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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
  • Bisphenol A is negative for mutagenicity in bacterial reverse mutation assay. 
  • Bisphenol A is not mutagenic in mouse lymphoma cells 
  • Bisphenol A does not induce Sister Chromatid Exchanges or Chromosome Abberations 

There is conclusive evidence that Bisphenol A is not mutagenic or genotoxic in vitro.

EU RAR 2008, SCOEL recommendation 2014 and EFSA opinion 2015 concluded that Bisphenol A is not mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration; DNA damage
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 which meets basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chinese hamster ovary (CHO-WBL) cells were used for detection of sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). Bisphenol A was tested in each assay with and without exogenous metabolic activation.

For the SCE trials without metabolic activation, cells were exposed to Bisphenol A (0, 0.6, 2.4, 8.0, 15, 20, 25, 30, 40, or 50 µg/mL) for approximately 25 hours; for the trails with metabolic activation, exposure was for two hours. For both testing conditions, BrdU was added two hours after dosing and Colcemid was added during the last 2 to 2.5 hours of incubation. Mitotic cells were fixed on slides, stained with Hoechst 33258, and examined for SCEs by fluorescence microscopy.

In the CA assay without metabolic activation, cells were exposed to Bisphenol A (0, 20, 30, 40, or 50 µg/mL) for eight hours, then cells were washed and treated with Colcemid for 2 to 2.5 hours. With metabolic activation, the cells were exposed to Bisphenol A and the metabolic activation mixture for two hours, washed, incubated for eight hours, then treated with Colcemid for 2 to 2.5 hours. Cells were harvested, fixed on slides, and stained with Giemsa for analysis and classification of CAs.
GLP compliance:
no
Type of assay:
other: in vitro mammalian chromosome aberration test; sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
15 µL/mL S9 (obtained from livers of Aroclor 1254-treated male Sprague Dawley rats), 1.5 mg/mL NADP, and 2.7 mg/mL isocitric acid in serum-free medium
Test concentrations with justification for top dose:
For SCE assay: 0, 0.6, 2.4, 8.0, 15, 20, 25, 30, 40, or 50 µg/mL
For CA assay: 0, 20, 30, 40, or 50 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C for assays without metabolic activation; cyclophosphamide for assays with metabolic activation
Details on test system and experimental conditions:
If a treatment resulted in a delay of the cell cycle progression, a later harvest was performed in an attempt to collect a sufficient number of metaphase II cells for scoring. In these cases, the reincubated cells were harvested after an additional 6 to 8 hour incubation for the SCE assay and the cell growth period was extended to about 20 hours for the CA assay.
Evaluation criteria:
For the SCE assay, 50 cells were scored per dose in the initial trial, and 25 were scored in the repeat trials.
For the CA assay, 100 to 200 cells from each of the three highest scorable doses were analyzed. All aberrations were individually classified (e.g., chromatid breaks, triradials, etc., as described by Galloway et al. [1985, 1987]); however, these data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex, and other) aberrations. Only the total percent cells with aberrations was considered in the statistical evaluation. Gaps and endoreduplications were recorded but were not included in the statistical analyses.
Statistics:
In the SCE assay, an increase of 20% or greater in SCEs per chromosome over the solvent control was considered significant. A significant increase in the CA assay was based on a binomial sampling assumption; the P values were adjusted according to Dunnett's method to take into account multiple dose comparisons. The trend test for both assays used a linear regression analysis: SCEs per chromosome vs. the log dose, and the percentage of cells with aberrations vs. the log dose.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
BPA, at 8 ug/mL, produced a small increase (22%) in the first SCE trial without metabolic activation. Cell cycle delay was observed at this dose, and this response was not observed in the second trial, even though higher doses (15 to 30 ug/mL) were analyzed. The trial with metabolic activation did not induce SCEs when tested up to toxic levels of BPA, and it was concluded that BPA does not induce SCE.

