Weight of evidence studies
In this guide
In this guide26. The following are tables summarising new in vitro and in vivo genotoxicity studies on BPA identified in the literature (2013 –2021) and studies considered in the ‘Scientific Opinion on the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs’ (EFSA CEF Panel, 2015). Key : *Indicates that more than one assay is reported/indicates when papers belong to more than one table. **Indicates that both in vitro and in vivo assays are reported in the same paper.
27. The studies have been evaluated based on the criteria described above in Annex A.
Bacterial reverse mutation assay
Table 1. Bacterial reverse mutation assay (OECD TG 471 was considered for the evaluation of reliability.
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Bacterial reverse mutation assay Salmonella Typhimurium strains TA 98 and TA 100 In vivo micronucleus assay (Table 7)** |
BPA 1–10 μmoles/plate with or without S9; 3 replicates |
BPA (Tokyo Kasei Kogyo Co., Ltd) Purity 99% not reported in the study but available in the website of the company |
Negative |
Reliability: 2 Only 2 strains Data on negative controls subtracted (but not shown) No positive control |
Limited |
Masuda et al., 20051** |
|
Bacterial reverse mutation assay Salmonella Typhimurium strains TA98, TA100, TA102 In vivo chromosomal aberration (Table 6) (Table 7) comet assay (Table 8)** |
BPA 0, 6.25, 12.5, 25, 50, 100, 150 and 200 μg/plate for 48 h; with or without S9; preincubation method |
BPA, purity 99% (Sigma Chemical Company) |
Negative |
Reliability: 2 Only 3 strains used |
Limited |
Tiwari et al., 20121** |
|
Bacterial reverse mutation assay Salmonella Typhimurium strains TA98 and TA 100 In vitro comet assay (Table 5)* |
BPA 0, 4, 20, 100, 500 μg/plate for 48 h (TA100) and 72 h (TA98); 3 replicates; with or without S9 |
BPA, purity >99% (Sigma-Aldrich) |
Negative |
Reliability: 2 Only 2 strains |
Limited |
Fic et al., 20131* |
|
Bacterial reverse mutation assay Salmonella Typhimurium strains TA1535, TA97, TA98, TA100 and TA102 In vitro chromosomal aberration (Table 3) micronucleus assay (Table 4) comet assay (Table 5) in CHO cells* |
BPA 10–5000 μg/plate; 48 h incubation; with or without S9; preincubation method in triplicates; 3 independent experiments |
BPA (purity 99%)2, was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China) |
Negative |
Reliability: 1 |
High |
Xin et al., 2015* |
|
Bacterial reverse mutation assay Salmonella Typhimurium strains TA98 and TA100 |
BPA 0.1, 1, 10 and 100 μg/plate with or without S9; plate incorporation assay in triplicates; 2 independent experiments |
BPA (Merck) Purity >97% not reported in the study but available on the website of the company |
Negative |
Reliability: 2 Only 2 bacterial strains used |
Limited |
Zemheri and Uguz, 2016 |
|
SOS/umuC assay in Salmonella Typhimurium TA1535 pSK1002 In vitro comet assay (Table 5)* |
BPA 0, 1, 10, 100, 1000 μg/L, without or with metabolic activation (S9) |
BPA (Sigma-Aldrich) Purity >97% not reported in the study but available on the website of the company |
Negative |
Reliability: 2 Non-standard test applied as a preliminary analysis of toxicity and mutagenicity |
Limited |
Balabanič et al., 2021* |
1Studies considered in the Scientific Opinion on the Risks to Public Health Related to the Presence of Bisphenol A (BPA) in 2Foodstuffs (EFSA CEF Panel, 2015) Information on BPA purity provided by the study authors on 11 October 2021, upon EFSA request
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vitro gene mutation in mammalian cells
Table 2: In vitro gene mutation in mammalian cells.
