4,4'-isopropylidenediphenol

Endpoint summary

Description of key information

Oral: 

Multi generational studies in mice and rats have shown that Bisphenol A affects kidney and liver weight in parental animals dosed with 50 – 600 mg/kg bw/d and its progeny. This finding is supported by the recently conducted CLARITY Core study. It revealed no conclusive evidence for systemic toxicity in rats exposed up to 25 mg BPA/kg bw/d (Stop-Dose & Continuous Dose). Given that kidney and liver weight are considered as relevant systemic effects for human risk assessment a BMDL10 for mean relative kidney weight of 8960 µg/kg bw/d in male mice of the F0 generation was taken as starting point according to EFSA’s Scientific opinion on BPA in 2015.

Inhalation: 

In rats exposed daily to airborne Bisphenol A for 13 weeks there was a NOAEC of 10 mg/m3, with mild olfactory epithelium inflammation at 50 and 150 mg/m3. There was no evidence of systemic toxicity in this study. 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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 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.

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.

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.

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).

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

Critical 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).

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Three-generation study

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Three-generation reproductive toxicity study.

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 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 gestation, 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.

Remarks:

Doses / Concentrations:
0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
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.

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.

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.

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.

Dose descriptor:

NOEL

Remarks:

Systemic

Effect level:

75 ppm

Sex:

female

Basis for effect level:

other: Based on body weight reduction in adult females at 750 ppm

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

750 ppm

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

Dose descriptor:

NOAEL

Remarks:

Reproductive / development

Effect level:

750 ppm

Sex:

male/female

Basis for effect level:

other: Based on reduced body weights in males and females, delayed vaginal patency in females and preputial separation in males (associated with the reduced body weights) and reduced litter size at 7500 ppm

Critical 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. 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 500 mg/kg/day.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted under OECD test guidelines and Good Laboratory Practice (GLP) guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Shiga, Japan)
- Age at study initiation: 6 weeks
- Housing: Animals were housed individually in stainless steel, wire-mesh cages
- Diet: ad libitum, commercial diet (MF, Oriental Yeast Co., Tokyo, Japan)
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 50-60
- Photoperiod: 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

The doses selected were 0, 40, 200, and 1000 mg/kg-day, based on a previous repeated dose toxicity study and uterotrophic assay data (NTP, 1982; Ashby and Tinwell, 1998; Yamasaki et al., 2000). One male rat in the 1000 mg/kg-day group died with toxic clinical signs within one week of administration. Thereafter, the highest dose was decreased to 600 mg/kg-day from day 8 onward. Two males and one female subsequently died within two weeks of administration of this dose.

Animals were dosed from 7 weeks of age for at least 28 days.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Oral gavage once per day for at least 28 days

Frequency of treatment:

Once per day

Remarks:

Doses / Concentrations:
0, 40, 200, and 1000 mg/kg-day (high dose was decreased to 600 mg/kg-day after the first seven days of exposure)
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rats were given oral gavage doses of BPA from 7 weeks of age for at least 28 days. A vehicle control group was given only olive oil. Clinical signs were recorded daily. Estrous cycles in females were determined daily. At autopsy, blood samples were obtained from the abdominal aorta and examined for hematology, clinical biochemistry, and hormonal analysis. Sperm count and motility were also examined.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice per week

FOOD CONSUMPTION:
- Time schedule: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At sacrifice

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At sacrifice

OTHER:
- Sperm motility and viability: examined at sacrifice
- Estrous cyclicity: determined daily
- Functional observation battery (FOB), such as sensory reactivity to stimuli and motor activity assessment: Conducted during fourth week of study

Sacrifice and pathology:

Males were necropsied on day 29. Females were necropsied after having been treated at least 29 days, being killed on day 30, 31, or 32 to allow observation of estrous cycling. All females were killed at the diestrous stage. Animals were killed by exsanguination under anesthesia.

Statistics:

Body weight, food consumption, hematological, clinical biochemical, organ weight, spermatological, and FOB data were analyzed using Bartlett's test for homogeneity of variance. When the variances were homogeneous at a significance level of 5%, one-way ANOVA was performed. Following a significant difference, the difference between the control group and each treatment group was analyzed by Dunnett's test. When the variances were not homogeneous, the Kruskal-Wallis test was used. Following a significant difference, the difference between the control group and each treatment group was analyzed by the nonparametric Dunnett's test. FOB counted data and spermatological data were analyzed using the Kruskal-Wallis test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

One female and three males in the highest dose group died within two weeks after administration of BPA. Body weights were also reduced in high dose group animals. Relative weights of testes and adrenals were increased and of ventral prostate were decreased in males of the high dose group. In females, the relative weights of the thyroids and liver were increased in the high dose group and the relative weight of the heart was decreased in both the middle and high dose groups. The effects on relative organ weights were not considered to be adverse according to the authors. The diestrous stage was continued for over 4 days in three female rats of the high dose group. No other abnormalities were detected.

Dose descriptor:

LOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Based on mortality and decreased body weight in males and females and on increased duration of diestrous cycle in females at the highest dose (600 mg/kg-day)

Critical effects observed:

not specified

Conclusions:

The authors concluded that the highest dose of 600 mg/kg-day was an apparent LOAEL because death or decreased body weight gain were observed in this group, and the only endocrine-mediated effect was an increase in estrous cycling.

Executive summary:

A 28-day repeated dose toxicity study of BPA was performed in rats. The study was based on the draft protocol of the 'Enhanced OECD Test Guideline 407.' Doses of 0, 40, 200, or 1000 mg/kg-day BPA were administered by oral gavage to male and female Sprague Dawley rats. The highest dose was decreased to 600 mg/kg-day from the second week of administration because a male rat given 1000 mg/kg-day had died within one week with toxic clinical signs. Two males and one female in this group subsequently died within two weeks of BPA administration. Body weights were reduced in males and females and the diestrous stage was prolonged in females at the 600 mg/kg-day dose. No other adverse effects were observed. The authors concluded that the highest dose of 600 mg/kg-day was an apparent LOAEL because death or decreased body weight gain were observed in this group, and the only endocrine-mediated effect was an increase in estrous cycling.

Reference 4

Endpoint:

repeated dose toxicity: oral, other

Remarks:

A study to characterize and evaluate the toxicologic potential of BPA following perinatal only or chronic exposure (see also chapter 7.7) in rats under the conditions of a chronic, extended-dose response design.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

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:

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 chapter 7.7) 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 included 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 (continuous-dose 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.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

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 NCTR.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animals and environmental conditions
- Source: Animals from the litters of the BPA exposed dams (for the in utero exposure see chapter 7.8.2) - pups without evident malformations randomly culled to a maximum of five males and five females on PND 1.
- Diet : ad libitum (after weaning)
- Water : ad libitum
-- Housing: litters were kept with their dams until weaning at PND 21. Animals were pair housed after weaning in same-sex pairs within dose groups. If an animal died or was removed as moribund, the cage mate remained single housed.

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:

oral: gavage

Details on route of administration:

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.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Aqueous CMC solution (0.3 % w/v)

Details on oral 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: Nutrients and Contaminants, Including Background BPA:
Pelleted rodent chow, verified casein diet 10 IF, irradiated, 5K96. The 5K96 diet, which is low in soyderived
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 f
or selected nutritive quality attributes (including protein, fat, crude fiber, calcium, phosphorous, and vit
amins A, B1, and E) and contaminants (including nitrosamines, fumonisins, arsenic, cadmium, lead, m
ercury, aflatoxins, organochlorine and organophosphate pesticides, butylated hydroxyanisole, butyla
ted 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 room s 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:

From PND1 to PND21 (stop-dose arm study)
From PND 1 to 1 year or 2 years (continuous-dose arm study)

Frequency of treatment:

once daily

Dose / conc.:

2.5 other: µg/kg bw/day

Remarks:

solution

Dose / conc.:

25 other: µg/kg bw/day

Remarks:

solution

Dose / conc.:

250 other: µg/kg bw/day

Remarks:

solution

Dose / conc.:

2 500 other: µg/kg bw/day

Remarks:

suspension

Dose / conc.:

25 000 other: µg/kg bw/day

Remarks:

suspension

No. of animals per sex per dose:

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 assessment
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 the hypothesis-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