In the CA trial without metabolic activation, BPA did not induce CAs when tested with delayed harvest and up to toxic levels. In the first CA trial with metabolic activation, a positive response was observed at 50 ug/mL, a dose where cell confluence was reduced by approximately 70%. This response did not repeat in the second trial, and it was concluded that BPA was negative in the CA assay.
Remarks on result:
other: strain/cell type: CHO-WBL
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The authors concluded that BPA does not induce SCEs or CAs in CHO cells.
Executive summary:

Chinese hamster ovary (CHO-WBL) cells were used for detection of sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). Bisphenol A was tested in each assay with and without exogenous metabolic activation. For the SCE trials without metabolic activation, cells were exposed to Bisphenol A (0, 0.6, 2.4, 8.0, 15, 20, 25, 30, 40, or 50 µg/mL) for approximately 25 hours; for the trails with metabolic activation, exposure was for two hours. For both testing conditions, BrdU was added two hours after dosing and Colcemid was added during the last 2 to 2.5 hours of incubation. Mitotic cells were fixed on slides, stained with Hoechst 33258, and examined for SCEs by fluorescence microscopy. Bisphenol A, at 8 µg/mL, produced a small increase (22%) in the first SCE trial without metabolic activation. Cell cycle delay was observed at this dose, and this response was not observed in the second trial, even though higher doses (15 to 30 µg/mL) were analyzed. The trial with metabolic activation did not induce SCEs when tested up to toxic levels of BPA, and it was concluded that BPA does not induce SCE.

In the CA assay without metabolic activation, cells were exposed to Bisphenol A (0, 20, 30, 40, or 50 ug/mL) for eight hours, then cells were washed and treated with Colcemid for 2 to 2.5 hours. With metabolic activation, the cells were exposed to Bisphenol A and the metabolic activation mixture for two hours, washed, incubated for eight hours, then treated with Colcemid for 2 to 2.5 hours. Cells were harvested, fixed on slides, and stained with Giemsa for analysis and classification of CAs. In the CA trial without metabolic activation, Bisphenol A did not induce CAs when tested with delayed harvest and up to toxic levels. In the first CA trial with metabolic activation, a positive response was observed at 50 ug/mL, a dose where cell confluence was reduced by approximately 70%. This response did not repeat in the second trial, and it was concluded that Bisphenol A was negative in the CA assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study report which meets basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The bacterial reverse mutation assay was performed with various concentrations of Bisphenol A in several strains of S. typhimurium (TA 100, TA 1535, TA 98, and TA 1537) and in E. coli (WP2 uvr A).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium (TA 1535, TA 1537, TA 98, TA 100) and E. coli (WP2 uvr A)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (no other details provided)
Test concentrations with justification for top dose:
0, 5, 10, 20, 39, 78, 156, 313, 625, and 1250 µg/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; 2-acetylaminofluorene; sodium azide; 9-aminoacridine
Details on test system and experimental conditions:
No details provided.
Evaluation criteria:
Not provided.
Statistics:
Not provided.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
BPA at any dose, and both with or without metabolic activation, did not increase the number of revertants per plate over those of the vehicle controls in any strain. Toxicity was observed in some strains at 313 and 625 µg BPA/plate, and in all strains at 1250 µg BPA/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that BPA was negative for mutagenicity in the bacterial test.
Executive summary:

