein diet 10 IF, irradiated, 5K96. The 5K96 diet, which is low in soy-derived phytoestrogens, was selected to ensure a consistent and low level of these phytoestrogens to minimize any impact on the endpoints measured in this study.The manufacturer provided analyses for selected nutritive quality attributes (including protein, fat, crude fiber, calcium, phosphorous, and vitamins A, B1, and E) and contaminants (including nitrosamines, fumonisins, arsenic, cadmium, lead, mercury, aflatoxins, organochlorine and organophosphate pesticides, butylated hydroxyanisole, butylated hydroxytoluene, and tert-butyl hydroquinone).
On arrival at NCTR, each lot of diet used in the present study was sampled and assayed by the Chemistry Support Group for background BPA and selected phyto- and myco-estrogens (genistein, daidzein, coumestrol, and zearalenone).
In addition to the dosing vehicle and diet, the rodent drinking water and extracts of animal cages and bedding (hardwood chip and cellulose) were assayed for background BPA levels. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- At the start of the study and approximately every 8–10 weeks over the course of the study, all dose level preparations were assayed by the Chemistry Support Group prior to delivery to the animal rooms and certified to be within 10% of the target concentration with a % CV of ≤10%. In addition, at intervals spaced 4-7 months apart, BPA and EE2 dosing preparations from the animal rooms were assayed at the end of their use to verify the dose concentrations
- Details on mating procedure:
- Females were placed in solid-bottomed polysulfone cages with hardwood chip bedding with the assigned males and were assessed daily for up to 10 days for sperm-positive vaginal smears or a copulation plug that precluded a vaginal smear.
- Duration of treatment / exposure:
- F0 dams: on GD 6 (GD 0 = sperm- or plug-positive day) and continued until initiation of parturition (GD 21)
F1 pubs: from PND 1 (for further information see chapter 7.5.1 and 7.7) - Frequency of treatment:
- once daily
- Duration of test:
- Until litters were weaned (PND 21) or after GD 26 if no litter was delivered.
- Dose / conc.:
- 2.5 other: µg/kg bw/day
- Dose / conc.:
- 25 other: µg/kg bw/day
- Dose / conc.:
- 250 other: µg/kg bw/day
- Dose / conc.:
- 2 500 other: µg/kg bw/day
- Dose / conc.:
- 25 000 other: µg/kg bw/day
- No. of animals per sex per dose:
- 70-78 dams/dose from which data were collected
- Control animals:
- yes
- yes, concurrent vehicle
- Details on study design:
- This study is designed to provide pubs with prenatal exposure to the test substance followed by daily treatment from PND 1.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
BODY WEIGHT: Yes
- Time schedule for examinations: daily from GD 6 through parturition
POST-MORTEM EXAMINATIONS (dead and moribund dams): Yes
- Weighed organs : adrenals, brain, heart, kidneys, liver, ovaries (with oviducts), pituitary (after 48-hour fixation), spleen, thymus, thyroid with parathyroid (after 48-hour fixation), uterus (blotted), ovarian and parametrial (combined), and retroperitoneal fat pads.
-Histopatholgy: adrenals, aorta (thoracic), bone marrow (femur), brain, heart, kidneys, liver, 5th left mammary gland (inguinal), ovaries, oviduct, pancreas, parathyroid, pituitary, spleen, thymus, thyroid, uterus, and vagina. In addition the following tissues were examined microscopically to assess possible cause of death in all dead or moribund animals: esophagus, colon, ileum, lung, nose, stomach, trachea, and any gross lesions.
- Ovaries and uterine content:
- The uterine was examined after termination: Yes
Examinations included: implantation sites were counted
- Number of implantations: Yes - Fetal examinations:
- - External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No - Statistics:
- All analyses and adjustments for multiple comparisons were performed separately for the BPA and EE2 treatments. Dunnett’s method was used to adjust for multiple comparisons. Tests of trend, for increasing treatment effect with increasing dose, were performed for thevehicle and BPA groups. All tests were performed as two-sided tests at the 0.05 significance level.
Litter size (number alive) and numbers of males and females were analyzed using Poisson regression.
Unsexed pups (i.e., pups that could not be definitively assigned as male or female) were assigned as males for analysis of sex proportions and of female and male counts.
Sex proportions were analyzed using logistic regression.
Implantation site counts and litter weight data were analyzed using a one-way ANOVA and litter mean pup weights were analyzed using contrasts within an ANOCOVA, with litter size as a covariate, to test for treatment effect. Dunnett’s test was used for comparisons to the vehicle control group to adjust for multiple comparisons. Sex ratios of pups within litters were analyzed for treatment effects using logistic regression. Pup counts (number alive, males, females, number unsexed (i.e., pups that could not be definitively assigned as male or female), and number born dead) were analyzed using Poisson regression. For analyses of sex proportions and female and male counts, unsexed pups were assigned either as male sex or female sex in separate runs with comparable results - Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- not specified
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- The number of implantation sites in mated dams did not differ across BPA or EE2 treatment groups and control groups as expected given that treatment did not begin until after implantation.
- Total litter losses by resorption:
- not specified
- Early or late resorptions:
- not specified
- Dead fetuses:
- not specified
- Changes in pregnancy duration:
- not specified
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified - Changes in number of pregnant:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- 25 000 other: µg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- Fetal body weight changes:
- not specified
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed - Reduction in number of live offspring:
- not specified
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- not specified
- External malformations:
- not examined
- Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- 25 000 other: µg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- changes in sex ratio
- fetal/pup body weight changes
- changes in litter size and weights
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
This part of the 2 Year BPA Toxicity Study with Sprague-Dawley rats is designed to provide pubs with prenatal exposure to the test substance for the “core” guideline-compliant chronic study (see chapter 7.7 and 7.8.1). For that purpose dams were daily dosed by gavage with 2.5, 25, 250, 2,500, and 25,000 μg BPA/kg body weight (bw)/day. Also reference estrogen groups (0.05 and 0.5 µg ethinyl estradiol/kg bw/day) and vehicle control group ( 0.3% CMC) were included. Dosing of the dams began on GD 6 and continued until the initiation of parturition.
Dam body weights during pregnancy were not affected by BPA or EE2 treatment. The number of implantation sites in mated dams did not differ across BPA or EE2 treatment groups and control groups as expected given that treatment did not begin until after implantation. There were no treatment effects on litter size, sex ratio, litter weight by sex, or mean pup weight at birth by sex up to and including
25,000 μg BPA/kg body weight (bw)/day.