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Prior to the design of this chronic toxicity study and the conception of the CLARITY-BPA project, a subchronic study was conducted at NCTR using the same animal model and dosing regimen used in the current chronic study. BPA in 0.3% carboxymethylcellulose (CMC) was administered by oral gavage to pregnant female Sprague-Dawley rats from gestation day (GD) 6 through the start of parturition, with seven doses between 2.5 and 2,700 μg BPA/kg bw/day spaced at half-log intervals. Two high doses of BPA (100,000 and 300,000 μg/kg bw/day) were added as doses expected to produce adverse effects based on published guideline studies. Vehicle and naïve (not dosed by gavage) controls were included. Dams were not dosed after their litters were born, but pups were directly dosed by oral gavage from PND 1 until termination (PND 90 ± 5). Pups were directly dosed because of the demonstrated low transmission of BPA to pups via milk. BPA showed clearly adverse effects in F1 females at the highest doses of 100,000 and 300,000 μg BPA/kg bw/day. Statistically significant differences at lower BPA doses were few and sporadic and were judged not to provide evidence of BPA-induced toxicity. There were no BPA effects observed in the males.
The design of the CLARITY-BPA core study was discussed and finalized at a series of meetings in late 2011 and early 2012. These meetings included NTP, NCTR, NIEHS/DERT, and FDA product center representatives, as well as NIEHS-funded CLARITY-BPA grantees. It was proposed that, because of the literature concerning permanent adverse effects resulting from developmental exposures to hormonally active agents, the study would include groups of animals for which exposure would terminate at weaning. For discussion of dose selection, a summary of data obtained from the NCTR BPA subchronic study was presented and discussed. It was initially agreed that a vehicle control and six log-spaced doses between 2.5 and 250,000 μg BPA/kg bw/day would provide an adequate dose range from reasonably close to human exposure on the low end to a dose expected to produce clear adverse effects at the high end. Serum measurements of BPA across the postnatal life span of animals in the subchronic study showed that the high dose of 300,000 μg BPA/kg bw/day gave rise to approximately 50 μM active unconjugated BPA in PND 4 animals and approximately 1 μM in PND 80 animals, which were clearly out of the range of attainable human internal exposure from dietary sources, estimated to be in the low to sub-pM range. There was general agreement that the current concern was restricted to a lower dose range, below the previously reported no-observed-adverse-effect level in guideline-compliant regulatory toxicity assays, which was 5,000 μg/kg bw/day. The 250,000 μg BPA/kg bw/day dose group would provide little additional information to influence regulatory action. A high dose of 25,000 μg BPA/kg bw/day was viewed as providing an adequate margin of human exposure, greater than 25,000-fold based on the aggregate human exposure estimates of <0.5 μg BPA/kg bw/day mentioned above. The low dose selected, 2.5 μg BPA/kg bw/day, provided a margin of exposure at least 10-fold higher than the maximum allowed background dietary exposure. Much of the research on BPA, particularly early in the investigations of the potential toxicity of low doses of BPA, focused on its estrogenic activity, although the involvement of mechanisms other than classical estrogen receptors have been increasingly implicated in BPA actions. Both doses of EE2 used in the NCTR BPA subchronic study (0.5 and 5 μg EE2/kg bw/day) produced strong effects in female reproductive organs and on estrous cyclicity. In males, the low dose of 0.5 μg/kg bw/day had little effect, with an increase in male mammary hyperplasia as the only apparent effect at PND 9019. The 5 μg EE2/kg bw/day dose also stimulated male mammary gland hyperplasia at PND 90, and increased hyperplasia in the coagulating gland, increased degeneration in the testicular germinal epithelium, and increased exfoliated germ cells and hypospermia in the epididymis. Based on the observed effects in the NCTR BPA subchronic study, two levels of EE2, one of which was lower than the doses used in the subchronic study, were selected for use in the current study to expand information on the sensitivity of the animal model to EE2. In the absence of data from this exposure model to guide the selection of the EE2 lower dose, a 10-fold lower dose was chosen. The CLARITY-BPA consortium consensus for the two doses of EE2 used in the current study was 0.05 and 0.5 μg/kg bw/day. Resource limitations did not allow for the inclusion of stop-dose EE2 groups. Likewise, although the NCTR BPA subchronic study had included a naïve control group that was not dosed by gavage, this group could not be included in the chronic study. The responses of the naïve and vehicle control groups in the NCTR BPA subchronic study were similar.


F0 females from the NCTR breeding colony were randomly allocated to dose groups prior to mating to give approximately equal mean body weights per dose group. Sires were selected randomly with the specification that there would be no brother/sister or first cousin matings. After the first mating, the numbers of mating pairs assigned to dose groups were adjusted to meet any deficits in pups available in a particular dose group. Pups were randomly culled to a maximum of 5 males and 5 females on PND 1, no fostering was conducted. The minimum litter size for keeping a litter on study was 3 males and 3 females. Up to 3 pups per sex per litter were assigned to the study at weaning; additional pups were assigned to studies conducted by CLARITY-BPA academic investigators and reported elsewhere. The rotating order of assignment of pups of a given sex to the study was as follows: 1) continuous-dose, 2-year sacrifice; 2) stop-dose, 2-year sacrifice; 3) continuous-dose, 1-year sacrifice; 4) continuous-dose, 2-year sacrifice; 5) stop-dose, 2-year sacrifice; and 6) stop-dose, 1-year sacrifice.

Positive control:

ethinyl estradiol (EE2) (0.05 and 0.5 µg/kg bw/day)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly or when a significant clinical observation was noted.

BODY WEIGHT: Yes
- Time schedule for examinations: Pups weighed daily from PND 1 to PND 21. Pups in continuous-dose arm were weighed daily until PND 90 ± 3 and weekly thereafter. Pups in the stop-dose arm were weighed weekly after weaning.

FOOD CONSUMPTION:
- Time schedule: weekly for approximately the first 13 weeks and monthly afterwards.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Interim sacrifice, PND 365 ± 20
- The following endpoints were evaluated in an aliquot of whole blood: hematocrit, hemoglobin concentration, erythrocyte, leukocyte, reticulocyte, and platelet counts, leukocyte differential count, mean corpuscular volume, and mean corpuscular hemoglobin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Interim sacrifice, PND 365 ± 20
- The following endpoints were measured in serum: total protein, albumin, urea nitrogen, creatinine, alanine aminotransferase, gamma glutamyl transpeptidase, sorbitol dehydrogenase, aspartate aminotransferase, alkaline phosphatase, total bile acids, glucose, cholesterol, triglycerides, insulin, leptin, cardiac troponins T and I, T3, T4, and TSH.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

- Interim sacrifice, PND 365 ± 20: Animals were fasted overnight. Animals were anesthetized with carbon dioxide and blood was collected from the retro-orbital sinus prior to euthanasia and complete necropsy.
Weighed organs (males): adrenals, brain, epididymides, heart, kidneys, liver, pituitary (after 48-hour fixation), seminal vesicles with coagulating gland, spleen, testes, thymus, thyroid with parathyroid (after 48-hour fixation), epididymal, and retroperitoneal fat pads.
Weighed organs (females): 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.
- Terminal sacrifice, 104 ± 3 weeks: Procedures were similar to those used for the interim sacrifice, except that the animals were not fasted, blood was not collected, and there were no hematology, clinical chemistry, sperm, or organ weight data collected.

HISTOPATHOLOGY: Yes

-Interim sacrifice, PND 365 ± 20 and terminal sacrifice, 104 ± 3:
The following organs, as well as all gross lesions, were evaluated microscopically: Males—adrenals, aorta (thoracic), bone marrow (femur), brain, right epididymis, heart, kidneys, liver, 5th left mammary gland (inguinal), pancreas, parathyroid, pituitary, prostate (dorsal/lateral and ventral), seminal vesicles with coagulating gland, spleen, right testis, thymus, and thyroid. For the dorsal/lateral prostate, 6 step sections cut at 100 μm intervals were evaluated. Subsets of intermediate sections were collected and stored unstained for potential additional evaluation.
Females—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.
All tissues, except testes and eyes, were fixed in 10 % NBF and stained with H&E for microscopic evaluation. Testes and eyes were fixed in modified Davidson’s fixative and testes were stained with periodic acid-Schiff (PAS) stain. Fixation times for the tissues listed for histopathology, except the brain, were limited to 96–120 hours. Brain remained in fixative until processing.
Tissues removed, fixed in 10% NBF, processed to block and stored: Clitoral gland, esophagus, epididymal fat pad, ovarian/parametrial fat pad, retroperitoneal fat pad, Harderian gland, cecum, colon, rectum, duodenum, ileum, jejunum, lung with bronchi, lymph nodes (mandibular and mesenteric), nose, penis, preputial gland, salivary glands, skin, forestomach, glandular stomach, trachea, and urinary bladder.

Other examinations:

- Vaginal opening from PND 22: Females (26 animals from 13 randomly selected cages per dose group) were monitored daily

- Vaginal smears: Collected for 14 consecutive days from these same animals beginning at 16 ± 2 weeks of age. One month after these vaginal smears were completed, the same animals had vaginal smears collected for 5 consecutive days monthly until the animal did not show evidence of cycling (three or more consecutive days of estrus (E, E/D or P/E) or five consecutive days that did not include an E) for two consecutive months.

- Sperm analysis (Interim sacrifice, PND 365 ± 20): Testicular spermatid head counts (left testis); epididymal sperm counts, morphology, and motility evaluations (left epididymis).

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.

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- Preweaning Survival

- Females
The survival of female pups between PND 1 and PND 21 ranged from 91–95% in vehicle control and BPA dose groups. There were no significant BPA treatment effects. The survival of female pups in the 0.05 and 0.5 μg EE2/kg bw/day groups was 85% and 91%, respectively. Survival in the lower EE2 group was significantly lower than that in the vehicle control group. The study protocol did not call for detailed evaluation of the cause of morbidity/death in pups removed from the study prior to weaning.
- Males
The survival of male pups between PND 1 and PND 21 ranged from 91–95% in vehicle control and BPA dose groups. There were no significant BPA treatment effects. The survival of male pups in the 0.05 and 0.5 μg EE2/kg bw/day groups was 85% and 91%, respectively, and did not differ significantly from the vehicle control. The study protocol did not call for detailed evaluation of the cause of morbidity/death in pups removed from the study prior to weaning.