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Analysis of mutational spectra in immortalised human embryonic kidney cells HEK 293T using whole genome sequencing (WGS)
DNA double strand breaks as measured using γH2AX immunofluorescence staining |
100 μM for 24 h exposure and WGS of clonally expanded cells populations
No metabolic Activation
Cell viability analysed in HEK 293T cells, treated for 24 h with 0.1, 1 and 100 μM BPA; cells were stained with crystal violet and results reported as colony area percentage |
BPA from TCI (B04 94) purity ≥ 99% not reported in the study but available on the website of the company |
Positive
Increased levels of single base substitutions, doublestrand breaks and small insertions/deletions in BPA-treated HEK 293T cells in comparison with DMSO-treated controls
Single base substitutions (C>A transversions) in BPAtreated cells preferentially occur at guanines Mutations at A:T bp were also reported
Colony formation assay: concentration dependent decrease in % colony area
Concentration dependent increase in DNA double strand breaks as increased number of nuclei with > 5 γH2AX foci |
Reliability: 2 Although there is no TG for this type of study, the research was adequately conducted and reported However, there is uncertainty in the level of toxicity of the BPA treatment |
Limited |
Hu et al., 2021 |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vitro chromosomal aberrations test
Table 3: In vitro chromosomal aberrations test (OECD TG 473 was considered for the evaluation of reliability).
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Chromosomal aberrations and SCE assays CHO-K1 cell line Cytotoxicity: cellcycle delay ‘recognised by the metaphases without differently staining sister chromatids’ In vitro comet assay (Table 5)* |
BPA 0, 0.1 to 0.6 mM for 3 h followed by 27 h recovery 100 metaphases SCE: 50 metaphases Without metabolic activation |
BPA, purity > 99% (Tokyo Kasei Kogyo Co., Ltd) |
Positive Only in presence of severe Cytotoxicity Increased CA (0.5, 0.55, 0.6 mM, % of differently staining sister chromatids 29%, 11%, and 0%, respectively) Increased endoreduplications (0.45 and 0.55 mM) Increased frequency of cmitosis- like figures (above 0.3 mM) Increased SCE (0.4 and 0.5 mM) |
Reliability: 3 Only short-term treatment; high level of cytotoxicity The recovery time exceeded the recommended (18–21 h) Cells recovered in the presence of BrdU |
Low |
Tayama et al., 20081* |
|
Chromosomal aberration assay CHO cells Cytotoxicity: MTT assay Bacterial reverse mutation assay (Table 1) In vitro micronucleus assay (Table 4), comet assay (Table 5)* |
BPA 0, 80, 100 and 120 μM for 24 h 500 metaphases/group; without metabolic activation MTT assay: BPA 0, 40, 80, 100 and 120 μM for 12 and 24 h |
BPA (purity 99%)2, was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China) |
Positive Increase of structural chromosomal aberrations from 80 μM, with significant decrease in cell viability (but not lower than 50%) MTT assay: increase of cell proliferation at 40 μM; cytotoxicity from 80 μM |
Reliability: 2 No short-term Treatment No positive control |
Limted |
Xin et al., 2015* |
|
Chromosomal aberration assay in: - MCF-7 human breast cancer line; - human amniocytes from male [oestrogen receptors (ER) negative] and from female (ER positive) Cytotoxicity: MTT test |
BPA 0, 0.4, 1, 4, 40 and 100 μg/mL for 48 h; 200 cells analysed for each treatment (less at highest concentrations in amniocytes for high toxicity) Without metabolic activation MTT test: BPA 0, 0.4, 1, 4, 40, 100 and 400 μg/mL for 48 h. |
BPA, no information on purity or the supplier company |