.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Acceptable, well-documented study report which meets basic scientific principles
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Developmental toxicity was evaluated in CD rat dams (at least 20 per group) dosed daily via gavage with Bisphenol A (0, 160, 320, 640, or 1280 mg/kg-day) on gestational days (GD) 6 to 15. Dams were sacrificed on GD 20 and all fetuses were examined for external, visceral, and skeletal malformations.
- GLP compliance:
- no
- Species:
- rat
- Strain:
- other: COBS CD (SD)BR outbred albino
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, New York, United States)
- Weight at study initiation: 200-275 g
- Housing: Animals were housed four per cage in solid-bottom polypropylene or polycarbonate cages with stainless steel lids. Ab-Sorb-Dri cage bedding (Laboratory Products, Garfield, New Jersey, United States) was used.
- Diet: Purina Certified Rodent Chow, ad libitum
- Water: Deionized/filtered water, ad libitum
- Acclimation period: Seven days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 20-62
- Air changes (per hr): 12-14
- Photoperiod: 12 hours light/12 hours dark - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- All treatments were administered by gavage in a dose volume of 5 ml/kg body weight using a 16-gauge 2-inch curved stainless steel dosing tube (Perfektum, Popper and Sons, New Hyde Park, New York, United States).
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- BPA was suspended in food grade corn oil. A 5 ml aliquot of each suspension was analyzed to verify the concentration prior to dosing on GD 6 and additional samples were analyzed at the end of the dosing period to confirm stability.
- Details on mating procedure:
- Individual females in estrus or proestrus were placed overnight in the home cage of singly-housed males of the same strain and source. The next morning, the presence of sperm was examimed via vaginal smear. Sperm-positive females were group housed (four per cage) with other sperm-positive females of the same treatment group. The day on which sperm were found was designated as GD 0.
- Duration of treatment / exposure:
- Single oral gavage treatment per day
- Frequency of treatment:
- Daily
- Duration of test:
- GD 6 to 15
- No. of animals per sex per dose:
- At least 20
- Control animals:
- yes, concurrent vehicle
- Maternal examinations:
- Body weight and clinical signs during dosing. Body weight, liver weight, and clinical signs at sacrifice.
- Ovaries and uterine content:
- Number of corpora lutea, gravid uterine weight, number of implantation sites, resorptions, dead fetuses, and live fetuses.
- Fetal examinations:
- Weight, gross morphological abnormalities, visceral malformations, skeletal malformations.
- Statistics:
- Parametric evaluation of the dose effect, replicate effect, and dose x replicate interaction was conducted on selected measures using appropriate ANOVA designs. General linear model analysis was used to determine the significance of the dose-response relationship. When significant (p<0.05) main effects for dose occurred, Williams' and/or Dunnett's multiple comparison test was used for comparison of BPA-exposed groups versus controls. A one-tailed test was used for all parameters except measures of maternal body and organ weight, fetal body weight, and percent males per litter for which a two-tailed test was used. Nominal scale measures were analyzed by Chi-Square Test for Independence for differences among treatment groups. When Chi-Square revealed significant differences among treatment groups, a one-tailed Fisher's Exact Test was used for pairwise comparisons between each BPA-treated group and control.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Dams in the high dose group (1280 mg/kg-day) exhibited an unexpectedly hight mortality rate of 26% , and this dose group was excluded from data used to make conclusions regarding the teratogenic potential of BPA. For doses up to 640 mg/kg-day, maternal body weight exhibited a statistically significant downward trend on GD 11, 15, and 20, with a statistically significant dose effect in all BPA-treated groups on GD 11 and 15. Measures of maternal weight gain displayed a dose-related downward trend, with a statistically significant dose effect on maternal weight gain during gestation and treatment and on absolute weight gain in all three BPA-treated groups. No other maternal effects were observed. - Dose descriptor:
- LOAEL
- Effect level:
- 160 mg/kg bw/day (nominal)
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 640 mg/kg bw/day (nominal)
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
BPA had no effect on the various parameters examined in fetuses. - Dose descriptor:
- NOAEL
- Effect level:
- 640 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remakt
- Remarks on result:
- other:
- Remarks:
- Dams in the high dose group (1280 mg/kg-day) exhibited an unexpectedly hight mortality rate of 26% , and this dose group was excluded from data used to make conclusions regarding the teratogenic potential of BPA. The authors concluded that BPA did not increase fetotoxicity and did not affect the incidence of malformations in CD rats, even at doses which caused significant maternal toxicity, and that BPA does not present a selective risk to the fetus.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The authors concluded that BPA did not increase fetotoxicity and did not affect the incidence of malformations in CD rats, even at doses which caused significant maternal toxicity, and that BPA does not present a selective risk to the fetus.
- Executive summary:
Developmental toxicity was evaluated in CD rat dams (>20 per group) dosed daily via gavage with BPA (0, 160, 320, 640, or 1280 mg/kg-day) on gestational days (GD) 6 to 15. Dams were sacrificed on GD 20 and all fetuses were examined for external, visceral, and skeletal malformations. Dams in the high dose group (1280 mg/kg-day) exhibited an unexpectedly hight mortality rate of 26% , and this dose group was excluded from data used to make conclusions regarding the teratogenic potential of BPA. For doses up to 640 mg/kg-day, maternal body weight exhibited a statistically significant downward trend on GD 11, 15, and 20, with a statistically significant dose effect in all BPA-treated groups on GD 11 and 15. Measures of maternal weight gain displayed a dose-related downward trend, with a statistically significant dose effect on maternal weight gain during gestation and treatment and on absolute weight gain in all three BPA-treated groups. No other maternal effects were observed, and BPA had no effect on the various parameters examined in fetuses. The authors concluded that BPA did not increase fetotoxicity and did not affect the incidence of malformations in CD rats, even at doses which caused significant maternal toxicity, and that BPA does not present a selective risk to the fetus.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at FDA Laboratory in Jefferson, AR
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- Dehavioural endpoints investigated in offspring; motor activity, Barnes maze, arcustic startle and Water maze.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- After the pairing, the female and the cage bottom were visually examined for a sperm plug. Because all breeding occurred prior to any treatment, breeding success or failure was unrelated to treatment.
- Duration of treatment / exposure:
- Dam: GD6 to delivery; offspring PND1 to 21.