- Postweaning Survival in Interim Sacrifice Animals

- Females, Continuous-Dose Arm
The survival of females dosed daily with vehicle or BPA until the scheduled one-year sacrifice ranged from 91–100% and there were no treatment effects. Over the same period, the survival of females in the low and high EE2 dose groups was 92 and 100%, respectively, and did not differ from vehicle controls.
- Females, Stop-Dose Arm
In the stop-dose study arm, there was 100% survival in all dose groups of females scheduled for sacrifice at one year, except for the 25,000 μg BPA/kg bw/day dose group where survival was 91%. There was no treatment effect. Causes of morbidity/death in animals that did not survive to the interim sacrifice, when known, are noted.
- Males, Continuous-Dose Arm
Survival of males dosed daily with vehicle or various BPA doses until the scheduled one-year sacrifice ranged from 82–100%, with the lowest percent survival seen in the vehicle control group. There were no significant treatment effects on survival after this one-year exposure. Over the same period, the survival of males in the low and high EE2 dose groups was 85 and 88%, respectively, and did not differ significantly from vehicle controls. Causes of morbidity/death in animals that did not survive to the interim sacrifice, when known, are noted.
- Males, Stop-Dose Arm
In the stop-dose study arm, there was 100% survival in males of all BPA dose groups sacrificed at one year, except for the 25 μg BPA/kg bw/day dose group where survival was 95%. There was no treatment effect. The cause of death in the one animal in the 25 μg BPA/kg bw/day dose group that died early was uncertain .

- Postweaning Survival in Terminal Sacrifice Animals

- Females, Continuous-Dose Arm
Survival at the end of the study in the BPA dose groups ranged from 17–40%. Survival in the low and high EE2 dose groups was 27 and 15%, respectively. No significant treatment effects were seen for this chronic exposure to BPA or EE2 at the doses administered. Most of the animals that did not survive until the terminal sacrifice were removed as moribund between one and two years of age. Causes of death/morbidity are listed in an appendix to the pathology report; mammary gland fibroadenomas and pituitary adenomas accounted for most of the early removals.
- Females, Stop-Dose Arm
Survival at the end of the study in the BPA dose groups ranged from 24-34%; survival in the vehicle control group was 22%. There were no significant treatment effects in female rats after only gestational and preweaning exposure to BPA. Most animals that did not survive until the terminal sacrifice were removed as moribund between one and two years of age. Causes of death/morbidity are listed in an appendix to the pathology report; as was the case in the two-year continuous-dose arm females, mammary gland fibroadenomas and pituitary adenomas accounted for most of the early removals.
- Males, Continuous-Dose Arm
Survival at the end of the study in the BPA dose groups ranged from 24–35%. Survival in the low and high EE2 dose groups was 35 and 46%, respectively. There were no significant treatment effects. Most animals that did not survive until the terminal sacrifice were removed as moribund between one and two years of age. Causes of death/morbidity are listed in an appendix to the pathology report. Many primary and contributory conditions in various organs were diagnosed, with pituitary adenomas, nephropathy, preputial gland carcinoma, and malignant lymphoma indicated as primary causes of death/morbidity in multiple animals across all dose groups.
- Males, Stop-Dose Arm
Survival at the end of the study in the BPA dose groups ranged from 20–33% in comparison to the 34% survival seen in the vehicle controls. There were no significant treatment effects. Most animals that did not survive until the terminal sacrifice were removed as moribund between one and two years of age. Causes of death/morbidity are listed in an appendix to the pathology report and were similar to those diagnosed for the continuous-dose arm males.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Body Weights in Preweaning Animals

- Females
There were no BPA treatment-related effects on female pup body weights at these time points. On PNDs 4 and 7, the low EE2 dose group females had significantly lower mean body weight than vehicle controls, with body weights in the treated animals approximately 5% lower than controls on both days. At PND 4, both the 2.5 μg BPA/kg bw/day and the 0.05 μg EE2/bw/day dose groups had identical means and standard errors, but only the latter was statistically significant, suggesting that the smaller sample size in that group contributed to this marginal difference.
- Males
Body weights on PNDs 1, 4, 7, 14, and 21 in male pups treated daily with vehicle, BPA, or EE2 are shown in Table 23. There were no BPA or EE2 treatment-related effects on male pup body weights at these time points.

- Body Weights in Interim and Terminal Sacrifice Animals

- Females, Continuous-Dose Arm
There were no significant differences from the vehicle controls in any of the BPA or EE2 dose groups. The mean body weights in the 2.5 μg BPA/kg bw/day group in weeks 36–52 were 10–13% higher than vehicle control means; however, these differences were not statistically significant.
Mean body weights of females in the 250 μg BPA/kg bw/day dose group were significantly higher by 16−18% than those of the vehicle control group for weeks 96–104. In this same period, the mean body weights of females in the 2.5 and 25 μg BPA/kg bw/day dose groups were 11–16% higher than those of vehicle controls, but these differences were not significant. Animals in the 2.5 μg BPA/kg bw/day dose group did not have higher mean body weights than vehicle controls in the earlier weeks noted above for interim sacrifice animals. In the terminal sacrifice high EE2 dose group, transiently higher mean body weights were observed at 4 and 8 weeks (7% and 8%, respectively). The same tendency in the high EE2 dose group was seen in the interim sacrifice animals at 4 and 8 weeks, although the differences in mean body weights were not statistically significant.
- Females, Stop-Dose Arm
There were no significant treatment effects. In the stop-dose females scheduled for terminal sacrifice, there was a significant decreasing trend at week 4, but no other treatment effects. Although statistical comparisons were not conducted between continuous- and stop-dose arms, the stop-dose arm females had higher mean weights over the course of the study (vehicle controls, mean stop-dose compared to continuous-dose: interim, 104%, range 98–109% from weeks 8–52; terminal, 107%, range 97–114% from weeks 8–104).
- Males, Continuous-Dose Arm
There were no significant treatment effects for either compound, although means were 10–16% higher than vehicle control means in the 250 μg BPA/kg bw/day dose group from weeks 92 through 104.
- Males, Stop-Dose Arm
The sole statistically significant treatment effect was a decreasing dose trend at week 4 in the terminal sacrifice animals. Although statistical comparisons were not conducted between continuous- and stop-dose arms, the stop-dose arm males had higher mean weights over the course of the study (vehicle controls, mean stop-dose compared to continuous-dose: interim, 102%, range 92–105% from weeks 8–52; terminal, 107%, range 92–113% from weeks 8–104).

Food consumption and compound intake (if feeding study):

not specified

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):

Hematology Endpoints in Interim Sacrifice Animals
- Females, Continuous-Dose Arm
Platelet counts were significantly lower (~10%) than vehicle controls in the 25,000 μg BPA/kg bw/day dose group. Eosinophils were decreased (~25%) in the 250 μg BPA/kg bw/day dose group relative to the vehicle control group, and mean corpuscular hemoglobin concentration was significantly higher (~1.4%) than vehicle controls in the 25 μg BPA/kg bw/day dose group. There were significant trends over increasing levels of BPA dose concentrations for hemoglobin concentration (p = 0.023), monocytes (p = 0.045), and platelet counts (p = 0.008). In female rats continuously dosed with 0.5 μg EE2/kg bw/day, lower eosinophil counts (~25%), % eosinophils (~ 21%), and platelet counts (~8%) were observed.
- Females, Stop-Dose Arm
No statistically significant differences in values were observed in pairwise comparisons between BPA dose groups and vehicle controls. Significant trends were noted for % basophils (p = 0.031), mean corpuscular hemoglobin (p = 0.013), and red blood cells (p = 0.044).
- Males, Continuous-Dose Arm
Hemoglobin levels were significantly higher (~4%) in the 25,000 μg BPA/kg bw/day group and the percentage of eosinophils lower (~28%) in the 250 μg BPA/kg bw/day group relative to vehicle controls. Significant trends were observed for hematocrit (p = 0.006), hemoglobin concentration (p = 0.016), PCV (p = 0.008), mean corpuscular hemoglobin (p = 0.018), mean corpuscular hemoglobin volume (p = 0.016), and platelet counts (p = 0.011). The sole observed statistically significant effect in the EE2 groups was an elevated hemoglobin concentration (~3%) relative to the vehicle control level in the 0.05 μg EE2/kg bw/day group.
- Males, Stop-Dose Arm
No statistically significant effects were observed in pairwise comparisons between BPA dose groups and vehicle controls. A significant trend for % neutrophils (p = 0.045) was noted over the levels of BPA dose concentrations in the stop-dose arm.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Clinical Chemistry Endpoints in Interim Sacrifice Animals

