4,4'-isopropylidenediphenol

Neurotoxicity

Description of key information

 A Guideline compliant developmental neurotoxicity study indicated no effect up to the highest dose tested; 2250 ppm (corresponding to 164 mg/kg/day during gestation and 410 mg/kg/day during lactation). This is in line with recently published studies in the course of the CLARITY-BPA program, in which no consistent effect of Bisphenol A exposure on Exploratory and Anxiety behavior or spatial navigational learning and memory was observed.   

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: oral

Remarks:

developmental

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted under OECD and US EPA guidelines and Good Laboratory Practices

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animal strain: Female Sprague Dawley Crl:CD(SD) rats were obtained from Charles River Laboratories (Raleigh, North Carolina, United States).
- Housing: Upon arrival and until pairing, all animals were housed individually in stainless steel wire-mesh cages suspended above cage-board. Animals were paired for mating in the home cage of the resident male. After mating, females were individually housed in stainless steel wire-mesh cages until gestation day 18, then were housed in polyphenylsulfone RaTEMP thermoplastic rat cages (Allentown Inc., Allentown, New Jersey, United States) with ground corncob bedding (The Andersons, Cob Products Division, Maumee, Ohio, United States). Pups were housed by litter in the polyphenylsulfone RaTEMP thermoplastic cages with corncob bedding until PND 28, when surviving offspring were housed individually in suspended wire-mesh stainless steel cages until euthanasia.
- Diet: Ad libitum. Feed was PMI International, LLC, Certified Rodent LabDiet 5002 (meal). No contaminants were present at concentrations sufficient to interfere with the objectives of the study.
- Drinking water: Ad libitum. Reverse osmosis-purified (on-site) drinking water was delivered by an automatic watering system. No contaminants were present at concentrations sufficient to interfere with the objectives of the study.
- Acclimation period: Thirteen days, during which the rats were observed twice daily for general changes in appearance and behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 degrees C
- Relative humidity: 30-70%
- Photoperiod: 12 hours light/12 hours dark

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

The test substance was administered as a constant concentration (ppm) in the diet. Individual maternal food consumption was recorded on GD 0, 3, 7, 10, 14, 17, and 20 and on lactation days 1, 4, 7, 11, 14, 17, and 21. Group mean body weights were calculated for each of these days. Group mean body weight changes were calculated for each corresponding interval of gestation and lactation and also for gestation days 0-7, 7-14, 14-20, and 0-20 and for lactation days 1-21.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity was analysed prior to the initiation of exposure in duplicate samples that were collected from the top, middle, and bottom of each test diet formulation. Stability of the test substance in the diet was established in a previous study (Bodle, 2009; WIL-186057) and was not analysed in this study.

Duration of treatment / exposure:

P generation females were administered test diets ad libitum from GD 0 to lactation day 21. F1 animals were potentially exposed to the test substance in utero as well as via milk while nursing and via direct consumption of the diet during the latter portion of the lactation period.

Frequency of treatment:

Animals ate Bisphenol A-containing food ad libitum.

Remarks:

Doses / Concentrations:
0, 0.15, 1.5, 75, 750, and 2250 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

The experimental design consisted of five test substance-treated groups and one control group. Each group was composed of 24 rats each, consisting of two cohorts starting approximately four weeks apart. Each cohort was composed of 12 females per group. On each GD 0, the bred females were assigned to groups using a WIL Toxicology Data Management System (WTDMS) computer program which randomised the animals based on stratification of the GD 0 body weights in a block design. Body weight values ranged from 231 g to 297 g on GD 0. To reduce variability among the litters of F1 animals, eight pups per litter with four per sex (if possible) were randomly selected for study inclusion on PND 4 and the remaining pups were euthanised. If a litter consisted of six pups or fewer or did not meet the sex ratio criteria (at least three/sex) for study inclusion, the litter was not used for neurobehavioural or neuropathological evaluation and the pups were discarded. Each dam and litter remained together until weaning on lactation day 21. Following standardisation of litters on PND4, a subset (Subset A) consisting of one pup/sex/litter was assigned to detailed clinical observations on PND 4, 11, 21, 35, 45, and 60; auditory startle response (PND 20 and 60); motor activity (PND 13, 17, 21, and 61); and learning and memory (PND 62). These same animals were selected for brain weight evaluation on PND72; of these, one pup/litter for a total of 10 animals/sex/group was selected for neuropathological and morphometric evaluations on PND 72. A second subset (Subset B) consisting of one pup/sex/litter was selected for learning and memory (PND 22). A third subset (Subset C) consisting of one pup/sex/litter was selected for brain weight evaluations on PND 21; of these, one pup/litter for a total of 10 animals/sex/group was selected for neuropathological and morphometric evaluations on PND 21. All F1 animals not selected for behavioural evaluations (Subset D) were euthanised on PND 21.