- Frequency of treatment:
- daily
- Duration of test:
- Novelty preference: PND 26-29; open field activity: PND40-42; motor coordination: PND43-33; Barnes maze: PND 47–50; Arcustic startle: PND 54; water maze performance: PND 75-79
- No. of animals per sex per dose:
- n=11-12/sex/group
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- other: Positive control 5 µg/kg/day EE2... (see attached file)
- Dose descriptor:
- other: behavioural observations in opffsprinig
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- other: behavioural observations in opffsprinig
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: behavioural observations in opffsprinig
- Remarks on result:
- other:
- Remarks:
- results indicate few consistent or dose-related effects resulting from developmental treatment with Bisphenol A at these doses.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
Widespread Bisphenol A (BPA) exposure necessitates increased knowledge of its potential effects for better risk assessment and regulatory guidance. Here, female Sprague-Dawley rats, reared in low exogenous estrogen environments and bred at adulthood, were gavaged on gestational days 6-21 with vehicle (VEH), 2.5 or 25.0 μg/kg/day BPA, or 5.0 or 10.0 μg/kg/day ethinyl estradiol (EE2). Offspring were orally treated on postnatal days (PNDs) 1-21 with the same dose their dam received. A naïve control group (NC) was not gavaged. Postweaning, one offspring/sex/litter (n=11-12/sex/group) was assessed for the typical behaviors measured in developmental neurotoxicology studies. At PND 29, novelty preference was unaffected by treatment; however, relative to the VEH group, males and females of both EE2 groups were more active. VEH males appeared somewhat hypoactive in open field assessments at PNDs 40-42 and, as a result, males of the BPA and EE2 groups were significantly more active. Latency to locate the Barnes maze escape box at PNDs 47-50 was increased in males and females of the 5.0 μg/kg/day EE2 group. Relative to other male groups, VEH males exhibited an increased startle response on the first trial block at PND 54 and thus, males of both BPA groups and the 10.0 μg/kg/day EE2 group exhibited a significantly decreased startle response. PNDs 43- 44 motor coordination and PNDs 75-79 water maze performance were unaffected by treatment. These results indicate few consistent or dose-related effects resulting from developmental treatment with BPA at these doses. Few of these behaviors, however, were sexually dimorphic which may prove more sensitive.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20014
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at FDA Laboratory in Jefferson, AR
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- Body weight, food, and water intake: until PND 69; Pubertal landmarks; Estrous cyclicity on PNDs 82–109; Serum hormone measures Levels were measured in males of replicates 1–3 at PND 110 and the remaining replicates (i.e., 4–9) at PND 90. Levels were measured in all females at PND 110 (Total thyroxine (T4), total triiodothyronine (T3), total estradiol (E2), total testosterone, corticosterone, and luteinizing hormone (LH) were analyzed). Whole and regional brain weights PND34. Play behavior on PND 34. Residential running wheel activity: PNDs 58–69; Intake of flavored solutions: PNDs 59–62; Manual lordosis measures: PNDs 96–100. Female sex behavior. Female no. 2 was assessed for maleelicited sex behavior on PNDs 96–100.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- Following pairing, the female and the cage bottom were visually examined for a sperm plug. Breeding occurred prior to any treatment; thus, success or failure was not treatment-related. Females for which a sperm plug was detected were randomly assigned to one of the six treatment groups within their body weight stratum. This assignment yields approximately equal dam body weight distributions across treatments.
- Duration of treatment / exposure:
- dams: GD6-parturition; offspring: PND1-21
- Frequency of treatment:
- daily
- Duration of test:
- Up to PND 110
- No. of animals per sex per dose:
- n=12/sex; vehicle control n = 12/sex; 2.5 BPA n = 11/sex; 25.0 BPA n = 12/sex; 5.0 EE2 n = 11/sex; 10.0 EE2 n = 12/sex.
- Dose descriptor:
- NOAEL
- Remarks:
- Bisphenol A doses and design used here produced few alterations.
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- other: Bisphenol A doses and design used here produced few alterations.
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- Only EE2 treatment caused significant effects. Bisphenol A treatment did not alter any measured endpoint. Authors indicate "Similar to our previous reports (Ferguson, S. A., Law, C. D. Jr and Abshire, J. S. (2011) Developmental treatment with Bisphenol A or ethinyl estradiol causes few alterations on early preweaning measures. Toxicol. Sci. 124, 149–160; Ferguson, S. A., Law, C. D. and Abshire, J. S. (2012) Developmental treatment with Bisphenol A causes few alterations on measures of postweaning activity and learning. Neurotoxicol. Teratol. 34, 598–606), the Bisphenol A doses and design used here produced few alterations."
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
The developing central nervous system may be particularly sensitive to bisphenol A (BPA)-induced alterations. Here, pregnant Sprague Dawley rats (n = 11–12/group) were gavaged daily with vehicle, 2.5 or 25.0 μg/kg BPA, or 5.0 or 10.0 μg/kg ethinyl estradiol (EE2) on gestational days 6–21. The BPA doses were selected to be below the no-observed-adverse-effect level (NOAEL) of 5 mg/kg/day. On postnatal days 1–21, all offspring/litter were orally treated with the same dose. A naïve control group was not gavaged. Body weight, pubertal age, estrous cyclicity, and adult serum hormone levels were measured. Adolescent play, running wheel activity, flavored solution intake, female sex behavior, and manually elicited lordosis were assessed. No significant differences existed between the vehicle and naïve control groups. Vehicle controls exhibited significant sexual dimorphism for most behaviors, indicating these evaluations were sensitive to sex differences. However, only EE2 treatment caused significant effects. Relative to female controls, EE2-treated females were heavier, exhibited delayed vaginal opening, aberrant estrous cyclicity, increased play behavior, decreased running wheel activity, and increased aggression toward the stimulus male during sexual behavior assessments. Relative to male controls, EE2-treated males were older at testes descent and preputial separation and had lower testosterone levels. These results suggest EE2-induced masculinization/defeminization of females and are consistent with increased volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) at weaning in female siblings of these subjects (He, Z., Paule, M. G. and Ferguson, S. A. (2012) Low oral doses of bisphenol A increase volume of the sexually dimorphic nucleus of the preoptic area in male, but not female, rats at postnatal day 21.Neurotoxicol. Teratol. 34, 331–337). Although EE2 treatment caused pubertal delays and decreased testosterone levels in males, their behaviors were within the range of control males. Conversely, BPA treatment did not alter any measured endpoint. Similar to our previous reports (Ferguson, S. A., Law, C. D. Jr and Abshire, J. S. (2011) Developmental treatment with bisphenol A or ethinyl estradiol causes few alterations on early preweaning measures. Toxicol. Sci. 124, 149–160; Ferguson, S. A., Law, C. D. and Abshire, J. S. (2012) Developmental treatment with bisphenol A causes few alterations on measures of postweaning activity and learning. Neurotoxicol. Teratol. 34, 598–606), the BPA doses and design used here produced few alterations.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at FDA Laboratory in Jefferson, AR
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- On PND 21, 1/sex/litter was perfused and the brain removed. TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. In a separate group anteroventral periventricular nucleus (AVPV) volume was quantified.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- See Ferguson et al.,2011, 2012, 2014
- Duration of treatment / exposure:
- Dams: GD6-parturition; offspring: PND1-21
- Frequency of treatment:
- daily
- Duration of test:
- PND21
- No. of animals per sex per dose:
- naïve control (12/sex), vehicle control (15/sex) ,BPA 2.5 (11 males,14 females), BPA25.0 (13/sex), EE2 5.0 (10 males,12 females),EE2 10.0 (15 males,13 females).