- Females, Continuous-Dose Arm
Alkaline phosphatase levels were significantly higher (~31%) in the 250 μg BPA/kg bw/day group than levels in the vehicle control group. Although mean levels of alkaline phosphatase were higher than controls in most of the BPA groups, none of the others were statistically significant. There were no other statistically significant treatment effects on clinical chemistry endpoints in any continuous BPA dose group. The female rats in the continuous 0.5 μg EE2/kg bw/day dose group had higher (~38%) mean levels of TSH, while rats in the 0.05 μg EE2/kg bw/day dose group had higher (~24%) mean levels of alkaline phosphatase than the vehicle control group. There were no EE2 treatment effects on T3 or T4.
- Females, Stop-Dose Arm
No statistically significant differences were noted in pairwise comparisons between stop-dose BPA-treated female rats and the vehicle controls. There was a significant trend over levels of BPA dose concentrations for albumin (p = 0.004).
- Males, Continuous-Dose Arm
No statistically significant differences were noted in pairwise comparisons between any BPA treatment group and vehicle controls. Significant trends were noted for albumin (p = 0.007), T4 (p = 0.002), total bile acids (p = 0.026), and troponin T (p = 0.003). For EE2, mean insulin levels were significantly lower (~35%, p = 0.047) in the 0.05 μg EE2/kg bw/day dose group and mean triglyceride levels were significantly higher (~26%) in the 0.5 μg EE2/kg bw/day dose group than those in vehicle controls.
- Males, Stop-Dose Arm
In the 25 μg BPA/kg bw/day dose group, decreases in the mean levels of total protein (~4%) and total bile acids (~31%) relative to vehicle controls were observed. Significant trends were noted over the levels of BPA dose concentrations for T4 (p = 0.046) and for total bile acids (p = 0.024).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ Weights in Interim Sacrifice Animals

- Females, Continuous-Dose Arm
There were few sporadic significant differences between BPA groups and the vehicle control group. In the 2.5 μg BPA/kg bw/day dose group, the mean absolute retroperitoneal fat pad weight was significantly higher (40%) than the mean weight in the vehicle control group. The brain weight-adjusted retroperitoneal fat pad weight was similarly significantly greater than the vehicle control. The mean retroperitoneal fat pad weight adjusted for body weight was higher (23%) than the vehicle control group, but this was not a statistically significant difference. The only other significant BPA treatment effect was a dose trend for liver adjusted for body weight.
Multiple organ weights were significantly affected by the 0.5 μg EE2/kg bw/day treatment. Mean absolute adrenal weights, as well as adrenal weights adjusted for brain and body weights, were increased by 27%, 28%, and 22%, respectively. Mean ovarian/parametrial fat pad weight was decreased, with mean weight adjusted for body weight significantly decreased (~17%) relative to the vehicle control group mean. Mean heart weight was also increased in the high EE2 dose group, with mean heart weight adjusted for body weight significantly increased (~6%) relative to the vehicle control group mean. Mean absolute kidney weights, as well as kidney weights adjusted for brain and body weights, were increased relative to the vehicle control mean by 15%, 16%, and 13%, respectively. Mean absolute liver weights, as well as liver weights adjusted for brain and body weights, were increased relative to the vehicle control mean by 20%, 20%, and 18%, respectively. Mean absolute ovary weights, as well as ovary weights adjusted for brain and body weights, were decreased relative to the vehicle control mean by 18%, 16%, and 15%, respectively. Mean absolute pituitary weights, as well as pituitary weights adjusted for brain and body weights, were increased by 31%, 32%, and 20%, respectively.
- Females, Stop-Dose Arm
There were significant dose trends for absolute ovary weight and ovary weight adjusted for brain and body weight. The mean absolute ovary weight, as well as ovary weight adjusted for brain weight, in the 25,000 μg BPA/kg bw/day dose group were significantly lower than the vehicle control group by approximately 13% and 12%, respectively. Mean ovary weight adjusted for body weight was also 9% lower than the controls, but this difference was not statistically different.
- Males, Continuous-Dose Arm
The sole significant BPA treatment effect was a lower (~7%) mean liver weight adjusted for body weight relative to the vehicle control in the 2.5 μg BPA/kg bw/day dose group. There were no significant treatment effects of either EE2 dose.
- Males, Stop-Dose Arm
The sole significant treatment effect was a significant dose trend for liver weight adjusted for body weight.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were also no treatment-related non-neoplastic effects in stop-dose interim or terminal sacrifice males. 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.

In the uterus, cystic endometrial hyperplasia was elevated in interim and terminal sacrifice animals in the stop-dose study arm at 25,000 μg BPA/kg bw/day (interim) and at 2,500 and 25,000 μg BPA/kg bw/day (terminal). The incidence in the terminal stop-dose vehicle control was low compared to all other terminal sacrifice groups. An increase in this lesion was not observed in the continuous-dose BPA animals, but did occur at the interim sacrifice with the high dose EE2 animals. Uterine squamous metaplasia was also increased in interim stop-dose animals at 25,000 μg BPA/kg bw/day. None of these uterine BPA effects were significant using the CAFE or Poly-3 tests that were considered the primary statistical tests in the study and, in the case of the cystic endometrial hyperplasia, severity was not increased in the treated groups relative to the vehicle control. An increase in squamous metaplasia of the uterus also occurred at the interim sacrifice with the high dose EE2 animals. In interim continuous-dose females, there were significant trends for increased apoptosis in luminal epithelial cells of the endometrium and squamous metaplasia, with a statistically significant elevation of the apoptosis at 25,000 μg BPA/kg bw/day. Apoptosis of the endometrial luminal epithelial cells also was significantly increased by the high EE2 dose in interim sacrifice animals. Taken together, these data, 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, there were trends in interim sacrifice animals for depletion of corpora lutea and interstitial cell hypertrophy in the continuous-dose arm and a trend and increase in follicular cysts at 25,000 μg BPA/kg bw/day in stop-dose BPA groups. 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. In the ovaries of interim sacrifice high dose EE2 females, there was a 100% incidence of cystic follicles.

There was an increasing trend for vaginal epithelial hyperplasia in interim sacrifice continuous BPA dose females with an increased incidence at 25,000 μg BPA/kg bw/day, although not in the primary CAFE test pairwise comparison. Vaginal epithelial hyperplasia was increased to nearly the same magnitude at all doses from 25–25,000 μg BPA/kg bw/day in terminal continuous-dose BPA animals. There were no significant BPA effects on vaginal epithelial hyperplasia in stop-dose animals. A more pronounced increased incidence of vaginal epithelial hyperplasia was also indicated by primary and secondary statistical tests with the interim sacrifice 0.5 μg EE2/kg bw/day dose group. This was accompanied by an increased incidence of epithelial mucification in the EE2 animals. When considered together with the observations mentioned above on the uterus in continuous-dose animals, the increased vaginal epithelial hyperplasia observed at 25,000 μg BPA/kg bw/day may be a plausible estrogenic effect.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No clearly interpretable impact on female mammary gland (for details see chapter 7.7)

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.

Dose descriptor:

LOAEL

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 female reproductive tract (ovary, uterus, and vagina) and in the male pituitary.

Dose descriptor:

NOAEL

Effect level:

2 500 other: µg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see information on LOAEL

Critical effects observed:

yes

Lowest effective dose / conc.:

25 000 other: µg/kg bw/day

System:

other: 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.

Organ:

ovary

pituitary gland

uterus

vagina

Treatment related:

yes

Dose response relationship:

no

Executive summary:

The toxicity of BPA administered by oral gavage to pups from postnatal day (PND) 1 (day of birth = PND 0) until termination at one year or two years was examined in Sprague-Dawley rats from the NCTR breeding colony (Sprague-Dawley/CD23/NctrBR). BPA doses were 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.I

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 conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

Comprehensive sub-acute to chronic studies, including multi-generation studies are available in rats and mice.

System:

other: General Toxicity

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles and was conducted in compliance with good laboratory practice (GLP) standards.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 30 male and 30 female rats were exposed to 0, 10, 50, or 150 mg/m3 aerosolised Bisphenol A via whole body exposure under dynamic airflow conditions for 6 hours per day, 5 days per week, for 13 weeks. Animals were observed daily and weighed weekly. Ten animals/sex/exposure group were necropsied on the day after the last exposure, and 10 animals/sex/exposure group were necropsied 4 and 12 weeks after the last exposure. Blood samples were obtained at each sacrifice for hematologic and clinical chemistry determinations. Major organs were weighed and tissues were evaluated histopathologically.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York, United States.
- Age at study initiation: 7 weeks
- Housing: Animals were individually housed in stainless steel wire cages throughout the study.
- Diet: Ad libitum (except during exposure) Ground Purina Certified Rodent Chow #5002 (Ralston Purina Company, St. Louis, Missouri, United States).
- Water: Ad libitum (except during exposure) municipal tap water.
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The APS 33-measured MMAD was 1.5-2.9 microns; the gravimetrically-measured MMAD was 2.2-5.2 microns.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 liter stainless steel and glass Rochester-type chambers operated under dynamic airflow conditions.
- Method of conditioning air: Air was filtered with an Absolute Filter (Cambridge Filter Corporation, Syracuse, New York, United States).
- System of generating particulates/aerosols: Modified Marple dust generator (Marple et al., 1978). Approximately 60 liters/minute of dry, compressed air was used to aerosolize the particles in the dust generator.
- Temperature, humidity in air chamber: 22 degrees C, 50% relative humidity
- Air flow rate: 225 liters/minute
- Air change rate: 13.5 air changes/hour
- Method of particle size determination: Aerodynamic particle sizer (APS 33, TSI Incorporated, St. Paul, Minnesota, United States)

