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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Two-generation study
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 and OECD good laboratory practices

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study) TG 416 Enhanced
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- purity: ≥99.7 %

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals 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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on oral 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.
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 at Bisphenol A-containing food ad libitum.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.018, 0.18, 1.8, 30, 300, and 3500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
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:
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)
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.

Examinations

Observations and examinations performed and frequency:
PARENTAL ANIMAL OBSERVATIONS

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.

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.
Sacrifice and pathology:
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.

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.
Other examinations:
Estrous cyclicity of parental animals 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 estrus at demise.

Sperm 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.

The following reproductive indices were measured: 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, estrous cycle.

The ofspring viability indices that were measured were: number of live litters on PND 0, live birth index, number of total/live/dead pups, sex ratio (% males) per litter on PND 0.

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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
PARENTAL ANIMALS

REPRODUCTIVE FUNCTION: SPERM MEASURES: Slight but significant decrease in epidydimal sperm concentration at 3500 ppm. The authors noted that this was not treatment-related because there were no effects in the F1 generation, no effects for other andrological endpoints, and no effects on male fertility.

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 consdier 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).

OFFSPRING

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: 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: 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: 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: 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).

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
Systemic
Effect level:
30 ppm
Sex:
male
Basis for effect level:
other: Based on centrilobular hypertrophy in adult males at 300 ppm
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 ppm
Sex:
male/female
Basis for effect level:
other: The EU RAR Update 2008 concluded that NOAEL is 300 ppm based on reduced body weight and effects on liver and kidney weight and histopathology at 3500 ppm
Dose descriptor:
NOAEL
Remarks:
Reproductive/developmental
Effect level:
300 ppm
Sex:
male/female
Basis for effect level:
other: Based on reduced body weight spleen and testes weights and delay in acquisition of puberty in F1 males at 3500 ppm

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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).