The bacterial reverse mutation assay was performed with various concentrations of BPA in several strains of S. typhimurium (TA 100, TA 1535, TA 98, and TA 1537) and in E. coli (WP2 uvr A). BPA at any dose, and both with or without metabolic activation, did not increase the number of revertants per plate over those of the vehicle controls in any strain. Toxicity was observed in some strains at 313 and 625 µg BPA/plate, and in all strains at 1250 µg BPA/plate. It was concluded that BPA was negative for mutagenicity in the bacterial test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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 which meets basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bisphenol A was tested for mutagenicity at the TK locus in L5178Y mouse lymphoma cells. Cells were treated with solvent or with Bisphenol A at doses from 5 to 60 µg/mL without S9 activation, and with doses from 25 to 100 µg/mL (trial 1) or 5 to 50 µg/mL (trial 2) with S9 activation. Treatment lasted four hours, then cells were washed and incubated for two days. Cells were cloned in soft agar medium and incubated for 11-12 days, then colonies were counted and mutant frequencies calculated.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from the livers of Aroclor 1254-induced male Fischer 344 rats.
Test concentrations with justification for top dose:
Without S9 activation: 0, 5, 10, 20, 30, 40, 50, or 60 µg/mL
With S9 activation: 0, 25, 50, or 100 µg/mL (trial 1) or 0, 5, 10, 20, 30, 40, or 50 µg/mL (trial 2)
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methyl methanesulfonate (MMS) for experiments without S9 activation; 3-methyl-cholanthrene (MCA) for experiments with S9 activation
Details on test system and experimental conditions:
An appropriate range of doses was chosen for the first mutation experiment such that the relative total growth values for clonable cultures would fall in an expected range of approximately 10-100%. Doses for subsequent mutation experiments were adjusted to allow better coverage of the toxicity range of greatest interest.
Evaluation criteria:
Colonies in soft agar were counted with an Artek 880 electronic counter. The mutation experiment consisted of four solvent controls, three positive controls, and duplicate or triplicate cultures of each BPA concenration. The overall evaluation was based on the test condition yielding the greatest response.
Statistics:
Statistical analyses were performed by computer for both the mutant frequency trend and comparisons between each dose level and the solvent controls in each experiment.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the absence or presence of S9 mix, BPA was not mutagenic. Toxicity was observed in the 40 to 50 ug/mL concentration range, which was well below the solubility limit of approximately 250 ug/mL in the cell culture medium. Treatment with 60 ug/mL was usually lethal. Treatment with 100 ug/mL in trial 1 of the S9-activated experiments was not lethal, but in this experiment, water suspensions of BPA were used rather than ethanol solutions. Only one treatment (50 ug/mL without S9 activation, trial 2) resulted in an increase in mutant frequency, but the authors noted that this response was very suspect because it occurred for only one of the triplicate cultures, and highly toxic treatments in other experiments failed to cause increases in the mutant frequency.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The authors concluded that BPA is not mutagenic in L5178Y cells in the absence or presence of S9 mix.
Executive summary:

BPA was tested for mutagenicity at the TK locus in L5178Y mouse lymphoma cells. Cells were treated with solvent or with BPA at doses from 5 to 60 µg/mL without S9 activation, and with doses from 25 to 100 µg/mL (trial 1) or 5 to 50 µg/mL (trial 2) with S9 activation. Treatment lasted four hours, then cells were washed and incubated for two days. Cells were cloned in soft agar medium and incubated for 11-12 days, then colonies were counted and mutant frequencies calculated. In the absence or presence of S9 mix, BPA was not mutagenic. Toxicity was observed in the 40 to 50 µg/mL concentration range, which was well below the solubility limit of approximately 250 µg/mL in the cell culture medium. Treatment with 60 µg/mL was usually lethal. Treatment with 100 µg/mL in trial 1 of the S9-activated experiments was not lethal, but in this experiment, water suspensions of BPA were used rather than ethanol solutions. Only one treatment (50 µg/mL without S9 activation, trial 2) resulted in an increase in mutant frequency, but the authors noted that this response was very suspect because it occurred for only one of the triplicate cultures, and highly toxic treatments in other experiments failed to cause increases in the mutant frequency.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information
  • Bisphenol A is negative in the micronucleus test 
  • Bisphenol A does not increase hyperploidy at meioisis II 
  • Bisphenol A exposure is not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome 

There is conclusive evidence that Bisphenol A is not mutagenic or genotoxic in vivo. 