TEST ATMOSPHERE
- Brief description of analytical method used: BPA concentration was determined either gravimetrically or with a mass monitor at least three times per day for each chamber. Aerodynamic particle size was measured approximately weekly with a cascade impactor (Model 266, Sierra Instruments, Carmel Valley, California, United States) and aerodynamic particle sizer (APS 33) during the 13-week exposure period.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure, BPA concentration was determined either gravimetrically or with a mass monitor (RamS, MIE Corporation, Bedford, Massachusetts, United States) at least three times per day for each chamber. The mass monitors were calibrated at least bi-weekly with gravimetric measurements of BPA. The mass monitors were evaluated with one gravimetric measurement on each day of exposure.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
0, 10, 50, or 150 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

30

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: After each exposure period

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined once per week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the initial exposure to BPA.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: hematocrit, hemoglobin, erythrocyte count (RBC), red blood cell indices (MCV, MCH, MCHC), total leukocyte (WBC) and platelet (PLAT) count, morphology of leukocytes, erythrocytes, and platelets.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: serum urea nitrogen (UN), glutamic pyruvic transaminase activity (SGPT), glutamic oxaloacetic transaminase activity (SGOT), alkaline phosphatase activity (AP), glucose, total protein, albumin, and globulins.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: After each exposure period.
- Battery of functions tested: Specific observations for lethargy, tremors, convulsions, salivation, lacrimation, diarrhea, and other signs of altered central nervous system function.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: lung, brain, liver, kidneys, testes
- A comprehensive gross pathological examination was peformed on all animals.

HISTOPATHOLOGY: Yes
- A comprehensive list of tissues were examined from animals sacrificed immediately after exposure to 0 or 150 mg/m3 BPA.
- Nasal tissues, lungs, cecum, and any grossly visible lesions were examined from rats sacrificed immediately after exposure to 10 or 50 mg/m3.
- After a 4-week recovery period, the nasal tissues, lungs, mediastinal tissues, mediastinal lymph nodes, trachea, cecum, and any grossly visible lesions from the control and 150 mg/m3 groups and the nasal tissues, lungs, cecum, and grossly visible lesions from rats exposed to 10 or 50 mg/m3 were examined.
- After a 12-week recovery period, the nasal tissues, lungs, mediastinal tissues, mediastinal lymph nodes, trachea, kidneys, and any grossly visible lesions from the control and 150 mg/m3 groups and the nasal tissues, kidneys, and grossly visible lesions from rats in the 10 and 50 mg/m3 groups were examined.

Statistics:

Descriptive statistics (mean and standard deviation) were used to report chamber concentrations, temperature, relative humidity, airflow, food consumption, red blood cell indices, and white blood cell differential counts. Body weights, organ weights, clinical chemistry data, and appropriate hematology data were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was peformed by parametric or non-parametric analysis of variance (ANOVA), followed by Dunnett's test or Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: A slight amount of red material, likely porphyrin, around the nose and perineal soiling was observed in male and female rats exposed to 50 or 150 mg/m3 BPA after 13 weeks of exposure. These effects were assumed to be stress-related.

BODY WEIGHT: Body weight decreased in male and female rats in the 50 and 150 mg/m3 dose groups after 13 weeks of exposure, in males and females in the 150 mg/m3 dose groups after the 4-week recovery period, and in males in the 150 mg/m3 dose group during the final 8 weeks of the 12-week recovery period.

FOOD CONSUMPTION: No effects.

HAEMATOLOGY: Male rats exposed to 10 mg/m3 had increased hemoglobin concentration but this slight increase was of no biological significance.

CLINICAL CHEMISTRY: Several effects were observed that were not considered to be biologically significant. For the 13-week exposure groups, these were decreased SGPT, SGOT, and glucose in males at 150 mg/m3 BPA; decreased total protein and albumin and increased AP in females at 150 mg/m3; and increased AP in females at 50 mg/m3. For the 13-week exposure plus 4-week recovery groups, these were increased glucose in males at 150 mg/m3; increased AP and decreased SGPT in females at 150 mg/m3; and increased AP in females at 10 mg/m3. For the 13-week exposure plus 12-week recovery group, these were increased UN in males at 10 or 150 mg/m3 and decreased total protein and globulin in females at 150 mg/m3.

ORGAN WEIGHTS: In the 13-week exposure groups, decreased absolute liver weight in males at 10 or 150 mg/m3, decreased absolute liver and kidney weights in females at 150 mg/m3, increased relative brain weights in females at 50 or 150 mg/m3, and increased relative lung weight in females at 150 mg/m3 were observed. In rats sacrificed 4 weeks after exposure, males exposed to 150 mg/m3 BPA had increased relative brain weight. In rats sacrificed 12 weeks after exposure, decreased absolute kidney and lung weights were observed in males at 150 mg/m3 and decreased absolute and relative kidney weights were observed in females at 150 mg/m3. These changes were not associated with microscopic changes and were not considered to be toxicologically significant.

GROSS PATHOLOGY: Increased cecal size attributable to distention of the cecum with food ingesta was observed in males and females exposed to 50 and 150 mg/m3 BPA for 13 weeks and in males exposed to 150 mg/m3 BPA for 13 weeks with a 4-week recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC: In the nasal cavity, slight hyperplasia of the stratified squamous epithelium, slight hyperplasia of the respiratory epithelium, and slight chronic inflammation of the underlying submucosa were observed in all animals exposed to 50 or 150 mg/m3 for 13 weeks. These changes were also observed in all animals exposed to 150 mg/m3 BPA for 13 weeks plus a 4-week recovery period, but with significanly less magnitude and severity.

Dose descriptor:

LOEC

Remarks:

Systemic

Effect level:

10 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Based on decreased absolute liver weight in males, increased AP in females, and increased UN in males at 10 mg/m3 Bisphenol A. The authors noted that all of these effects are not toxicologically significant, however.

Dose descriptor:

NOEC

Remarks:

Respiratory

Effect level:

10 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Based on reversible epithelial hyperplasia and chronic inflammation in the nasal cavity in males and females at 50 mg/m3.

Critical effects observed:

not specified

Conclusions:

The authors concluded that inhalation exposure to BPA did not result in any lower respiratory tract effects at any concentration tested.

Executive summary:

Groups of 30 male and 30 female rats were exposed to 0, 10, 50, or 150 mg/m3 aerosolised Bisphenol A via whole body exposure under dynamic airflow conditions for 6 hours per day, 5 days per week, for 13 weeks. Animals were observed daily and weighed weekly. Ten animals/sex/exposure group were necropsied on the day after the last exposure, and 10 animals/sex/exposure group were necropsied 4 and 12 weeks after the last exposure. Blood samples were obtained at each sacrifice for hematologic and clinical chemistry determinations. Major organs were weighed and tissues were evaluated histopathologically. Slight stress-related effects were observed at all concentrations of Bisphenol A, but except for decreased body weight of male rats at the highest dose, these effects disappeared relatively quickly after cessation of exposure. Enlarged ceca observed in rats necropsied the day after exposure to the two highest doses were not apparent in rats sacrificed 12 weeks later. Examination of the respiratory tract revealed slight epithelial hyperplasia and chronic inflammation in the submucosa in the nasal cavity at the two highest doses. Similar but less severe changes were observed in rats after 4 weeks of recovery but only in the highest dose group. The minor morphological changes were fully reversible within 12 weeks following exposure. In conclusion, exposure to Bisphenol A by inhalation did not result in any lower respiratory tract effects at any concentration tested.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 10 male and 10 female Sprague-Dawley rats were exposed to 0, 10, 30, and 90 mg/m³ BPA, 6 hr/day, 5 days/week for 8 weeks via whole-body inhalation. Mortality, clinical signs, body weight, hematology, serum chemistry, estrous cycle parameters, performance in the Morris water maze test, and organ weights, as well as gross and histopathological findings, were compared between the control and BPA exposure groups.

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bio Genomics Inc. (Gapyeong, Korea)
- Age at study initiation: 7 weeks
- Housing: Animals were individually housed in stainless steel wire mesh cages
- Diet: Ad libitum (except during exposure) commercial rodent diet (PMI Nutrition International, Inc. St. Louis, MO, USA)).
- Water: Ad libitum (except during exposure) filtered tap water.
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 50 +- 20
- Photoperiod (hrs dark/hrs light): 12/12
- Air changes (per hr): 10 - 15

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

The sizes of BPA particles mostly ranged from 2.10 to 7.0 μm, which accounted for 75.06% of the total distribution

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber (SIS-20RG, Shibata, Niigata, Japan)
- Method of conditioning air: no data
- System of generating particulates/aerosols: dust generator (DM-800 and DF-3, Shibata); no further data available
- Temperature, humidity in air chamber: not specified
- Air flow rate: not specified
- Air change rate: not specified
- Method of particle size determination: Anderson sampler (AN-200, Sibata, Tokyo, Japan) at a flow rate of 28.3 L/min for 20 min.