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- other: Positive control 5 µg/kg/day EE2... (see attached file)
- Dose descriptor:
- NOAEL
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. ANOVA results indicated significantly higher TH-ir cells/mm2 in females (main effect of sex: p<0.01); however, there was no significant effect of treatment or a significant interaction of treatment with sex. In a separate untreated group of PND 21 Sprague-Dawley pups, AVPV volume was quantified and no significant sexual dimorphism was apparent. Similar to our reported results of behavioral assessments, the Bisphenol A treatment paradigm used here (2.5 or 25.0 µg Bisphenol A/kg/day administered orally) does not appear to cause significant alterations in AVPV TH-ir.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
Exposure to Bisphenol A (BPA) may interfere with brain sexual differentiation. Altered numbers of tyrosine hydroxylase (TH) cells in the rodent anteroventral periventricular nucleus (AVPV) after developmental BPA treatment have been reported; however, definitive conclusions are lacking. The current study incorporated many of the guidelines suggested for endocrine disrupter research. Specifically, ethinyl estradiol (EE2) served as a reference estrogen, exogenous environmental estrogen exposure was controlled, BPA was administered orally, and subjects consumed a low phytoestrogen diet. Here, on gestational days 6–21, Sprague-Dawley rats (10–15/group) were gavaged with 2.5 or 25.0 µg BPA/kg/day or 5.0 or 10.0 µg EE2/kg/day or the vehicle (5 ml of 0.3% aqueous carboxymethylcellulose/kg/day). A naïve control group was weighed and restrained, but not gavaged. Beginning on postnatal day (PND) 1 and continuing until PND 21, the 4 pups/sex/litter were orally treated with the same dose their dam had received. On PND 21, 1/sex/litter was perfused and the brain removed. TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. ANOVA results indicated significantly higher TH-ir cells/mm2 in females (main effect of sex: p<0.01); however, there was no significant effect of treatment or a significant interaction of treatment with sex. In a separate untreated group of PND 21 Sprague-Dawley pups, AVPV volume was quantified and no significant sexual dimorphism was apparent. Similar to our reported results of behavioral assessments, the BPA treatment paradigm used here (2.5 or 25.0 µg BPA/kg/day administered orally) does not appear to cause significant alterations in AVPV TH-ir.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20111
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at FDA Laboratory in Jefferson, AR
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- SD rat dams were gavaged on gestational days (GDs) 6-21 with vehicle (VEH), 2.5 or 25.0 μg/kg/day Bisphenol A, or 5.0 or 10.0 μg/kg/day ethinyl estradiol (EE2). Offspring were orally treated on PNDs 1-21 with the same dose the dam received. Parameters investigated included: body weight, PND 1 absolute anogenital distance and anogenital index, ages at fur development and eye and ear opening, PNDs 3-6 righting latencies, PNDs 8-11 slant board behavior, PND 21 whole or regional brain weights and estradiol, testosterone, corticosterone, T3, T4, luteinizing hormone, ghrelin or leptin.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.3% (by weight) aqueous solution of carboxymethylcellulose sodium salt (CMC, high viscosity)
- Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- Beginning on PND 87 or 90, each female was placed into a wire bottom cage with a PND 90 male that had been placed into the cage 72 h earlier for habituation. The pair was housed together for 24 h, after which the female and the cage bottom were visually examined for a sperm plug. If a sperm plug was detected, the female was removed, weighed, and housed individually, and this day was termed as gestational day (GD) 0. If no sperm plug was detected, the female was returned to her home cage and used in the next breeding round (2 weeks later). If no sperm plug was detected after two breeding
rounds, the female was euthanized. Males were used for a maximum of three breeding rounds after which they were euthanized. Because all breeding occurred prior to any treatment, breeding success or failure was unrelated to treatment. - Duration of treatment / exposure:
- After breeding at adulthood, dams were gavaged on gestational days (GDs) 6-21 with vehicle (VEH), 2.5 or 25.0 μg/kg/day Bisphenol A, or 5.0 or 10.0 μg/kg/day ethinyl estradiol (EE2). Offspring were orally treated on PNDs 1-21 with the same dose the dam received.