EU RAR 2008, SCOEL recommendation 2014 and EFSA opinion 2015 concluded that Bisphenol A is not mutagenic 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Conducted in compliance with OECD Principles of Good Laboratory Practice (GLP), United States Food and Drug Administration GLP Regulations, United States Environmental Protection Agency GLP Standards, the United Kingdom GLP Compliance Programme, and the Japanese GLP Standard.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female ICR mice were dosed by oral gavage with 0, 500, 1000, or 2000 mg/kg Bisphenol A. Bone marrow cells were collected at 24 or 48 hours after treatment and were examined microscopically for the presence of micronucleated polychromatic erythrocytes.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Frederick, Maryland, United States)
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: males, 28.9 - 35.5 g; females, 26.2 - 32.2 g
- Assigned to test groups randomly: no, distributed according to body weight
- Fasting period before study: no
- Housing: Mice of the same sex were housed up to five per cage in plastic autoclavable cages maintained on stainless steel racks. Heat-treated hardwood chips were used for bedding.
- Diet: ad libitum, certified laboratory rodent chow (Harlan TEKLAD certified Rodent 7012C) which had been analyzed for environmental contaminants
- Water: ad libitum, tap water
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-80
- Humidity (%): 30-70
- Photoperiod: 12 hours light/12 hours dark
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Mice were assigned to seven experimental groups of five males and five females each according to a computer-generated program which is based on distribution according to body weight. The BPA-vehicle mixture, vehicle alone, or positive control were administered by oral gavage at a constant volume of 20 mL/kg body weight. All mice were weighed immediately prior to dose administration and the dose volume was based on individual body weights.
Duration of treatment / exposure:
Single administration by oral gavage
Frequency of treatment:
Single adminstration
Post exposure period:
24 or 48 hours
Remarks:
Doses / Concentrations:
0, 500, 1000, or 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
10 for vehicle controls and highest test dose (2000 mg/kg); 5 for the low and mid test doses (500 and 1000 mg/kg) and the positive controls
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide at 50 mg/kg
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
Bone marrow cells were isolated from femurs and suspensions of cells were spread on glass slides. Two slides were prepared from each mouse. Slides were fixed in methanol, stained with May-Gruenwald-Giemsa, and permanently mounted.
Evaluation criteria:
2000 polychromatic erythrocytes per slide were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated and the proportion of polychromatic to total erythrocytes was recorded per 1000 erythrocytes.

A positive response was induced if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treament group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered suspect of unconfirmed positive and a repeat assay recommended. A negative response was determined if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

The criteria for a valid test were described as follows: The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control group. The incidence in the positive control group must be significantly increased relative to the vehicle control group.
Statistics:
The incidence of micronucleated polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distrubution.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No mortality occurred at any dose level during the course of the study. Lethargy was noted in 2 of 5 male mice and 1 of 5 female mice at 500 mg/kg and in all mice at 1000 and 2000 mg/kg. Piloerection was observed in all mice at 1000 and 2000 mg/kg.

Reductions of 15 to 24% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in male and female dose groups 24 hours after treatment with all doses of BPA. Reductions of 26% and 36% were observed in male and female mice, respectively, 48 hours after treatment with 2000 mg/kg BPA.

The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in BPA-treated groups was not increased relative to their respective vehicle controls in either males or females, regardless of dose level or bone marrow collection time (p>0.05).
Conclusions:
Interpretation of results (migrated information): negative
The authors concluded that under the conditions of the assay, BPA was negative in the micronucleus test using male and female ICR mice.
Executive summary:

Male and female ICR mice were dosed by oral gavage with 0, 500, 1000, or 2000 mg/kg BPA. Bone marrow cells were collected at 24 or 48 hours after treatment and were examined microscopically for the presence of micronucleated polychromatic erythrocytes. No mortality occurred at any dose level during the course of the study. Lethargy was noted in 2 of 5 male mice and 1 of 5 female mice at 500 mg/kg and in all mice at 1000 and 2000 mg/kg. Piloerection was observed in all mice at 1000 and 2000 mg/kg. Reductions of 15 to 24% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in male and female dose groups 24 hours after treatment with all doses of BPA. Reductions of 26% and 36% were observed in male and female mice, respectively, 48 hours after treatment with 2000 mg/kg BPA. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in BPA-treated groups was not increased reltive to their respective vehicle controls in either males or females, regardless of dose level or bone marrow collection time (p>0.05).