Positive Increase of cells with chromosome aberrations (from 1 μg/mL) in all cell types; the increase in cells with aberrations was not clearly concentration related and decreased at the highest concentrations, possible due to cytotoxicity that was not concurrently evaluated; no clear association with ER expression In a preliminary evaluation of cytotoxicity by the MTT test, the IC50 of BPA was 100, 40 and 4 μg/mL in MCF-7 and ER-negative (male) and ERpositive (female) amniocytes, respectively |
Reliability: 2 Cells scored less than recommended in OECD TG 473 No short-term treatment No positive control No concurrent control of toxicity |
Low No information on source and purity of BPA |
Aghajanpour-Mir et al., 2016 |
|
Chromosomal aberration assay Human peripheral blood lymphocytes from 5 female subjects In vitro micronucleus assay (Table 4)* |
BPA 0, 0.20, 0.10, 0.05, 0.02 and 0.01 μg/mL for 24 h 1000 metaphases (200/subject)/concentration Without metabolic activation |
BPA (Sigma- Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Positive Increase from 0.05 μg/mL (prevalence of chromatid breaks) No numerical aberrations |
Reliability: 2 No short-term treatment |
Limited |
Santovito et al., 2018* |
|
Chromosomal Aberrations fibroblasts (MEF)
In vitro comet assay (Table 5)* |
BPA 150 μM for 24 h or co-exposure with camptothecin (CPT)
25 metaphases/treatment were analysed Without metabolic activation |
BPA (Sigma- Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Negative frequency Cytotoxicity of BPA alone was not measured but the authors refer to 150 μM as concentration with minimal toxic effect from a previous publication |
Reliability: 3
Single concentration tested; low number of metaphases analysed No short-term treatment |
Low |
Sonavane et al., 2018* |
|
Chromosomal aberrations Human peripheral blood mononuclear cells (PBMC) Cell proliferation: MTT test Cell-cycle analysis: FACS γH2AX: western blot and FACS analysis |
BPA 0, 25, 50, 100 nM, cells stimulated with PHA for 16h and then treated with BPA for 48 h 30 metaphases/treatment/subject (5 donors) MTT test: BPA 0, 5, 10, 25, 50, 100, 200 nM and BPA 25, 50, 100, 200 μM, cells were treated with or without PHA for 16 h and then treated with BPA for 24 and 48 h γH2AX: cells treated with PHA and then with BPA 50 nM for 24 h or 48 h (western blot) or only for 24 h (FACS analysis analysing T and B lymphocytes) Without metabolic activation |
BPA (Merck) Purity ≥97% not reported in the study but available on the website of the company |
Positive Increased number of aberrant cells, structural chromosomal aberrations and highly fragmented metaphases MTT test: - unstimulated PBMCs: decreased cell proliferation only at 200 μM at both 24 and 48 h PHA stimulated PBMCs: - increased cell proliferation from 10 nM to 100 nM; - concentration-dependent decreased cell proliferation from 25 to 200 μM Effect on cell proliferation confirmed using cell-cycle analysis γH2AX (western blot): - increase of protein phosphorylation only at 24 h (BPA 50 nM) γH2AX (FACS): increase in CD3+ and in CD4+ T cells |
Reliability: 2 No positive Control No short-term treatment |
Limited |
Di Pietro et al., 2020 |
|
Chromosomal aberrations assay in human peripheral blood lymphocytes |
BPA 0, 5, 10, 20 and 50 μg/mL for 24 and 48 h Mitomycin C (MMC) at 0.10 μg/mL ‘was added to the negative and a positive controls and to each concentration and chemical groups as well’ Without metabolic activation |
BPA, no information on purity or the supplier company |
No data on chromosome aberrations were reported |
Reliability: 3 MMC added to all Treatments No mitogenic Stimulation No short-term treatment |
Low No information on BPA purity |
Özgür et al., 2021 |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vitro mammalian cell micronucleus test
Table 4: In vitro mammalian cell micronucleus test (OECD TG 487 was considered for the evaluation of reliability).