Observations and clinical examinations performed and frequency:

P generation females were observed twice daily for moribundity and mortality. General clinical observations were recorded daily. Detailed clinical observations were performed on GD 10 and 15 and lactation days 10 and 21. Body weights were measured on GD 0, 3, 7, 10, 14, 17, and 20 and on lactation days 1, 4, 7, 11, 14, 17, and 21. During parturition, the dams were observed twice daily for initiation and completion of parturition and for signs of dystocia.

Neurobehavioural examinations performed and frequency:

F1 pups were evaluated for the following:
- Auditory startle response (Subset A; PND 20 and 60)
- Motor activity (Subset A; PND 13, 17, 21, and 61)
- Biel maze swimming trials (Subsets A and B; beginning on PND 22 and 62)

Sacrifice and (histo)pathology:

P GENERATION FEMALES:
All surviving females with viable pups on lactation day 21 that had six pups or fewer or failed to meet the pup sex ratio criteria (lactation day 4) and that did not deliver (post-mating day 25) were euthanised by carbon dioxide inhalation and a gross necropsy was performed. The liver and kidneys were weighed and microscopic evaluations were performed on these organs.

F1 GENERATION ANIMALS:
Intact pups that died from PND 0 to 4 were necropsied using a fresh dissection technique that included the heart and major vessels and a detailed gross necropsy was performed on any pup dying or euthanised after PND 4. Subset C and Subset A were macroscopically examined for neuropathology on PND 21 and PND 72, respectively. Brains from these pups were used for qualitative histopathological examination and morphometric analysis.

Statistics:

The litter was used as the experimental unit, where applicable.
The data for cohorts 1 and 2 were combined for all statistical analyses.
Analyses were conducted for Type I error, using two-tailed tests (unless otherwise noted) for minimum significance levels of 0.01 and 0.05, comparing each test substance-treated group to the control group.
Reproductive parameters and detailed clinical observation data were analysed by Dunnett's test.
Mean litter proportions (percent per litter) of pup viability and males per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.
All body weight and food consumption data were analysed separately with a repeated measures analysis of variance (RANOVA), except for cumulative gestation body weight gain, which was analysed with ANOVA, and pup body weight gain from PND 1-4, which was analysed with a nested ANOVA.
Maternal organ weights were analysed with analysis of covariance (ANCOVA).
The mean day of attainment for surface righting and for balanopreputial separation (male pups) and vaginal separation (female pups) were analysed with a nested ANOVA. For all developmental landmark nested ANOVA analyses, the Kenward Rogers adjustment for denominator degrees of freedom were applied and pairwise comparisons were made for each individual treatment group with the control group using Dunnett's test.
The neurobehavioural data were analysed with a RANOVA.
Brain measurement data were analysed using a MANOVA.