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: other: meiotic effects
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study which meets basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Experiment 1: Oocytes from untreated MF1 mice (n=4-12 per group) were matured in vitro with 0, 50, 100, 200, 400, or 800 ng/ml, 4 or 10 µg/ml Bisphenol A (0.22-43.8 µM) and evaluated for meiotic progression.
Experiment 2: C57Bl x CBA/Ca F1 hybrid female mouse pups at 22 days of age (n=4 per group, usually) were administered by gavage 0, 20, 40, or 100 ng/g Bisphenol A on 7 consecutive days. Oocytes were isolated and matured ex vivo, then evaluated for meiotic progression.
GLP compliance:
no
Type of assay:
other: assessment of meiotic progression
Species:
mouse
Strain:
other: MF1; C57Bl x CBA/Ca hybrid
Sex:
female
Route of administration:
other: in vitro; oral gavage
Vehicle:
corn oil
Sex:
female
Genotoxicity:
other: Experiment 1: Bisphenol A did not increase hyperploidy at meiosis II at any tested concentration.
Sex:
female
Genotoxicity:
other: Experiment 2: Bisphenol A exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.
Additional information on results:
Experiment 1: At the highest Bisphenol A dose (10 ug/ml), meiotic progression, nuclear maturation, and chromosomal constitution were increased. This dose also affected spindle formation, distribution of pericentriolar material, and chromosome alignment on the spindle, causing significant meiotic arrest. Bisphenol A did not increase hyperploidy at meiosis II at any tested concentration.
Experiment 2: Bisphenol A exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.

Experiment 1: BPA did not increase hyperploidy at meiosis II at any tested concentration.

Experiment 2: BPA exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.

Conclusions:
Experiment 1: Bisphenol A did not increase hyperploidy at meiosis II at any tested concentration.

Experiment 2: Bisphenol A exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.
Executive summary:

Experiment 1: Oocytes from untreated MF1 mice (n=4-12 per group) were matured in vitro with 0, 50, 100, 200, 400, or 800 ng/ml, 4 or 10 ug/ml BPA (0.22-43.8 µM) and evaluated for meiotic progression. At the highest BPA dose (10 µg/ml), meiotic progression, nuclear maturation, and chromosomal constitution were increased. This dose also affected spindle formation, distribution of pericentriolar material, and chromosome alignment on the spindle, causing significant meiotic arrest. BPA did not increase hyperploidy at meiosis II at any tested concentration.

Experiment 2: C57Bl x CBA/Ca F1 hybrid female mouse pups at 22 days of age (n=4 per group, usually) were administered by gavage 0, 20, 40, or 100 ng/g BPA on 7 consecutive days. Oocytes were isolated and matured ex vivo, then evaluated for meiotic progression. BPA exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour. The authors concluded that low chronic BPA exposure in vivo does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes.

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: other: oocyte development
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study which meets basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female C57BL/6J mice were fed one of two diets for at least one week prior to mating: 1) TestDiet AIN-93G, a casein diet that does not contain soy as a protein source but does contain soybean oil, and 2) Harlan Teklad Sterilizable Rodent Diet 8656, a soy-based diet. At 21 days of age, female offspring were treated orally with Bisphenol A (20, 40, 100, 200, or 500 µg/kg-day) for 7 days and effects on oocyte development were determined.
GLP compliance:
no
Type of assay:
other: assessment of meiotic progression
Species:
mouse
Strain:
C57BL
Sex:
female
Route of administration:
oral: gavage
Vehicle:
corn oil
Sex:
female
Genotoxicity:
other: The authors could not replicate their initial findings on “congression failure” (Hunt et al. 2003. Curr Biol. 13: 546 – 553) and report significant differences between the two diets investigated.
Additional information on results:
Abnormalities of metaphase II were observed in 2% of the eggs from the offspring of control group females on the casein diet, as compared to 8% of control group offspring of animals on the soy diet. The casein diet produced an apparent linear dose response, with increases in spindle/chromosome alignment abnormalities at the 200 ug/kg-day BPA dose level. The soy diet produced an apparent U-shaped dose response. Offspring from the 500 ug/kg-day dose group on the soy diet had a higher abnormal metaphase II rate than that of the combined data for the vehicle and baseline (no vehicle, no BPA dosing) control groups, but no difference was seen for this group as compared with either control group individually.