- Frequency of treatment:
- daily
- Duration of test:
- PND21
- No. of animals per sex per dose:
- Number of litter maintained until weaning: 13-19
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- other: Positive control EE2 5 µg/kg/day... (see attached file)
- Dose descriptor:
- NOAEL
- Effect level:
- > 0.025 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Basis for effect level:
- other: other:
- Dose descriptor:
- NOAEL
- Effect level:
- > 0.025 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- These results add to the literature indicating developmental BPA treatment at these doses has no effects on gestational or lactational body weight, offspring anogenital distance, preweaning behaviors or hormone levels and whole and regional brain weights measured at weaning.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
Since Bisphenol A (BPA) exposure is nearly ubiquitous, increased knowledge of its potential effects on development will enable better risk assessment and regulatory guidance. Here, Sprague-Dawley rats were reared in low exogenous estrogen environments. After breeding at adulthood, dams were gavaged on gestational days (GDs) 6-21 with vehicle (VEH), 2.5 or 25.0 μg/kg/day BPA, or 5.0 or 10.0 μg/kg/day ethinyl estradiol (EE2). Offspring were orally treated on PNDs 1-21 with the same dose the dam received. Relative to the VEH group, dams of both EE2-treated groups weighed less throughout gestation and lactation. PND 1 absolute anogenital distance and anogenital index were unaltered by any treatment. Ages at fur development and eye and ear opening were unaffected by any treatment. Despite a significant treatment effect, no group was significantly different from VEH in PNDs 3-6 righting latencies; although, males had shorter latencies and all latencies decreased with age. PNDs 8-11 slant board behavior was unaffected by any treatment; however, males had shorter turning latencies and latencies decreased with age. Preweaning body weights of BPA- and EE2-treated groups as well as naïve controls were less than VEH. No treatment affected PND 21 whole or regional brain weights or levels of estradiol, testosterone, corticosterone, T3, T4, luteinizing hormone, ghrelin or leptin. These results add to the literature indicating developmental BPA treatment at these doses has no effects on gestational or lactational body weight, offspring anogenital distance, preweaning behaviors or hormone levels and whole and regional brain weights measured at weaning.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Research Conducted at FDA Laboratory in Jefferson, AR
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Effects of pre- and postnatal treatment with low Bisphenol A doses on sexually dimorphic nucleus of the preoptic area (SDN-POA) volume was investigated in PND 21 rat offspring.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Details on mating procedure:
- Detailed methods concerning animals, housing, diet, breeding, and treatment have been published (Ferguson et al., 2011).
- Duration of treatment / exposure:
- Dams: GD6-parturition; offspring: PND1-21
- Frequency of treatment:
- daily
- Duration of test:
- PND21
- No. of animals per sex per dose:
- n=10–15/sex/group
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- other: Positive controlö 5 µg/kg/day EE2... (see attached file)
- Dose descriptor:
- NOAEL
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- No observation in maternal animals.
- Dose descriptor:
- LOEL
- Remarks:
- SDN-POA volume on PND21
- Effect level:
- 0.025 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- Pairwise comparisons of the significant treatment by sex interaction indicated that neither Bisphenol A dose affected female volume. However, females treated with 5.0 or 10.0 μg/kg EE2 exhibited volumes that were larger than same-sex controls, respectively (p<0.001). Males treated with either Bisphenol A dose or 10.0 μg/kg/day EE2 had larger volumes than same-sex controls (p<0.006). These treatment effects might lead to organizational changes within sexually dimorphic neuroendocrine pathways which, if persistent, could theoretically alter adult reproductive physiology and socio-sexual behavior in rats. LOEL0.0025 mg/kg
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
Perinatal treatment with relatively high doses of bisphenol A (BPA) appears to have little effect on volume of the rodent sexually dimorphic nucleus of the preoptic area (SDN-POA). However, doses more relevant to human exposures have not been examined. Here, effects of pre- and post-natal treatment with low BPA doses on SDN-POA volume of postnatal day (PND) 21 Sprague-Dawley rats were evaluated. Pregnant rats were orally gavaged with vehicle, 2.5 or 25.0 μg/kg BPA, or 5.0 or 10.0 μg/kg ethinyl estradiol (EE2) on gestational days 6-21. Beginning on the day after birth, offspring were orally treated with the same dose their dam had received. On PND 21, offspring (n=10-15/sex/group; 1/sex/litter) were perfused and volume evaluation was conducted blind to treatment. SDN-POA outline was delineated using calbindin D28K immunoreactivity. Pairwise comparisons of the significant treatment by sex interaction indicated that neither BPA dose affected female volume. However, females treated with 5.0 or 10.0 μg/kg EE2 exhibited volumes that were larger than same-sex controls, respectively (p<0.001). Males treated with either BPA dose or 10.0 μg/kg/day EE2 had larger volumes than same-sex controls (p<0.006). These data indicate that BPA can have sex-specific effects on SDN-POA volume and that these effects manifest as larger volumes in males. Sensitivity of the methodology as well as the treatment paradigm were confirmed by the expected EE2-induced increase in female volume. These treatment effects might lead to organizational changes within sexually dimorphic neuroendocrine pathways which, if persistent, could theoretically alter adult reproductive physiology and socio-sexual behavior in rats.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted at Division of Health Effects Research, National Institute of Occupational Safety and Health, Kawasaki, Japan
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- bw, food consumption, gestational length, litter size, Live births, sex ratio. Offspring: body weight, body length, tail length, AGD,AGD index, or the weight of liver, kidney, heart, spleen, thymus, testis, epididyis, uterus.
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- mouse
- Strain:
- other: C57BL/6J
- Route of administration:
- oral: feed
- Vehicle:
- other: diet
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- Pregnant (GD 3) female mice (C57BL/6J strain, 9 weeks of age) were purchased from Charles River Japan (Kanagawa, Japan). The presence of a copulatory plug defined GD 0. Mice were acclimated on GD 3–6 and were housed individually.
- Duration of treatment / exposure:
- The present study was conducted to examine the effects of low-dose exposure to bisphenol Aon reproduction and development in two generations of mice. Pregnant female C57BL/6J mice (F0) were fed a diet containing low doses of bisphenol A (0, 0.33, 3.3, or 33 ppm) from gestational day 6 through postnatal day 22, and the weanlings (F1 and F2) from each F0 and F1 dam group, respectively, were also fed these same concentrations of bisphenol A ad libitum until sacrifice.
- Frequency of treatment:
- diet (daily)
- Duration of test:
- F1 and F2 offspring were necropsied at 15 weeks of age
- No. of animals per sex per dose:
- 12/group/sex
- Control animals:
- yes
- Dose descriptor:
- NOAEL
- Effect level:
- 33 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- ~0.05, 0.5, or 5 mg/kg body weight/day; These findings indicate that dietary exposure to bisphenol A between 0.33 and 33 ppm does not adversely affect reproduction or development as assessed in two generations of mice.