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Cytokinesis block micronucleus assay (CBMN) AHH-1 cell line (human lymphoblastoid cells) Effects on mitotic spindle using staining: brilliant blue and safranin O; α- and ƴ-tubulin immunofluorescence staining |
BPA 0, 1.5, 3.1, 6.2, 7.7, 9.2, 10.8, 12.3, 18.5, 24.6, 37 μg/mL for a complete cell cycle (22–26 h), Five experiments: average of 8082 cells scored for each treatment Effects on mitotic spindle: BPA 0, 4.2–14 μg/mL for 20 h (one cell cycle); 100 cells undergoing mitosis scored in each experiment, 3 experiments Without metabolic activation |
BPA (Sigma-Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Positive increased BNMN cells from 12.3 μg/m Aberrant mitotic divisions (multiple spindle poles) |
Reliability: 1 BN cells % as parameter of cytotoxicity High number of analysed binucleated cells |
High |
Johnson and Parry, 20081 |
|
Micronucleus test in: - human umbilical vascular endothelial cells (HUVEC); - human colon adenocarcinoma (HT29) cell line Immunofluorescence analysis of cytoskeleton organisation of HUVEC
cells with anti-α-tubulin and anti-γ-tubulin Apoptosis using TUNEL assay and cell viability using CellTiter-Blue assay |
BPA 0, 44 nM and 4.4 μM, (i.e. 10 ng/mL and 1 μg/mL) for 72 h BPA 10 ng/mL and 1 μg/ml for 24 or 72 h CellTiter-Blue assay: BPA 10 ng/mL and 1 μg/mL for 24, 48 or 72 h
Without metabolic activation |
BPA, no information on purity or the supplier company |
Positive in HUVEC cells: slight increase of MN frequency Negative in HT29 cells Multipolar spindles and microtubule misalignment associated with BPA exposure
No effects on cell viability, proliferation and apoptosis in both cell lines |
Reliability: 2
No analysis of cell proliferation; no positive control; no short-term treatment |
Low
No information on source and purity of BPA |
Ribeiro-Varandas et al., 2013 |
|
Cytokinesis block micronucleus assay; bovine peripheral blood lymphocytes; cell proliferation: nuclear division index (NDI) |
BPA 1×10−4, 1×10−5, 1×10−6 and 1×10−7 mol/L for 48 h
Without metabolic activation |
BPA (Sigma-Aldrich) Purity ≥97% not reported in the study but available on the website of the company |
Positive
concentration-related increase in MN frequency, statistically significant at the highest concentration; no effect on NDI at any concentration |
Reliability: 2
No short-term treatment; bovine lymphocytes are not commonly used in the micronucleus test, and their use has not been validate. However the study appears to be adequately performed and reported |
Limited |
Šutiaková et al., 2014 |
|
Micronucleus assay CHO cells Cytotoxicity: MTT test Bacterial reverse mutation assay (Table 1) In vitro chromosomal aberration (Table 3) comet assay (Table 5)* |
BPA 0, 80, 100 and 120 μM for 24 h, without cytochalasin B; 1000 cells were scored for each sample; 3 independent experiments Without metabolic activation MTT test: - BPA 0, 40, 80, 100 and 120 μM for 12 and 24 h |
BPA (purity 99%)2, was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China) |
Positive
increase in MN frequency at 100 and 120 μM MTT assay: concentration-related decrease in cell viability from 100 μM |
Reliability: 2
No short-term Treatment
No positive control |
Limited |
Xin et al., 2015* |
|
Cytokinesis-blocked micronucleus assay in murine macrophage RAW264.7 cells 1000 binucleated cells/concentration Cell viability: MTT test
In vitro comet assay (Table 5)* |