The authors could not replicate their initial findings on “congression failure” (Hunt et al. 2003. Curr Biol. 13: 546 – 553) and report significant differences between the two diets investigated.

Conclusions:
The authors could not replicate their initial findings on “congression failure” (Hunt et al. 2003. Curr Biol. 13: 546 – 553) and report significant differences between the two diets investigated.
Executive summary:

Female C57BL/6J mice were fed one of two diets for at least one week prior to mating: 1) TestDiet AIN-93G, a casein diet that does not contain soy as a protein source but does contain soybean oil, and 2) Harlan Teklad Sterilizable Rodent Diet 8656, a soy-based diet. At 21 days of age, female offspring were treated orally with BPA (20, 40, 100, 200, or 500 µg/kg-day) for 7 days and effects on oocyte development were determined. Abnormalities of metaphase II were observed in 2% of the eggs from the offspring of control group females on the casein diet, as compared to 8% of control group offspring of animals on the soy diet. The casein diet produced an apparent linear dose response, with increases in spindle/chromosome alignment abnormalities at the 200 ug/kg-day BPA dose level. The soy diet produced an apparent U-shaped dose response. Offspring from the 500 µg/kg-day dose group on the soy diet had a higher abnormal metaphase II rate than that of the combined data for the vehicle and baseline (no vehicle, no BPA dosing) control groups, but no difference was seen for this group as compared with either control group individually.

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: other: chromosome aberration; aneugenic effects
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study which meets basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Experiment 1: Superovulated C57Bl/6 female mice of either four or nine weeks of age were injected with human chorionic gonadotropin (HCG) then immediately treated by gavage with 0.2 or 20 mg/kg Bisphenol A. Metaphase II oocytes were harvested 17 hours later and cytogenetically analyzed after C-banding. To study effects of subchronic or chronic Bisphenol A exposures, two further groups of female mice, four weeks of age, were either treated with daily oral gavage administrations of 0.04 mg/kg Bisphenol A for seven days or exposed for seven weeks to Bisphenol A at 0.5 mg/L in drinking water. Two days before the end of the seven-day or seven-week exposure, females were superovulated, then 48 hours later were injected with HCG. The females were mated overnight, and unplugged females were sacrificed to harvest oocytes for cytogenetic analysis and plugged females were used to prepare zygote metaphases to evaluate effects on the second meiotic division. Zygote metaphases were also prepared from plugged females that received a single oral dose of 0.2 mg/kg Bisphenol A.

Experiment 2: Meiotic delay in spermatocytes was assessed using 102/ElxC3H/E1 F1 males that were intraperitoneally injected with BrdU in saline and randomly assigned to receive either (1) an oral dose of 0.2 mg/kg Bisphenol A for six consecutive days starting on day 8 after BrdU, or (2) a comparable volume of vehicle (corn oil) for the same number of days. Five males from each group were sacrificed on days 21, 22, 23, 24, and 25 after the end of the Bisphenol A treatment. Sperm were collected and examined for BrdU incorporation. Twenty more male mice were then treated orally with 0, 0.002, 0.02, or 0.2 mg/kg Bisphenol A on six consecutive days. Sperm were collected for multicolor FISH analysis to assess the induction of aneuploidy during the first and second meioitc division.

Experiment 3: Male mice were treated with 0. 0.002, 0.02, or 0.2 mg/kg Bisphenol A by gavage on two consecutive days, then 24 hours later, bone marrow smears were prepared for the micronucleus assay.
GLP compliance:
no
Type of assay:
other: micronucleus assay; assessment of meiotic progression
Species:
mouse
Strain:
other: C57Bl/6; 102/E1xC3H/E1
Sex:
male/female
Route of administration:
other: oral gavage; oral: drinking water
Vehicle:
corn oil or water
Sex:
female
Genotoxicity:
other: Experiment 1: negative; Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II
Sex:
male
Genotoxicity:
other: Experiment 2: negative; the proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of Bisphenol A.
Sex:
male
Genotoxicity:
other: Experiment 3: negative; Bisphenol A did not induce micronuclei in bone marrow erythrocytes at any dose.
Additional information on results:
Experiment 1: Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in more than two dyads, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: The proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Experiment 1: negative; Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in the group treated for 7 weeks, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: negative; the proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: negative; BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Conclusions:
Experiment 1: negative; Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in the group treated for 7 weeks, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: negative; the proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: negative; BPA did not induce micronuclei in bone marrow erythrocytes at any dose.
Executive summary:

