Reaction mass of 4,4'-isopropylidenediphenol and phenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

3 genreration reproductive toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study designed and conducted using an internationally accepted reproductive toxicity protocol under Good Laboratory Practice (GLP) regulations (U.S. EPA, 1989). BPA is a constituent of the reaction mass so that its hazard data are relevant for the assessment of the reaction mass.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 837.3800, 1998

Principles of method if other than guideline:

Three-generation reproductive toxicity study.

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:

2,2-bis(4-hydroxyphenyl)propane

Constituent 2

Reference substance name:

BPA commercially available

IUPAC Name:

BPA commercially available

Details on test material:

- Name of test material (as cited in study report): BPA (4,4'-isopropylidene-2'-diphenol)
- Purity: 99.5%
- Physical state: White crystalline solid
- Other: Obtained in one shipment and lot number from Acros Organics NV (Fairlawn, NJ)

Test animals

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral 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.

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:

BPA in feed was available ad libitum.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm
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.

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.

Examinations

Observations and examinations performed and frequency:

PARENTAL ANIMALS

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 estrous was also determined at necropsy for P, F1, and F2 parental and F3 retained females.

SPERM PARAMETERS: Yes
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.

OFFSPRING

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 and pathology:

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.

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.

Other examinations:

- 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.

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.

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

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

PARENTAL ANIMALS

Body weight: Body weights during the pre-breed and mating period were significantly reduced for F1 males at 750 or 7500 ppm and 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 (males and females) at 7500 ppm, and in F1 females and F1/F2 males at 750 ppm.

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.

OFFSPRING

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.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

75 ppm

Sex:

female

Basis for effect level:

other: Based on body weight reduction in P, F1, and F2 females at 750 ppm.

2

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

ca. 5 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: dose estimated from food consumption

3

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

750 ppm

Sex:

male/female

Basis for effect level:

other: Based on delayed vaginal patency in F1, F2, and F3 females and preputial separation in F1, F2, and F3 males (associated with reduced body weights) at 7500 ppm.

4

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

ca. 50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: dose estimated from food consumption

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.