BPA 0, 3, 10, 30, or 50 μM for 24 h BPA 10 μM tested for MN assay and cell viability, in the presence or absence of pretreatment with N-acetyl- L-cysteine (NAC) at the concentration of 10 μM for 30 min Without metabolic activation MTT test: BPA 0, 3, 10, 30, or 50 μM for 12 or 24 h |
BPA (Sigma-Aldrich) Purity ≥97% not reported in the study but available on the website of the company |
Positive
Concentration dependent increase in MN frequency from 10 μM In the presence of NAC, MN frequencyand cytotoxicity were statistically significantly reduced (see also data on ROS in Table 5) MTT test: concentration- and time-dependent decrease of cell viability |
Reliability: 2
No short-term treatments; no positive controls; no data on cell proliferation |
Limited |
Huang et al., 2018* |
|
Cytokinesis block micronucleus assay Human peripheral blood lymphocytes from 5 female subjects 1000 binucleated lymphocytes/subject (5000 binucleated cells per concentration) In vitro chromosomal aberrations assay (Table 3)* |
BPA 0, 0.20, 0.10, 0.05, 0.02 and 0.01 μg/mL for 48 h
Without metabolic activation |
BPA (Sigma-Aldrich) Purity ≥97% not reported in the study but available on the website of the company |
Positive
Increase in MN frequency from 0.02 μg/mL. At 0.2 μg/mL 4-fold increase with respect to the vehicle control (DMSO) level No significant reduction of the CBPI value |
Reliability: 2
No short-term treatment |
Limited |
Santovito et al., 2018* |
|
Mitotic abnormalities and micronuclei evaluated in DAPI stained cells: - Hep-2 cells (human epithelial cells from laryngeal carcinoma); - MRC-5 cells (human lung fibroblasts) Cell viability using CellTiter-Blue assay, after 48 h exposure In vitro comet assay (Table 5)* |
BPA 0.44 nM, 4.4 nM, 4.4 μM (0.1 ng/mL, 1 ng/mL, 1 μg/mL) for 48 h; 1000 cells scored for each treatment |
BPA (Sigma) purity ≥97% not reported in the study but available on the website of the company |
Positive
Slight (two-fold) increase in MN frequency from BPA 4.4 nM in both cell lines Mitotic index: - in Hep-2 cells, no effects; - in MRC-5 cells, statistically significant increase Cytotoxicity: no effects on cell viability |
Reliability: 3
No short-term treatment Proliferation of the cell population not determined; extremely low % of mitosis is indicative of a very low rate of cell division, which is not appropriate to measure MN formation
Protocol of MN assay not reported; no positive control |
Low |
Ramos et al., 2019* |
|
Micronucleus assay in Chinese hamster V79- derived cell lines expressing various human CYP enzymes Micronucleus assay in C3A cells (human hepatoma cell line, endogenously express various CYP enzymes, including CYP1A1, 1A2, 1B1, 2E1, 3A4, and phase II metabolic enzymes, such as UGTs and SULTs) 2000 cells analysed for each treatment Cytotoxicity: CCK-8 Assay γ-H2AX in V79-Mz, V79- hCYP1A1 cells and in C3A cells; analysis using In- Cell Western Blot Immunofluorescence staining of CENP-B of MN induced in C3A cells |
1) Micronucleus assay in V79-derived cell lines: - BPA 0, 40, 80, 160 μM for 9 h + 15 h; (recovery period); - 2000 cells analysed for each treatment 2) Micronucleus assay in: - V79-Mz, V79- hCYP1A1 cells: BPA 0 to 80 μM for 24 h + 0 h ; with or without ABT; - C3A cells: BPA 0 to 80 μM for 72 h + 0 h; with or without ABT or 7-HF 3) Micronucleus assay in C3A cells: BPA 0 to 5 μM for 72 h + 0 h, with or without KET or PCP (phase II enzyme inhibitors), an inhibitor of UGT1 and SULT1, respectively Immunofluorescence staining of CENP-B was applied Cytotoxicity performed for each test using the same testing conditions of the MN assay or of γH2AX analysis γH2AX: BPA 0, 10, 20, 40, 80, 160 μM for 9 h; ABT (1- aminobenzotriazole a CYP inhibitor) or 7-HF (a selective CYP1A1 inhibitor) were added from 2 h ahead of test compound exposure to the end of cell culture