Experiment 1: Superovulated C57Bl/6 female mice of either four or nine weeks of age were injected with human chorionic gonadotropin (HCG) then immediately treated by gavage with 0.2 or 20 mg/kg BPA. Metaphase II oocytes were harvested 17 hours later and cytogenetically analyzed after C-banding. To study effects of subchronic or chronic BPA exposures, two further groups of female mice, four weeks of age, were either treated with daily oral gavage administrations of 0.04 mg/kg BPA for seven days or exposed for seven weeks to BPA at 0.5 mg/L in drinking water. Two days before the end of the seven-day or seven-week exposure, females were superovulated, then 48 hours later were injected with HCG. The females were mated overnight, and unplugged females were sacrificed to harvest oocytes for cytogenetic analysis and plugged females were used to prepare zygote metaphases to evaluate effects on the second meiotic division. Zygote metaphases were also prepared from plugged females that received a single oral dose of 0.2 mg/kg BPA. Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in the group treated for 7 weeks, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: Meiotic delay in spermatocytes was assessed using 102/ElxC3H/E1 F1 males that were intraperitoneally injected with BrdU in saline and randomly assigned to receive either (1) an oral dose of 0.2 mg/kg BPA for six consecutive days starting on day 8 after BrdU, or (2) a comparable volume of vehicle (corn oil) for the same number of days. Five males from each group were sacrificed on days 21, 22, 23, 24, and 25 after the end of the BPA treatment. Sperm were collected and examined for BrdU incorporation. Twenty more male mice were then treated orally with 0, 0.002, 0.02, or 0.2 mg/kg BPA on six consecutive days. Sperm were collected for multicolor FISH analysis to assess the induction of aneuploidy during the first and second meioitc division. The proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: Male mice were treated with 0. 0.002, 0.02, or 0.2 mg/kg BPA by gavage on two consecutive days, then 24 hours later, bonem arrow smears were prepared for the micronucleus assay. BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

EFSA Opinion 2015 concluded on genotoxicity:

“The available data support that Bisphenol A is not mutagenic (in bacteria or mammalian cells), or clastogenic (micronuclei and chromosomal aberrations). The potential of Bisphenol A to produce aneuploidy in vitro was not expressed in vivo. The positive finding in the postlabelling assays in vitro and in vivo is unlikely to be of concern, given the lack of mutagenicity and clastogenicity of Bisphenol A in vitro and in vivo. 

Using a WoE approach, the CEF Panel assigned a likelihood level of “unlikely” to Bisphenol A genotoxicity.” 

 

SCOEL Recommendation 2014 concluded on genotoxicity: 

“Considering all of the available genotoxicity data and the absence of significant tumour findings in animal carcinogenicity studies (see Section 3.8), it does not appear that Bisphenol A has significant mutagenic or genotoxic potential in vivo.” 

 

The 2008 updated EU RAR concluded: 

"New data from a study indicating effects of Bisphenol A on meiosis in female mice cannot be taken as conclusive evidence of an effect of Bisphenol A on germ cell meiosis because of the several methodological weaknesses and flaws identified in the study, the reporting inadequacies, and the known mutagenicity and toxicity profile of Bisphenol A. In addition, these findings have not been confirmed in more recent publications. Thus, the original conclusion that Bisphenol A has no significant mutagenic potential in vivo is still valid." 

