4,4'-isopropylidenediphenol

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study) TG 416 Enhanced

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-isopropylidenediphenol

EC Number:

201-245-8

EC Name:

4,4'-isopropylidenediphenol

Cas Number:

80-05-7

Molecular formula:

C15H16O2

IUPAC Name:

4-[2-(4-hydroxyphenyl)propan-2-yl]phenol

Test material form:

solid: crystalline

Details on test material:

- purity: ≥99.7 %

Test animals

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

Administration / exposure

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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

30 ppm

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

300 ppm

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

300 ppm

Basis for effect level:

other: developmental toxicity

Results (fetuses)

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).

Effect levels (fetuses)

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.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

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).