- Dose descriptor:
- NOAEL
- Effect level:
- 33 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- ~0.05, 0.5, or 5 mg/kg body weight/day; These findings indicate that dietary exposure to bisphenol A between 0.33 and 33 ppm does not adversely affect reproduction or development as assessed in two generations of mice.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
The present study was conducted to examine the effects of low-dose exposure to bisphenol Aon reproduction and development in two generations of mice. Pregnant female C57BL/6J mice (F0) were fed a diet containing low doses of bisphenol A (0, 0.33, 3.3, or 33 ppm) from gestational day 6 through postnatal day 22, and the weanlings (F1 and F2) from each F0 and F1 dam group, respectively, were also fed these same concentrations of bisphenol A ad libitum until sacrifice. There were no treatment-related changes in body weight, body weight gain, food consumption, gestation length, or the number of live births on postnatal day 1 in F0 dams between the control group and bisphenol A groups. Sex ratio and viability were similar in all F1 pups. No treatment-related changes were observed in body weight, food consumption, developmental parameters, anogenital distance, or weight of any of the organs (liver, kidney, heart, spleen, thymus, testis, ovary, or uterus) in F1 and F2 adults in either sex. The epididymis weight was slightly higher with 0.33 and 3.3 ppm in F1 males, but this slight increase was neither dose dependent nor seen across generations. There were no treatment-related effects of bisphenol A on cauda epididymal sperm count or sperm motility in F1 or F2 males. These findings indicate that dietary exposure to bisphenol A between 0.33 and 33 ppm does not adversely affect reproduction or development as assessed in two generations of mice.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted at the National Institute of Occupational Safety and Health, Kawasaki, Japan
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- Parameter investigated: bw, reproductive parameter (gestational length, total pups/litter, live birth, number and sex of offspring), AGD. At 3 months: sperm count and motility, serum testosterone, dihydrotestosterone, estradiol and progesterone concentrations: organ weights (females: ovary, uterus, vagina; males testes, epidermis, prostate).
- GLP compliance:
- not specified
- Remarks:
- The information on the GLP status is not given in the publication.
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Route of administration:
- oral: feed
- Vehicle:
- other: diet
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- Time mated females were purchased from CHarles River Japan.
- Duration of treatment / exposure:
- GD6 to PND21
- Frequency of treatment:
- diet
- Duration of test:
- up to 3 months
- No. of animals per sex per dose:
- 10 litter per does
- Control animals:
- yes
- Dose descriptor:
- NOAEL
- Effect level:
- 33 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- Low-dose exposure to Bisphenol A in the diet does not adversely affect reproductive development in rat offspring.
- Dose descriptor:
- NOAEL
- Effect level:
- 33 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see remarks
- Remarks on result:
- other:
- Remarks:
- Low-dose exposure to Bisphenol A in the diet does not adversely affect reproductive development in rat offspring.
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Executive summary:
The purpose of the present study was t oinvestigate the effects ofl ow-dose exposure to BPA on reproductive development in F1 rat offspring. Pregnant female Sprague-Dawley rats(F0) were fed a diet containing low doses of BPA (0,0.33, 3.3, or 33 ppm) from gestational day (GD) 6 through postnatal day (PND) 21. The weanlings (F 1) from all dose groups were fed anominal diet adlibitum after weaning and then were subjected to necropsy at 5 weeks or 3 months of age. No BPA-related changes were observed in body weight or weight of any of the major reproductive organs in F1 males and females. Epididymis weight was significantly lower only in 3 month-old F1males exposed to 33ppm BPA. Anogenital distance (AGD), the ratio of AGD to the cube root of body weight, and relative ovary weight were significantly lower in 5 -week-old F1females exposed to 3.3 and 33ppm BPA, but significant differences were not observed in 3 -month-old females.There were no BPA-related effects on cauda epididymal spermmotility in 3 -month-old F1 males. Plasma reproductive steroid hormone concentrations were not altered among groups in either sex. These outcomes indicate that low-dose exposure to BPA in the diet does not adversely affect reproductive development in F1 rat offspring.
Referenceopen allclose all
Postweaning, one offspring/sex/litter (n=11-12/sex/group) was assessed for the typical behaviors measured in developmental neurotoxicology studies. At PND 29, novelty preference was unaffected by treatment; however, relative to the VEH group, males and females of both EE2 groups were more active. VEH males appeared somewhat hypoactive in open field assessments at PNDs 40-42 and, as a result, males of the BPA and EE2 groups were significantly more active. Latency to locate the Barnes maze escape box at PNDs 47-50 was increased in males and females of the 5.0 μg/kg/day EE2 group. Relative to other male groups, VEH males exhibited an increased startle response on the first trial block at PND 54 and thus, males of both BPA groups and the 10.0 μg/kg/day EE2 group exhibited a significantly decreased startle response. PNDs 43- 44 motor coordination and PNDs 75-79 water maze performance were unaffected by treatment. These results indicate few consistent or dose-related effects resulting from developmental treatment with BPA at these doses. Few of these behaviors, however, were sexually dimorphic which may prove more sensitive.
The developing central nervous system may be particularly sensitive to bisphenol A (BPA)-induced alterations. Here, pregnant Sprague Dawley rats (n = 11–12/group) were gavaged daily with vehicle, 2.5 or 25.0 μg/kg BPA, or 5.0 or 10.0 μg/kg ethinyl estradiol (EE2) on gestational days 6–21. The BPA doses were selected to be below the no-observed-adverse-effect level (NOAEL) of 5 mg/kg/day. On postnatal days 1–21, all offspring/litter were orally treated with the same dose. A naïve control group was not gavaged. Body weight, pubertal age, estrous cyclicity, and adult serum hormone levels were measured. Adolescent play, running wheel activity, flavored solution intake, female sex behavior, and manually elicited lordosis were assessed. No significant differences existed between the vehicle and naïve control groups. Vehicle controls exhibited significant sexual dimorphism for most behaviors, indicating these evaluations were sensitive to sex differences. However, only EE2 treatment caused significant effects. Relative to female controls, EE2-treated females were heavier, exhibited delayed vaginal opening, aberrant estrous cyclicity, increased play behavior, decreased running wheel activity, and increased aggression toward the stimulus male during sexual behavior assessments. Relative to male controls, EE2-treated males were older at testes descent and preputial separation and had lower testosterone levels. These results suggest EE2-induced masculinization/defeminization of females and are consistent with increased volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) at weaning in female siblings of these subjects (He, Z., Paule, M. G. and Ferguson, S. A. (2012) Low oral doses of bisphenol A increase volume of the sexually dimorphic nucleus of the preoptic area in male, but not female, rats at postnatal day 21.Neurotoxicol. Teratol. 34, 331–337). Although EE2 treatment caused pubertal delays and decreased testosterone levels in males, their behaviors were within the range of control males. Conversely, BPA treatment did not alter any measured endpoint. Similar to our previous reports (Ferguson, S. A., Law, C. D. Jr and Abshire, J. S. (2011) Developmental treatment with bisphenol A or ethinyl estradiol causes few alterations on early preweaning measures. Toxicol. Sci. 124, 149–160; Ferguson, S. A., Law, C. D. and Abshire, J. S. (2012) Developmental treatment with bisphenol A causes few alterations on measures of postweaning activity and learning. Neurotoxicol. Teratol. 34, 598–606), the BPA doses and design used here produced few alterations.