BPA 0, 10, 20, 40, 80, 160 μM for 9 h; ABT (1- aminobenzotriazole a CYP inhibitor) or 7-HF (a selective CYP1A1 inhibitor) were added from 2 h ahead of test compound exposure to the end of cell culture |
BPA (99.6%), AccuStandard Inc. |
1) Micronucleus assay (9 h + 15 h): - Negative in V79- Mz; - Positive in V79- hCYP1A1 cells and in V79-hCYP1B1 cells; - Cytotoxicity: statistically significant decrease at the highest concentrations 2) Micronucleus assay (24 h + 0 h): - Negative in V79- Mz; - Positive in V79- hCYP1A1 cells, effect abrogated by ABT 2)Micronucleus assay (72 h + 0 h): - Positive in C3A cells, effect abrogated by ABT or 7-HF; - Cytotoxicity: statistically significant decrease at the highest concentrations 3)Micronucleus assay in C3A cells (72 h + 0 h): - Positive - Effects enhanced by KET or PCP; statistically significant increase of MN negative for CENP-B staining, (clastogenic mechanism) Cytotoxicity: statistically significant increase in cell viability from 2.5 μM γH2AX: - increase in V79-Mz, in V79-hCYP1A1 cells and in C3A cells (concentration dependent); effect reduced by ABT or 7- HF - Effects enhanced by KET or PCP; statistically significant increase of MN negative for CENP-B staining, (clastogenic mechanism) Cytotoxicity: statistically significant increase in cell viability from 2.5 μM γH2AX: - increase in V79-Mz, in V79-hCYP1A1 cells and in C3A cells (concentration dependent); effect reduced by ABT or 7- HF |
Reliability: 2 Micronucleus method poorly described No short-term treatment |
Limited |
Yu et al., 2020 |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vitro DNA damage (comet assay)
Table 5: In vitro DNA damage (comet assay).
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Alkaline comet assay MCF-7 (oestrogen receptor (ER) positive) and MDA-MB-231 (ER negative) γH2AX foci using immunofluorescence in MCF-7 cells |
MCF-7 cells exposure: - BPA 0, 0.1 10, 100 μM for 3 h; - BPA 100 μM for 1, 3, 24 h MDA-MB-231 cells exposure: - BPA 100 μM for 3, 24 h; 30 cells analysed (10 cells/slide) Immunofluorescence in MCF-7 cells: BPA 10 μM for 3 h Without metabolic activation |
BPA (Wako Pure Chemicals Industries, Ltd) purity ≥99% not reported in the study but available on the website of the company |
Positive
MCF-7: increased comet tail length after 3 h at 10, 100 μM and after all exposure times at 100 μM MDA-MB-231: increased comet tail length after 3 and 24 h exposure times at 100 μM No toxicity in comet assays Induction of γH2AX foci in MCF-7 cells (10 μM) ER-positive MCF-7 cells are more sensitive than ER-negative MDA-MB-231 cells to BPA-induced DNA damage |
Reliability: 2
Only 30 cells were Analysed
No positive control |
Limited |
Iso et al., 20061 |
|
Alkaline comet assay in CHO-K1 cell line In vitro chromosomal aberrations (Table 3)* |
BPA 0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mM; 1 h exposure Positive control: H2O2 200 cells were scored Quantification of DNA damage: a score of 0–3 (mean score value = mean comet points/cell, comet points) Cell viability (trypan blue) Without metabolic activation |
BPA, purity > 99% (Tokyo Kasei Kogyo Co., Ltd) |
Positive Increased DNA strand breaks only at the highest concentration tested (0.7 mM) |