TEST ATMOSPHERE
- Brief description of analytical method used: A personal sampler (Sensidyne, St. Petersburg, FL, USA) was used for sampling BPA at a flow rate of 2 L/min through the exposure chamber. The particles
were collected from the center of the chamber and captured on mixed cellulose ester membrane filters (pore size: 4.5 μm, diameter: 37 mm; Millipore, Billerica, MA, USA).
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure, BPA concentration was determined either gravimetrically or with a mass monitor (RamS, MIE Corporation, Bedford, Massachusetts, United States) at least three times per day for each chamber. The mass monitors were calibrated at least bi-weekly with gravimetric measurements of BPA. The mass monitors were evaluated with one gravimetric measurement on each day of exposure.

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

Dose / conc.:

90 mg/m³ air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week

FOOD CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes (5 male and 5 female rats in each group)
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: total erythrocyte counts, hemoglobin concentration, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, and whole leukocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: total protein, albumin, blood urea nitrogen, creatinine, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, glucose, and total cholesterol.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: brain, thymus, lung, heart, liver, spleen, kidneys, adrenal glands,
testes, and ovaries
- A comprehensive gross pathological examination was peformed on all animals.

HISTOPATHOLOGY: Yes
- The following tissues
were removed from each animal at necropsy: the liver, kidney, adrenal glands, heart, lungs, cerebrum, cerebellum, pituitary gland, spleen, seminal vesicles, prostate, testes, epididymides, ovaries, uterus, vagina, tongue, trachea, esophagus, thymus, thyroid gland, stomach, urinary bladder, small/large intestine, eyes/harderian glands, skeletal muscles, sciatic nerve, pancreas, aorta, mesenteric lymph node, femur, larynx,
and nasal cavity. The nasal cavity was sectioned at three levels: posterior to the upper incisors; incisive papilla; and first molar teeth. The eyes were fixed in Davidson’s solution, and the testes in Bouin’s solution. Other organs were preserved in 10% neutral buffered formalin. All organs were embedded in paraffin, sectioned at 3~4 μm, stained with hematoxylin and eosin, and examined microscopically under low - and high - power fields.

Other examinations:

- Vaginal cytology
Five female rats in each group were evaluated for changes in their estrus cycles for 9 days. Vaginal smears were collected every afternoon, stained with a Giemsa solution, and observed microscopically. The estrous cycle, reflected by the vaginal
smear, was characterized as proestrus, estrus, metestrus, or diestrus according to the features of each cell type.

- Morris water maze test
Five male and five female rats from the control and 90 mg/m3 groups were evaluated for their spatial learning ability and memory for 3 days as previously described (25). A circular swimming pool (150 cm in diameter and 50 cm in height) was filled to a depth of 30 cm with water that was maintained at 25 ± 2oC and dyed with a white colored dye (Tempera; Fine Art Supplies,Auckland, New Zealand). Four equally spaced points were designated as the east, south, west, and north around the edge of the pool. An escape platform was set 1 cm below the water surface. The paths of the mice were recorded using an HVS image-tracking system (HVS Image, Hampton, UK). Each subject performed the task three times daily at intervals of 1 min, and the trials started by placing a rat in the water so that the animal faced the wall surrounding the pool. The rats were allowed to swim to the platform, and the escape latency was recorded.

Statistics:

Differences among the groups in various parameters were determined using the SPSS (version 19.0, IBM, Chicago, IL, USA) or SigmaPlot (version 13.0, SYSTAT, San Jose, CA, USA) software. The homogeneity of variance was analyzed by the Levene’s test, followed
by either one-way analysis of variance for samples with homogenous variance or the Kruskal-Wallis test for samples with heterogeneous variance. Duncan’s or Dunnett’s multiple test was used to compare the result of each experimental group with that for the control group if the first result was statistically significant.

Clinical signs:

no effects observed

Description (incidence and severity):

data not shown

Mortality:

no mortality observed

Description (incidence):

data not shown

Body weight and weight changes:

no effects observed

Description (incidence and severity):

data not shown

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):

No statistically significant hematological changes was observed in any of the BPA-exposed groups compared with the control group (data not shown).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Total cholesterol significantly increased (P < 0.05) in female rats exposed to 10 and 90 mg/m³ BPA compared to those in the control group. There were no changes in the other serum biochemical parameters. This change was not considered an adverse effect because it was not observed in the males and was not associated with morphological alterations in the liver or thyroid gland

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant change was observed in the 90 mg/m³ BPA group compared to the control group (data not shown).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The relative organ weight of the adrenal gland significantly increased (P < 0.05) in the male rat exposed to 90 mg/m³ BPA compared to those in the control group. However, these changes were not regarded as adverse effects because they were not accompanied by an
increase in the absolute organ weight of the adrenal glands and the absolute and relative organ weight of the adrenal glands in the females from the 90 mg/m3 BPA group, nor were they associated with histopathological alterations in the adrenal glands.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related histopathological change was observed in any organs of the BPA exposed group. All the other lesions were determined to be spontaneous changes that were similar in the control and exposed animals, and were accidental changes not accompanied by any dose-response relationship.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Vaginal cytology: No significant change was observed in any of the BPA groups compared to the control group (data not shown).

Dose descriptor:

NOAEL

Effect level:

> 90 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the parameters investigated

Critical effects observed:

not specified

Conclusions:

The authors concluded that inhalation exposure to BPA did not result in any adverse effects at any concentration tested.

Executive summary:

Groups of 10 male and 10 female rats were exposed to 0, 10, 30, or 90 mg/m³ aerosolised Bisphenol A via whole body exposure for 6 hours per day, 5 days per week, for 8 weeks. Animals were observed daily and weighed weekly. Ten animals/sex/exposure group were necropsied on the day after the last exposure, and 10 animals/sex/exposure group were necropsied 4 and 12 weeks after the last exposure.

Mortality, clinical signs, body weight, hematology, serum chemistry, estrous cycle parameters, performance in the Morris water maze test, and organ weights, as well as gross and histopathological findings, were compared between the control and BPA exposure groups. Statistically significant changes were observed in serum chemistry and organ weights upon exposure to BPA. However, there was no BPA-related toxic effect on the body weight, food consumption, hematology, serum chemistry, organ weights, estrous cycle, performance in the Morris water maze test, or gross or histopathological lesions in any male or female rats in the BPA exposure groups. In conclusion, the results of this study suggested that the no observable adverse effect level (NOAEL) for BPA in rats is above 90 mg/m3/6 hr/day, 5 days/week upon 8-week exposure. Furthermore, BPA did not affect the estrous cycle, spatial learning, or memory in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

150 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

There are two comprehensive sub-chronic inhalation toxicity studies available. Comprehensive sub-acute to chronic oral studies, including multi-generation studies are available in rats and mice.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles and was conducted in compliance with good laboratory practice (GLP) standards.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 30 male and 30 female rats were exposed to 0, 10, 50, or 150 mg/m3 aerosolised Bisphenol A via whole body exposure under dynamic airflow conditions for 6 hours per day, 5 days per week, for 13 weeks. Animals were observed daily and weighed weekly. Ten animals/sex/exposure group were necropsied on the day after the last exposure, and 10 animals/sex/exposure group were necropsied 4 and 12 weeks after the last exposure. Blood samples were obtained at each sacrifice for hematologic and clinical chemistry determinations. Major organs were weighed and tissues were evaluated histopathologically.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York, United States.
- Age at study initiation: 7 weeks
- Housing: Animals were individually housed in stainless steel wire cages throughout the study.
- Diet: Ad libitum (except during exposure) Ground Purina Certified Rodent Chow #5002 (Ralston Purina Company, St. Louis, Missouri, United States).
- Water: Ad libitum (except during exposure) municipal tap water.
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The APS 33-measured MMAD was 1.5-2.9 microns; the gravimetrically-measured MMAD was 2.2-5.2 microns.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 liter stainless steel and glass Rochester-type chambers operated under dynamic airflow conditions.
- Method of conditioning air: Air was filtered with an Absolute Filter (Cambridge Filter Corporation, Syracuse, New York, United States).
- System of generating particulates/aerosols: Modified Marple dust generator (Marple et al., 1978). Approximately 60 liters/minute of dry, compressed air was used to aerosolize the particles in the dust generator.
- Temperature, humidity in air chamber: 22 degrees C, 50% relative humidity
- Air flow rate: 225 liters/minute
- Air change rate: 13.5 air changes/hour
- Method of particle size determination: Aerodynamic particle sizer (APS 33, TSI Incorporated, St. Paul, Minnesota, United States)

TEST ATMOSPHERE
- Brief description of analytical method used: BPA concentration was determined either gravimetrically or with a mass monitor at least three times per day for each chamber. Aerodynamic particle size was measured approximately weekly with a cascade impactor (Model 266, Sierra Instruments, Carmel Valley, California, United States) and aerodynamic particle sizer (APS 33) during the 13-week exposure period.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure, BPA concentration was determined either gravimetrically or with a mass monitor (RamS, MIE Corporation, Bedford, Massachusetts, United States) at least three times per day for each chamber. The mass monitors were calibrated at least bi-weekly with gravimetric measurements of BPA. The mass monitors were evaluated with one gravimetric measurement on each day of exposure.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
0, 10, 50, or 150 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

30

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: After each exposure period

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined once per week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the initial exposure to BPA.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: hematocrit, hemoglobin, erythrocyte count (RBC), red blood cell indices (MCV, MCH, MCHC), total leukocyte (WBC) and platelet (PLAT) count, morphology of leukocytes, erythrocytes, and platelets.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: serum urea nitrogen (UN), glutamic pyruvic transaminase activity (SGPT), glutamic oxaloacetic transaminase activity (SGOT), alkaline phosphatase activity (AP), glucose, total protein, albumin, and globulins.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: After each exposure period.
- Battery of functions tested: Specific observations for lethargy, tremors, convulsions, salivation, lacrimation, diarrhea, and other signs of altered central nervous system function.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: lung, brain, liver, kidneys, testes
- A comprehensive gross pathological examination was peformed on all animals.