Exposure to Bisphenol A (BPA) may interfere with brain sexual differentiation. Altered numbers of tyrosine hydroxylase (TH) cells in the rodent anteroventral periventricular nucleus (AVPV) after developmental BPA treatment have been reported; however, definitive conclusions are lacking. The current study incorporated many of the guidelines suggested for endocrine disrupter research. Specifically, ethinyl estradiol (EE2) served as a reference estrogen, exogenous environmental estrogen exposure was controlled, BPA was administered orally, and subjects consumed a low phytoestrogen diet. Here, on gestational days 6–21, Sprague-Dawley rats (10–15/group) were gavaged with 2.5 or 25.0 µg BPA/kg/day or 5.0 or 10.0 µg EE2/kg/day or the vehicle (5 ml of 0.3% aqueous carboxymethylcellulose/kg/day). A naïve control group was weighed and restrained, but not gavaged. Beginning on postnatal day (PND) 1 and continuing until PND 21, the 4 pups/sex/litter were orally treated with the same dose their dam had received. On PND 21, 1/sex/litter was perfused and the brain removed. TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. ANOVA results indicated significantly higher TH-ir cells/mm2 in females (main effect of sex: p<0.01); however, there was no significant effect of treatment or a significant interaction of treatment with sex. In a separate untreated group of PND 21 Sprague-Dawley pups, AVPV volume was quantified and no significant sexual dimorphism was apparent. Similar to our reported results of behavioral assessments, the BPA treatment paradigm used here (2.5 or 25.0 µg BPA/kg/day administered orally) does not appear to cause significant alterations in AVPV TH-ir.
Relative to the vehicle control (VEH) group, dams of both EE2-treated groups weighed less throughout gestation and lactation. PND 1 absolute anogenital distance and anogenital index were unaltered by any treatment. Ages at fur development and eye and ear opening were unaffected by any treatment. Despite a significant treatment effect, no group was significantly different from VEH in PNDs 3-6 righting latencies; although, males had shorter latencies and all latencies decreased with age. PNDs 8-11 slant board behavior was unaffected by any treatment; however, males had shorter turning latencies and latencies decreased with age. Preweaning body weights of BPA- and EE2-treated groups as well as naïve controls were less than VEH. No treatment affected PND 21 whole or regional brain weights or levels of estradiol, testosterone, corticosterone, T3, T4, luteinizing hormone, ghrelin or leptin. These results add to the literature indicating developmental BPA treatment at these doses has no effects on gestational or lactational body weight, offspring anogenital distance, preweaning behaviors or hormone levels and whole and regional brain weights measured at weaning.
In conclusion, gestational and direct postnatal treatment with low doses of BPA significantly increased SDN-POA volume in postnatal day 21male rats but did not have a similar effect in females. As expected, the reference estrogen (EE2) increased SDN-POA volume in females. Further, the higher EE2 dose (10.0 μg/kg/day) increased SDN-POA volumes of males. These effects were unrelated to postnatal day 1 anogenital distance or postnatal day 21 hormonal concentrations as these were not significantly affected by treatment (Ferguson et al., 2011). Treatmentinduced alterations in SDN-POA volume could conceivably lead to organizational changes in sexually dimorphic neuroendocrine pathways which, if of sufficient duration, could theoretically alter adult reproductive physiology and socio-sexual behavior.
There were no treatment-related changes in body weight, body weight gain, food consumption, gestation length, or the number of live births on postnatal day 1 in F0 dams between the control group and bisphenol A groups. Sex ratio and viability were similar in all F1 pups. No treatment-related changes were observed in body weight, food consumption, developmental parameters, anogenital distance, or weight of any of the organs (liver, kidney, heart, spleen, thymus, testis, ovary, or uterus) in F1 and F2 adults in either sex. The epididymis weight was slightly higher with 0.33 and 3.3 ppm in F1 males, but this slight increase was neither dose dependent nor seen across generations. There were no treatment-related effects of bisphenol A on cauda epididymal sperm count or sperm motility in F1 or F2 males. These findings indicate that dietary exposure to bisphenol A between 0.33 and 33 ppm does not adversely affect reproduction or development as assessed in two generations of mice.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 640 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Multiple comprehensive guideline studies, including multi-generation and lifelong exposure studies are available.
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
EFSA Opinion 2015 concluded on reproductive and developmental toxicity:
Overall, the better powered, better conducted studies in animals found few consistent effects of in-utero exposure to Bisphenol A on reproductive development at dose levels at or below 3.6 mg Bisphenol A/kg/day HED. On balance, the evidence remains contradictory and highly variable between studies. The CEF Panel noted that there is some evidence for effects of Bisphenol A exposure on several parameters indicative for changes in the reproductive system in adult male animals at dose levels below 3.6 mg/kg bw per day, although these effects were modest. It is not possible to conclude that these changes are reflective of changes in reproductive performance, since the studies rarely included a forced/continuous breeding phase in adulthood to establish reduced fertility. However, in several multigenerational studies no effects were observed at dose levels as low as 3mg/kg bw per day up to at least 50 mg/kg bw per day.
Using a WoE approach, the CEF Panel assigned a likelihood level of “as likely as not” to reproductive and developmental effects of Bisphenol A at low doses (below the HED of 3.6 mg/kg bw per day). Since the likelihood level for this endpoint is less than "likely" (see Appendix A), this endpoint was not taken forward for assessing the toxicological reference point, but was taken into account in the evaluation of uncertainty for hazard characterisation and risk characterisation (Section 4.3)."