Reliability: 3
Non-standard method of DNA damage quantification Data of cytotoxicity not clearly reported |
Low |
Tayama et al., 20081* |
|
Alkaline comet assay HepG2 cells Cell viability: MTT test Bacterial reverse mutation assay (Table 1)* |
BPA 0, 0.1, 1.0 and 10.0 μM for 4 and 24 h; 50 nuclei scored/treatment; at least 2 independent experiments; positive control: benzo[a]pyrene
MTT test: 12.5, 25, 50, 100 μM for 24 h |
BPA, purity >99% (Sigma-Aldrich) |
Negative after 4 h of exposure
Equivocal after 24 h exposure (no concentration related effect)
No cytotoxicity was observed |
Reliability: 2
Only 50 nuclei scored |
Limited |
Fic et al., 20131* |
|
Comet assay in rat INS-1 insulinoma cells Cell viability: Hoechst staining kit and trypan blue (apoptotic cells detection) Expression of nuclear p53 and p-Chk2 (T68) proteins: western blotting Intracellular (ROS): DCFH-DA Glutathione (GSH): detection with ophthalaldehyde (OPT) |
BPA 0, 25, 50, 100 μM for 24 h; or pretreatment with or without NAC (10 mM) for 1 h then BPA (100 μM) was added for 24 h; Without metabolic activation 50 cells/slide were analysed; 3 experiments ROS and GSH analysis: BPA 0, 25, 50, 100 μM for 24 h
ROS measurements also in cells pre-treated with NAC and exposed to 100 μM BPA |
BPA, purity 99% (Sigma-Aldrich) |
Positive concentration related increase in tail DNA %, tail moment and tail length at 50 and 100 μM
Significant decrease in tail DNA % in cells pre-treated with NAC
No apoptotic cells and 90% cell survival were used in comet assays (results are not shown)
Increase of expression of DNA damage-associated proteins: p53 (from 50 μM) and p-Chk2 (at 100 μM) Levels of p53 are reduced by NAC pre-treatment Intracellular ROS: increase at 50 and 100 μM Decrease of ROS upon NAC pretreatment
GSH: concentration related decrease
|
Reliability: 2
No positive control; results on cytotoxicity assessment are not reported |
Limited |
Xin et al., 2014 |
|
Alkaline comet assay in CHO cells Cytotoxicity: MTT assay Bacterial reverse mutation assay (Table 1) In vitro chromosomal aberration (Table 3) micronucleus assays (Table 4)* |
BPA 0, 40, 80, 100 and 120 μM for 12 and 24 h; 100 cells were analysed/sample. Without metabolic activation MTT assay: BPA 0, 40, 80, 100 and 120 μM for 12 and 24 h |
BPA (purity 99%)2, was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China) |
Positive Concentration related increase in (%) tail DNA from 80 μM with 12 h treatment, and at all tested concentrations after 24 h MTT assay: decrease in cell viability (but less than 50%) from 80 μM after 12 and 24 h |
Reliability: 2
No positive control |
Limited |
Xin et al., 2015* |
|
Alkaline comet assay NIH3T3 cells (mouse embryonic fibroblast cell line) At least 100 nucleoids/sample Cytotoxicity: CCK-8 assay and LDH release Intracellular ROS: DCFHDA 8-OHdG: EpiQuick 8- OHdG DNA damage quantification direct kit γH2AX: immunofluorescence and western blot |
BPA 0, 2, 10 and 50 μM (0.4–11 μg/mL) for 24 h CCK-8 and LDH assays, ROS, 8-OHdG, γH2AX analysis: BPA 0, 2, 10 and 50 μM for 24 h At least 100 nucleoids of each sample were obtained in 3 independent experiments without metabolic activation |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Positive
increase tail DNA% at 50 μM Cytotoxicity: 80% cell survival at 50 μM γH2AX, ROS and 8- OHdG: increase at 50 μM |
Reliability: 2
No positive control |
Limited |
Chen et al., 2016 |
|