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):

effects observed, treatment-related

Food efficiency:

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):There were no effects on reproductive performance in P generation animals.
In F1 generation animals, there were no effects on viability, developmental parameters, or the onset of sexual maturation as measured by the age of attainment of vaginal opening or preputial separation at any exposure level.
(migrated information)

Details on results:

Maternal survival, clinical signs, and detailed clinical observations were not affected at any exposure level.
Maternal toxicity was expressed at 750 and 2250 ppm by lower mean body weight gains during gestation of 9.5% and 22.4%, respectively, with an associated reduction in mean food consumption from the onset of test stubstance exposure through GD 10 and 20, respectively. No additional evidence of systemic toxicity was observed in P generation animals, including organ weights, gross findings, or microscopic changes.
Reduced mean pup body weights of 5.6% and 8.3% were observed in F1 animals at 750 ppm (during PND 14-17) and 2250 ppm (during PND 11-21), respectively.
For F1 males and females, there were no effects on detailed clinical observations, macroscopic examinations and measurements, motor activity, auditory startle, learning and memory, neuropathology, or brain morphometry at any exposure level.

Dose descriptor:

other: NOAEL - Systemic

Effect level:

ca. 75 ppm

Sex:

male/female

Basis for effect level:

other: Based on reductions in mean body weight and body weight gain in dams and offspring at 750 ppm.

Remarks on result:

other: Generation: other: - maternal and offspring (migrated information)

Dose descriptor:

other: NOAEL - Developmental neurotoxicity

Effect level:

ca. 2 250 ppm

Sex:

male/female

Basis for effect level:

other: There was no evidence of developmental neurotoxicity at any dietary concentration, so the NOAEL for this endpoint is 2250 ppm, the highest Bisphenol A concentration tested.

Remarks on result:

other: Generation: offspring (migrated information)

Conclusions:

The authors concluded that based on the conditions of this study, there was no evidence that BPA is a developmental neurotoxicant in rats.

Executive summary:

BPA was administered at concentrations of 0, 0.15, 1.5, 75, 750, and 2250 ppm on a continuous basis in the diet to six groups of 24 bred female Crl:CD(SD) rat dams daily from GD 0 through lactation day 21. Clinical observations and body weight and food consumption measurements were conducted on all dams in each group during gestation and lactation. All dams were allowed to deliver and rear their offspring until lactation day 21. Clinical observations, body weights, and sex were recorded for the F1 pups at appropriate intervals. Surface righting response was evaluated for all pups beginning on PND 2. On PND 4, litters were culled to eight pups/litter. Subsets of pups were assigned to detailed clinical observations, various neurobehavioral evaluations, brain weight measurements, and neuropathological and morphometric evaluations. Maternal survival, clinical signs, and detailed clinical observations were not affected at any exposure level. Maternal toxicity was expressed at 750 and 2250 ppm by lower mean body weight gains during gestation of 9.5% and 22.4%, respectively, with an associated reduction in mean food consumption from the onset of test stubstance exposure through GD 10 and 20, respectively. No additional evidence of systemic toxicity was observed at any exposure level in P generation animals, including organ weights, gross findings, or microscopic changes and there were no effects on reproductive performance. Reduced mean pup body weights of 5.6% and 8.3% were observed in F1 animals at 750 ppm (during PND 14-17) and 2250 ppm (during PND 11-21), respectively. For F1 males and females, there were no effects on viability, detailed clinical observations, motor activity, auditory startle, learning and memory, macroscopic examinations and measurements, neuropathology, brain morphometry, developmental parameters, or the onset of sexual maturation as measured by the age of attainment of vaginal opening or preputial separation at any exposure level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

164 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A robust study in rats conducted under OECD and US EPA guidelines (Stump, 2009) reported no evidence of developmental neurotoxicity at any of the tested concentrations of Bisphenol A. Bisphenol A was continuously administered to rat dams during gestation and lactation via test diets containing 0, 0.15, 1.5, 75, 750, and 2250 ppm Bisphenol A. Reduced body weight and body weight gain was observed in dams and F1 males and females at 750 and 2250 ppm Bisphenol A. There were no effects observed at any exposure level in the neurobehavioural and neuropathological evaluations of F1 animals. A systemic NOAEL of 75 ppm (corresponding to 5.85 mg/kg/day during gestation and 13.1 mg/kg/day during lactation) for reduction in body weight and weight gain of dams and offspring was determined. A NOAEL for developmental neurotoxicity of 2250 ppm (corresponding to 164 mg/kg/day during gestation and 410 mg/kg/day during lactation) in offspring was determined, as there was no evidence of developmental neurotoxicity at any tested dose, including the highest dose of 2250 ppm Bisphenol A.