EFSA Opinion 2015 concluded concerning potential Non-Monotonic-Dose-Responses (NMDR):
“The CEF Panel developed criteria for nonmonotonic dose-responses (NMDRs) and reviewed studies reporting a NMDR for Bisphenol A. None of the studies fulfil these criteria. Overall the CEF Panel concluded that the available data do not provide evidence that Bisphenol A exhibits a NMDR for the endpoints considered (reproductive/developmental toxicity, neurotoxicity/behavioural effects, metabolic effects, proliferative changes in mammary gland). “
SCOEL Recommendation 2014 concluded on reproductive and developmental toxicity:
"Overall, in standard reproductive and developmental studies in rodents, effects on reproduction have been seen only at high doses showing also other toxic effects. Even though several non-guideline studies suggest effects on reproductive and developmental parameters at lower dose levels (< 5 mg/kg bw), the data are contradictory and are not supported by the recent FDA/NTCR study with a wide-dose range (Delclos et al 2014). In humans, based on Chinese epidemiological studies, there is some concern for impaired sperm quality but, for example, the effect of other concurrent exposures cannot be excluded. In addition, there are some concerns on the potential developmental neurotoxicity of Bisphenol A based on animal studies suggesting effects on memory and learning and anxiety-like behaviour. However, since the data are very inconsistent it is difficult to conclude on the relevance of these findings."
SCOEL Recommendation 2014 concluded concerning potential low-dose effects:
“Even though there are some concerns related to the long-term effects of Bisphenol A at exposure levels lower than 5 mg/kg bw after exposure during the foetal and early postnatal period, the results of these studies are controversial and there is no clear support for these effects at low dose levels from good quality animal studies (including the recent study by Delcloset al2014). Therefore, at present SCOEL did not consider them relevant for deriving the recommended OEL.”
The 2008 updated EU RAR concluded:
"A new two-generation study in mice by Tyl et al. (published in 2008) provides a comprehensive and definitive investigation on the effects of Bispheol A on reproduction at exposure levels spanning the low (ug/kg/day) to high (mg/kg/day) ranges. This study showed that Bisphenol A causes adverse effects on pregnancy and the offspring at 600 mg/kg/day, an exposure level that also caused mild parental toxicity. Fertility was not affected by Bisphenol A exposure. A NOAEL for reproductive toxicity of 50 mg/kd/day was identified and should be used in the risk assessment."
The 2003 EU RAR concluded:
"No human data on reproductive toxicity of Bisphenol A are available. Bisphenol A has been shown to have endocrine modulating activity in a number of screening assays, with a potency that generally ranged from 3 to 5 orders of magnitude less than that of oestradiol. The effects of Bisphenol A on fertility and reproductive performance have been investigated in two-generation and multi-generation studies in the rat and a continuous breeding study in mice. Effects were seen in both species at approximately the same dose level and it is considered that the NOAEL of 50 mg/kg/day identified in the rat multi-generation study is also likely to produce no adverse effects in mice for which there is only a LOAEL of 300 mg/kg/day for a small decrease in epididymal weight in F1 males. The NOAEL of 50 mg/kg/day from the multi-generation study will be used for risk characterisation purposes, in relation to effects on fertility."
Toxicity to reproduction: other studies
Additional information
EFSA Opinion 2015 concluded concerning potential Non-Monotonic-Dose-Responses (NMDR):
“The CEF Panel developed criteria for nonmonotonic dose-responses (NMDRs) and reviewed studies reporting a NMDR for Bisphenol A. None of the studies fulfil these criteria. Overall the CEF Panel concluded that the available data do not provide evidence that Bisphenol A exhibits a NMDR for the endpoints considered (reproductive/developmental toxicity, neurotoxicity/behavioural effects, metabolic effects, proliferative changes in mammary gland).“
SCOEL Recommendation 2014 concluded concerning potential low-dose effects:
“Even though there are some concerns related to the long-term effects of Bisphenol A at exposure levels lower than 5 mg/kg bw after exposure during the foetal and early postnatal period, the results of these studies are controversial and there is no clear support for these effects at low dose levels from good quality animal studies (including the recent study by Delcloset al2014). Therefore, at present SCOEL did not consider them relevant for deriving the recommended OEL.”
Justification for classification or non-classification
Harmonised classification - Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation).
ATP Updated: CLP00/ATP09: Repr. 1B (H360F)
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General Information
The ECHA Risk Assessment Committee (RAC) recently proposed to strengthen the reproductive toxicity classification of Bisphenol A to a category 1B (H360 May damage fertility) reproductive toxicant. This conclusion is based on high-dose effects in the comprehensive studies. RAC (ECHA/RAC/RES-O-0000001412-86-56/F) concluded:“Based on the available studies, RAC considers that there is evidence of effects of Bisphenol A exposure on several parameters indicative of changes in the reproductive system. The multi-generation studies (Tyl et al. 2008 and 2002, NTP 1985, EMA et al. 2001) and a subchronic study (Delclos et al. 2014, also referred to as US FDA/NCTR 2013) were the basis of the CLP classification for fertility by RAC (2014). RAC’s opinion (RAC 2014) was based on adverse effects, such as disturbances in the oestrous cycle, at a dose of 600 mg/kg bw/day (Tyl et al. 2008) and at a dose of 100 mg/kg bw/day (Delclos et al. 2014). The ovarian toxicity reported in Tyl et al. (2002) included reduced absolute and relative ovarian weight at the two highest doses of 50 and 500 mg/kg bw/day and in Delclos et al. (2014) an increase in ovarian follicular cysts was observed at 300 mg/kg bw/day. In Delclos et al. (2014), an increase in cystic endometrial hyperplasia was observed in the uterus at the highest dose of 300 mg/kg bw/day.”
The above mentioned guideline generation studies in rats and mice show that Bisphenol A is not a selective reproductive toxicant.
• NOAEL for systemic toxicity 5 mg/kg bw/day in rats and mice; LOAEL >= 50 mg/kg bw/day.
• In rats no adverse effects on oestrus cycle, fertility, litter sizes, pre- and post-natal survival, growth and development at doses at doses up to ca. 200 mg/kg bw/day. With 500 mg/kg bw/day reduced litter size (at systemic toxic dose) in the Three Generation Reproduction Study.
• In mice no adverse effects on fertility, litter sizes, pre- and post-natal survival at doses up to ca. 600 mg/kg bw/day. With 600 mg/kg bw/day delayed offspring development in the Two Generation Reproduction Study. As the studies discussed in this chapter show, effects on animal fertility only occur at high doses of Bisphenol A and are a consequence of systemic toxicity.
Additional information
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