 

Additional recent information concerning the observations reported discussed in the 2008 updated EU RAR was discussed in the initial dossier submitted in 2010:

 

Two in vivo studies (Hunt et al.(2003) and Susiarjo et al. (2007)) evaluated during the 2008 EU RAR reported that short-term oral exposure to low doses of Bisphenol A (≥ 0.020 mg/kg bw/day) in peripubertal or pregnant mice can interfere with meiotic divisions in development of female germ cells (“egg” or “oocyte”). An increase in hyperploid (aneuploid) metaphase II oocytes was observed following treatment with 0.020 mg/kg bw/day. There was not a significant increase in aneuploid embryos. 

Two subsequent in vivo studies (Pacchierotti et al.(2008), Eichenlaub-Ritter et al. (2008)) attempted to replicate these findings. Consistent with the previous findings, they detected no significant effects of Bisphenol A exposure on the frequency of aneuploidy in “zygotes” (fertilised oocytes) produced from female mice treated before puberty or as adults with a similar range of doses. In addition, Eichenlaub-Ritter et al. (2008) found no effects of Bisphenol A exposure on aneuploid oocytes and Pacchierotti et al. (2008) found no increase in aneuploid or diploid sperm following exposure of male mice to Bisphenol A. The authors concluded that the aneuploidy predicted by the Hunt group could not be confirmed. 

In addition, in a recent study published by the Hunt group, Muhlhauser et al. (2009), the authors could not replicate their initial findings on “congression failure” but report effects on chromosome alignment and/or spindle formation. The authors state “After publishing our findings [Hunt et al., 2003], we initiated studies to assess the effect of long term Bisphenol A exposure on the growing follicle. To our surprise, levels of Bisphenol A that were sufficient to elicit an effect on meiotic chromosome dynamics during the previous two years of study suddenly produced little or no effect. In an analysis of possible changes in experimental protocol, the only change identified was the lot of animal feed.” The authors report frequencies of abnormal oocytes in the absence and presence of Bisphenol A in two different diets (casein based and soy based). The reported frequencies of abnormal oocytes of the Bisphenol A/casein group are lower than the background value reported in the soy-based diet. 

Overall, the initial observations reported by the Hunt laboratory were not reproduced in the same laboratory or in other independent laboratories. Therefore, the conclusion from the 2003 EU RAR and the 2008 EU RAR Update is still valid; Bisphenol A has no significant mutagenic potential in vivo. 

 

The 2003 EU RAR concluded:

"No human data regarding mutagenicity are available. However, Bisphenol A appears to have demonstrated aneugenic potential in vitro, positive results being observed without metabolic activation in a micronucleus test in Chinese hamster V79 cells and in a non-conventional aneuploidy assay in cultured Syrian hamster embryo cells. Additionally, in cell-free and cellular systems there is information that shows Bisphenol A disrupts microtubule formation. Bisphenol A has been shown to produce adduct spots in a post-labelling assay with isolated DNA and a peroxidase activation system, but it does not appear to produce either gene mutations or structural chromosome aberrations in bacteria, fungi or mammalian cells in vitro. However, some deficiencies in the conduct of these studies have been noted and the negative results cannot be taken as entirely conclusive. Bisphenol A does not appear to be aneugenic in vivo, since a recently conducted, standard mouse bone marrow micronucleus test has given a negative result. Bisphenol A was negative in a briefly reported dominant lethal study in rats but, given the limited details provided, this is not regarded as an adequate negative result. The only other data in somatic cells in vivo are from a 32P-postlabelling assay, which showed that Bisphenol A is capable of producing DNA adduct spots in rat liver following oral administration. These adduct spots were not characterised fully. 

Considering all of the available genotoxicity data, and the absence of significant tumour findings in animal carcinogenicity studies (see below), it does not appear that Bisphenol A has significant mutagenic potential in vivo. Any aneugenic potential of Bisphenol A seems to be limited to in vitro test systems and is not of concern. The relevance of the finding that Bisphenol A can produce rat hepatic DNA adduct spots in a postlabelling assay is not entirely clear. However, given the absence of positive results for gene mutation and clastogenicity in cultured mammalian cell tests, it seems unlikely that these are of concern for human health." 

Justification for classification or non-classification

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