HISTOPATHOLOGY: Yes
- A comprehensive list of tissues were examined from animals sacrificed immediately after exposure to 0 or 150 mg/m3 BPA.
- Nasal tissues, lungs, cecum, and any grossly visible lesions were examined from rats sacrificed immediately after exposure to 10 or 50 mg/m3.
- After a 4-week recovery period, the nasal tissues, lungs, mediastinal tissues, mediastinal lymph nodes, trachea, cecum, and any grossly visible lesions from the control and 150 mg/m3 groups and the nasal tissues, lungs, cecum, and grossly visible lesions from rats exposed to 10 or 50 mg/m3 were examined.
- After a 12-week recovery period, the nasal tissues, lungs, mediastinal tissues, mediastinal lymph nodes, trachea, kidneys, and any grossly visible lesions from the control and 150 mg/m3 groups and the nasal tissues, kidneys, and grossly visible lesions from rats in the 10 and 50 mg/m3 groups were examined.

Statistics:

Descriptive statistics (mean and standard deviation) were used to report chamber concentrations, temperature, relative humidity, airflow, food consumption, red blood cell indices, and white blood cell differential counts. Body weights, organ weights, clinical chemistry data, and appropriate hematology data were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was peformed by parametric or non-parametric analysis of variance (ANOVA), followed by Dunnett's test or Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: A slight amount of red material, likely porphyrin, around the nose and perineal soiling was observed in male and female rats exposed to 50 or 150 mg/m3 BPA after 13 weeks of exposure. These effects were assumed to be stress-related.

BODY WEIGHT: Body weight decreased in male and female rats in the 50 and 150 mg/m3 dose groups after 13 weeks of exposure, in males and females in the 150 mg/m3 dose groups after the 4-week recovery period, and in males in the 150 mg/m3 dose group during the final 8 weeks of the 12-week recovery period.

FOOD CONSUMPTION: No effects.

HAEMATOLOGY: Male rats exposed to 10 mg/m3 had increased hemoglobin concentration but this slight increase was of no biological significance.

CLINICAL CHEMISTRY: Several effects were observed that were not considered to be biologically significant. For the 13-week exposure groups, these were decreased SGPT, SGOT, and glucose in males at 150 mg/m3 BPA; decreased total protein and albumin and increased AP in females at 150 mg/m3; and increased AP in females at 50 mg/m3. For the 13-week exposure plus 4-week recovery groups, these were increased glucose in males at 150 mg/m3; increased AP and decreased SGPT in females at 150 mg/m3; and increased AP in females at 10 mg/m3. For the 13-week exposure plus 12-week recovery group, these were increased UN in males at 10 or 150 mg/m3 and decreased total protein and globulin in females at 150 mg/m3.

ORGAN WEIGHTS: In the 13-week exposure groups, decreased absolute liver weight in males at 10 or 150 mg/m3, decreased absolute liver and kidney weights in females at 150 mg/m3, increased relative brain weights in females at 50 or 150 mg/m3, and increased relative lung weight in females at 150 mg/m3 were observed. In rats sacrificed 4 weeks after exposure, males exposed to 150 mg/m3 BPA had increased relative brain weight. In rats sacrificed 12 weeks after exposure, decreased absolute kidney and lung weights were observed in males at 150 mg/m3 and decreased absolute and relative kidney weights were observed in females at 150 mg/m3. These changes were not associated with microscopic changes and were not considered to be toxicologically significant.

GROSS PATHOLOGY: Increased cecal size attributable to distention of the cecum with food ingesta was observed in males and females exposed to 50 and 150 mg/m3 BPA for 13 weeks and in males exposed to 150 mg/m3 BPA for 13 weeks with a 4-week recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC: In the nasal cavity, slight hyperplasia of the stratified squamous epithelium, slight hyperplasia of the respiratory epithelium, and slight chronic inflammation of the underlying submucosa were observed in all animals exposed to 50 or 150 mg/m3 for 13 weeks. These changes were also observed in all animals exposed to 150 mg/m3 BPA for 13 weeks plus a 4-week recovery period, but with significanly less magnitude and severity.

Dose descriptor:

LOEC

Remarks:

Systemic

Effect level:

10 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Based on decreased absolute liver weight in males, increased AP in females, and increased UN in males at 10 mg/m3 Bisphenol A. The authors noted that all of these effects are not toxicologically significant, however.

Dose descriptor:

NOEC

Remarks:

Respiratory

Effect level:

10 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Based on reversible epithelial hyperplasia and chronic inflammation in the nasal cavity in males and females at 50 mg/m3.

Critical effects observed:

not specified

Conclusions:

The authors concluded that inhalation exposure to BPA did not result in any lower respiratory tract effects at any concentration tested.

Executive summary:

Groups of 30 male and 30 female rats were exposed to 0, 10, 50, or 150 mg/m3 aerosolised Bisphenol A via whole body exposure under dynamic airflow conditions for 6 hours per day, 5 days per week, for 13 weeks. Animals were observed daily and weighed weekly. Ten animals/sex/exposure group were necropsied on the day after the last exposure, and 10 animals/sex/exposure group were necropsied 4 and 12 weeks after the last exposure. Blood samples were obtained at each sacrifice for hematologic and clinical chemistry determinations. Major organs were weighed and tissues were evaluated histopathologically. Slight stress-related effects were observed at all concentrations of Bisphenol A, but except for decreased body weight of male rats at the highest dose, these effects disappeared relatively quickly after cessation of exposure. Enlarged ceca observed in rats necropsied the day after exposure to the two highest doses were not apparent in rats sacrificed 12 weeks later. Examination of the respiratory tract revealed slight epithelial hyperplasia and chronic inflammation in the submucosa in the nasal cavity at the two highest doses. Similar but less severe changes were observed in rats after 4 weeks of recovery but only in the highest dose group. The minor morphological changes were fully reversible within 12 weeks following exposure. In conclusion, exposure to Bisphenol A by inhalation did not result in any lower respiratory tract effects at any concentration tested.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 10 male and 10 female Sprague-Dawley rats were exposed to 0, 10, 30, and 90 mg/m³ BPA, 6 hr/day, 5 days/week for 8 weeks via whole-body inhalation. Mortality, clinical signs, body weight, hematology, serum chemistry, estrous cycle parameters, performance in the Morris water maze test, and organ weights, as well as gross and histopathological findings, were compared between the control and BPA exposure groups.

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bio Genomics Inc. (Gapyeong, Korea)
- Age at study initiation: 7 weeks
- Housing: Animals were individually housed in stainless steel wire mesh cages
- Diet: Ad libitum (except during exposure) commercial rodent diet (PMI Nutrition International, Inc. St. Louis, MO, USA)).
- Water: Ad libitum (except during exposure) filtered tap water.
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 50 +- 20
- Photoperiod (hrs dark/hrs light): 12/12
- Air changes (per hr): 10 - 15

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

The sizes of BPA particles mostly ranged from 2.10 to 7.0 μm, which accounted for 75.06% of the total distribution

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber (SIS-20RG, Shibata, Niigata, Japan)
- Method of conditioning air: no data
- System of generating particulates/aerosols: dust generator (DM-800 and DF-3, Shibata); no further data available
- Temperature, humidity in air chamber: not specified
- Air flow rate: not specified
- Air change rate: not specified
- Method of particle size determination: Anderson sampler (AN-200, Sibata, Tokyo, Japan) at a flow rate of 28.3 L/min for 20 min.

TEST ATMOSPHERE
- Brief description of analytical method used: A personal sampler (Sensidyne, St. Petersburg, FL, USA) was used for sampling BPA at a flow rate of 2 L/min through the exposure chamber. The particles
were collected from the center of the chamber and captured on mixed cellulose ester membrane filters (pore size: 4.5 μm, diameter: 37 mm; Millipore, Billerica, MA, USA).
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure, BPA concentration was determined either gravimetrically or with a mass monitor (RamS, MIE Corporation, Bedford, Massachusetts, United States) at least three times per day for each chamber. The mass monitors were calibrated at least bi-weekly with gravimetric measurements of BPA. The mass monitors were evaluated with one gravimetric measurement on each day of exposure.