 

Recent information taken into account for the dossier update:

There are two recent studies in the context of the US NTP CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) program addressing potential neurobehavioral effects (Rebuli et al 2015 and Johnson et al 2015). The impact of perinatal Bisphenol A exposure (2.5, 25, or 2500 µg/kg body weight (bw)/day) on behaviors related to anxiety and exploratory activity was assessed in juvenile (pre-pubertal) and adult NCTR Sprague-Dawley rats of both sexes. Ethinyl estradiol (EE; 0.5 µg/kg bw/day) was used as a reference estrogen. Exposure spanned gestation and lactation with dams gavaged from gestational day 6 until birth, and then the offspring gavaged directly through weaning (n = 12/sex/group).

Behavioral assessments included open field, elevated plus maze, and zero maze. Anticipated sex differences in behavior were statistically identified or suggested in most cases. No consistent effects of Bisphenol A were observed for any endpoint, in either sex, at either age compared to vehicle controls; however, significant differences between Bisphenol A-exposed and EE-exposed groups were identified for some endpoints. These data do not indicate Bisphenol A-related effects on anxiety or exploratory activity in these developmentally exposed rats (Rebuli et al 2015; for further details on the study see chapter toxicity to reproduction)

At adulthood, offspring were tested for seven days in the Barnes maze. No consistent or robust effect was observed. The 2500 Bisphenol A group sniffed more incorrect holes on day 7 only than those in the control, 2.5 Bisphenol A, and EE groups. The 2500 Bisphenol A females were less likely than control females to locate the escape box in the allotted time (p value = 0.04). Although 2.5 Bisphenol A females exhibited a prolonged latency, the effect did not reach significance (p value = 0.06), whereas 2.5 Bisphenol A males showed improved latency compared to control males (p value = 0.04); the significance of this result is uncertain. No differences in serum testosterone concentration were detected in any male or female treatment groups (Johnson et al 2015; for further details on the study see chapter toxicity to reproduction).

 

EFSA Opinion 2015 Conclusions on neurological, neurodevelopmental and neuroendocrine effects.

"There are indications from prospective studies in humans that prenatal Bisphenol A exposure (Bisphenol A exposure during pregnancy) may be associated with altered child behaviour in a sex-dependent manner. However, the associations were not consistent across the studies and it cannot be ruled out that the results are confounded by diet or concurrent exposure factors. The associations reported do not provide sufficient evidence to infer a causal link between Bisphenol A exposure during pregnancy or childhood and neurodevelopmental effects in humans.

A number of new studies report changes that may indicate effects of Bisphenol A on brain development (effect on neurogenesis and on gene expression, neuroendocrine effects, effects on the morphology of certain brain regions, etc.). Whether such changes are mechanistically related to the neurobehavioral responses reported following exposure is attempted addressed by some studies but with inconsistent results.

Several new animal studies investigated anxiety-like behaviour, learning and memory, social behaviour and sensory-motor function. Some studies report changes in anxiety-like behaviour after Bisphenol A exposure. Some, but not all, studies reported significant impairment of either learning and/or memory capacities. A few studies also report effects on social behaviour and sensory-motor function. However, the studies present methodological shortcomings, such as small sample size, lack of consideration of the litter effect, not properly controlled variability of exposure through diet and inadequate statistics. Using a WoE approach, the CEF Panel assigned a likelihood level of “as likely as not” to neurological, neurodevelopmental and neuroendocrine effects of Bisphenol A level for this endpoint is less than "likely" (see Appendix A), this endpoint was not taken forward for assessing the toxicological reference point, but was taken into account in the evaluation of uncertainty for hazard characterisation and risk characterisation (Section 4.3)."

Justification for classification or non-classification

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