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

Dose / conc.:

90 mg/m³ air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week

FOOD CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes (5 male and 5 female rats in each group)
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: total erythrocyte counts, hemoglobin concentration, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, and whole leukocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At all scheduled necropsies.
- Animals fasted: Yes, overnight.
- Parameters examined: total protein, albumin, blood urea nitrogen, creatinine, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, glucose, and total cholesterol.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: brain, thymus, lung, heart, liver, spleen, kidneys, adrenal glands,
testes, and ovaries
- A comprehensive gross pathological examination was peformed on all animals.

HISTOPATHOLOGY: Yes
- The following tissues
were removed from each animal at necropsy: the liver, kidney, adrenal glands, heart, lungs, cerebrum, cerebellum, pituitary gland, spleen, seminal vesicles, prostate, testes, epididymides, ovaries, uterus, vagina, tongue, trachea, esophagus, thymus, thyroid gland, stomach, urinary bladder, small/large intestine, eyes/harderian glands, skeletal muscles, sciatic nerve, pancreas, aorta, mesenteric lymph node, femur, larynx,
and nasal cavity. The nasal cavity was sectioned at three levels: posterior to the upper incisors; incisive papilla; and first molar teeth. The eyes were fixed in Davidson’s solution, and the testes in Bouin’s solution. Other organs were preserved in 10% neutral buffered formalin. All organs were embedded in paraffin, sectioned at 3~4 μm, stained with hematoxylin and eosin, and examined microscopically under low - and high - power fields.

Other examinations:

- Vaginal cytology
Five female rats in each group were evaluated for changes in their estrus cycles for 9 days. Vaginal smears were collected every afternoon, stained with a Giemsa solution, and observed microscopically. The estrous cycle, reflected by the vaginal
smear, was characterized as proestrus, estrus, metestrus, or diestrus according to the features of each cell type.

- Morris water maze test
Five male and five female rats from the control and 90 mg/m3 groups were evaluated for their spatial learning ability and memory for 3 days as previously described (25). A circular swimming pool (150 cm in diameter and 50 cm in height) was filled to a depth of 30 cm with water that was maintained at 25 ± 2oC and dyed with a white colored dye (Tempera; Fine Art Supplies,Auckland, New Zealand). Four equally spaced points were designated as the east, south, west, and north around the edge of the pool. An escape platform was set 1 cm below the water surface. The paths of the mice were recorded using an HVS image-tracking system (HVS Image, Hampton, UK). Each subject performed the task three times daily at intervals of 1 min, and the trials started by placing a rat in the water so that the animal faced the wall surrounding the pool. The rats were allowed to swim to the platform, and the escape latency was recorded.

Statistics:

Differences among the groups in various parameters were determined using the SPSS (version 19.0, IBM, Chicago, IL, USA) or SigmaPlot (version 13.0, SYSTAT, San Jose, CA, USA) software. The homogeneity of variance was analyzed by the Levene’s test, followed
by either one-way analysis of variance for samples with homogenous variance or the Kruskal-Wallis test for samples with heterogeneous variance. Duncan’s or Dunnett’s multiple test was used to compare the result of each experimental group with that for the control group if the first result was statistically significant.

Clinical signs:

no effects observed

Description (incidence and severity):

data not shown

Mortality:

no mortality observed

Description (incidence):

data not shown

Body weight and weight changes:

no effects observed

Description (incidence and severity):

data not shown

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):

No statistically significant hematological changes was observed in any of the BPA-exposed groups compared with the control group (data not shown).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Total cholesterol significantly increased (P < 0.05) in female rats exposed to 10 and 90 mg/m³ BPA compared to those in the control group. There were no changes in the other serum biochemical parameters. This change was not considered an adverse effect because it was not observed in the males and was not associated with morphological alterations in the liver or thyroid gland

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant change was observed in the 90 mg/m³ BPA group compared to the control group (data not shown).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The relative organ weight of the adrenal gland significantly increased (P < 0.05) in the male rat exposed to 90 mg/m³ BPA compared to those in the control group. However, these changes were not regarded as adverse effects because they were not accompanied by an
increase in the absolute organ weight of the adrenal glands and the absolute and relative organ weight of the adrenal glands in the females from the 90 mg/m3 BPA group, nor were they associated with histopathological alterations in the adrenal glands.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related histopathological change was observed in any organs of the BPA exposed group. All the other lesions were determined to be spontaneous changes that were similar in the control and exposed animals, and were accidental changes not accompanied by any dose-response relationship.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Vaginal cytology: No significant change was observed in any of the BPA groups compared to the control group (data not shown).

Dose descriptor:

NOAEL

Effect level:

> 90 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the parameters investigated

Critical effects observed:

not specified

Conclusions:

The authors concluded that inhalation exposure to BPA did not result in any adverse effects at any concentration tested.

Executive summary:

Groups of 10 male and 10 female rats were exposed to 0, 10, 30, or 90 mg/m³ aerosolised Bisphenol A via whole body exposure for 6 hours per day, 5 days per week, for 8 weeks. Animals were observed daily and weighed weekly. Ten animals/sex/exposure group were necropsied on the day after the last exposure, and 10 animals/sex/exposure group were necropsied 4 and 12 weeks after the last exposure.

Mortality, clinical signs, body weight, hematology, serum chemistry, estrous cycle parameters, performance in the Morris water maze test, and organ weights, as well as gross and histopathological findings, were compared between the control and BPA exposure groups. Statistically significant changes were observed in serum chemistry and organ weights upon exposure to BPA. However, there was no BPA-related toxic effect on the body weight, food consumption, hematology, serum chemistry, organ weights, estrous cycle, performance in the Morris water maze test, or gross or histopathological lesions in any male or female rats in the BPA exposure groups. In conclusion, the results of this study suggested that the no observable adverse effect level (NOAEL) for BPA in rats is above 90 mg/m3/6 hr/day, 5 days/week upon 8-week exposure. Furthermore, BPA did not affect the estrous cycle, spatial learning, or memory in rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

There are two comprehensive sub-chronic inhalation toxicity studies available. Comprehensive sub-acute to chronic oral studies, including multi-generation studies are available in rats and mice.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

EFSA reevaluated Bisphenol A and published a Scientific Opinion in 2015: 

“Bisphenol A was found to affect kidney and liver weight in parental animals and in all the generations of rats and mice examined in multi-generation studies. These effects were considered by EFSA (2006, 2010) as relevant systemic effects for the identification of a NOAEL. In mice the increased kidney weight was associated with nephropathy at the highest Bisphenol A dose. Liver weight was increased in rats (relative weight) and mice (both absolute and relative weight). The latter species also showed hepatocellular hypertrophy. The CEF Panel considered the endpoint “general toxicity” for hazard characterisation, using a reference point from the two-generation study in mice, which provided a BMDL10 for mean relative kidney weight of 8 960 μg/kg bw per day in male mice of the F0 generation.” 

In 2014 SCOEL published a Recommendation on Bisphenol A:

 

“To establish a recommended occupational exposure limit (OEL), SCOEL began by considering the available data relating to inhalation exposure. In rats exposed daily to airborne Bisphenol A for 13 weeks there was a NOAEC of 10 mg/m3, with mild olfactoryepithelium inflammation at 50 and 150 mg/m3. There was no evidence of systemic toxicity in this study (Nitschke 1988).” 

 

The 2008 updated EU RAR concluded:

 

"Oral studies in rats and mice have shown that the repeated dose toxicity of Bisphenol A involve[s] effects on bodyweight gain, liver and kidney. A NOAEL of 50 mg/kg/day has been identified in a recent 2-generation study in mice for these effects. This NOAEL rather than the original NOAEL of 120 mg/kg/day for liver effects from the published report is taken forward to the risk characterisation." 

 

The 2003 EU RAR concluded:

"No useful information on the effects of repeated exposure to Bisphenol A in humans is available, but experimental studies in rats, mice, and dogs are available. In rat inhalation studies, the principal effect of repeated exposure was the same as observed following a single exposure: slight upper respiratory tract epithelium inflammation, with a NOAEC of 10 mg/m3and a LOAEC of 50 mg/m3. Dietary studies in rats have reported reductions in reproductive organ weights and testicular toxicity at 235 mg/kg and a NOAEL of 74 mg/kg was established in a two-year study based on marginal effects on body weight gain at the next dose level of 148 mg/kg. In mice, the LOAELs of 120 mg/kg in males for multinuclear giant hepatocytes and 650 mg/kg in females for a reduction in body weight gain of unknown magnitude were identifed in a two-year study. There are no animal data available for repeated dermal exposure." 

 
Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:  
Study was selected by EFSA in 2015 as a starting point for TDI calculation  
 
Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:  
Study was selected by SCOEL in 2014 as a starting point for TWA calculation  
 
Justification for selection of repeated dose toxicity inhalation - local effects endpoint:  
Study was selected by SCOEL in 2014 as a starting point for TWA calculation  
 
Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys 

Justification for classification or non-classification

Bisphenol A is included in Annex VI of Regulation (EC) No 1272/2008. No classification regarding repeated dose toxicity is required.