Alkaline comet assay in FRTL-5 rat immortalised thyrocyte cell line Cell proliferation (population doubling) Transcriptome analysis (microarray) Intracellular ROS: H2DCFDA |
BPA 10−9 M for 6h, 48h, 96 h; 100 cells for each condition Transcriptome analysis and intracellular ROS: cells exposed for 1, 3, and 7 days to 10−9 M BPA Without metabolic activation |
BPA (Sigma-Aldrich), purity ≥97% not reported in the study but available on the website of the company |
Comet assay on BPA alone: Negative Intracellular ROS: statistically significant increase after 1 and 3 days exposure Transcriptome analysis: decreased expression of genes involved in DNA replication, recombination and repair (confirmed by RT-PCR) (after 3 and 7 days BPA exposure) |
Reliability: 3
Comet assay: - one low concentration tested; - no positive control Small effects on transcription Large variations in DNA strand breaks in the comet assay |
Low |
Porreca et al., 2016 |
|
Comet assay MCF-7 cells (from human breast adenocarcinoma) Cell viability: CCK-8 assay Cell membrane damage: LDH ROS |
BPA 0, 1, 10, 25, 50 μM; 24 h Positive control: tBHP (tert-butyl hydroperoxide); 300 cells from each sample were analysed Without metabolic activation CCK-8 assay: 0, 0.01, 0.1, 1, 10, 25, 50, 100 μM for 24 h LDH: 0, 1, 10, 25, 50, 100 μM for 24 h ROS: 0, 0.01, 0.1, 1, 10, 25, 50 μM for 24 h |
BPA, purity > 98% (Tokyo Chemical Industry) |
Positive
Concentration dependent increase in % tail DNA from 10 μM Cell viability: at 1 μM increase in cell viability; inhibition of cell viability at concentrations from 10 μM (70%) to 100 μM (80%) Cell membrane damage: increase in LDH release in a concentration dependent manner from 10 μM ROS formation: concentration dependent increase in ROS levels No measurement at 50 μM, because of excessive cell death (90%) |
Reliability: 3
Excessive toxicity at the analysed positive concentrations
Results of positive control are not reported
Comet methods are not described in detail |
Low |
Lei et al., 2017 |
|
Alkaline comet assay HepG2 cells Cytotoxicity: MTT assay Oxidative stress: intracellular ROS: DCFHDA in the same cells, also MDA and SOD |
BPA from 10–8 to 10–6 mol/L (0.02–22.8 μg/mL) for 24 h
MTT: BPA from 10–8 to 10–4 mol/L for 24 h ROS, MDA and SOD analysis: BPA from 10–8 to 10–4 mol/L for 6 h Positive control: H2O2 |
BPA purity > 99.8% (Sigma-Aldrich) |
Positive
Concentration related increase of tail DNA (%) MTT: concentration related increase of cytotoxicity; increase of ROS and MDA; decrease of SOD |
Reliability: 2
No sufficient details on the comet method
(e.g. number of cells analysed is not specified) |
Limited |
Li et al., 2017 |
|
Alkaline and neutral comet assay Human PBMC (3 donors) 450 cells/concentration Cytotoxicity using flow cytometry |
Alkaline comet assay: - BPA 0.1, 1 and 10 μg/mL for 1 h; - 0.01, 0.1, 1 and 10 μg/mL for 4 h Neutral comet assay: - BPA 0.1, 1 and 10 μg/mL for 1 h DNA repair: BPA at 10 μg/mL Without metabolic activation |
BPA, 99–99.5% purity (Sigma-Aldrich) |
Positive
Both alkaline and neutral comet DNA repair of DNA breaks: decrease at 60 min, but the repair was not complete after 120 min |
Reliability: 2
unusual software for comet analysis
No positive control |
Limited |
Mokra et al., 2017 |
|
Alkaline comet assay and modified comet assay with Fpg enzyme in human peripheral blood lymphocytes |
1 h exposure to BPA: 0.001 mM, 0.1 mM, 2.5 mM Three experiments |
BPA (Sigma-Aldrich) Purity ≥97% not reported in the study but available on the website of the company |
Positive
Increase of % tail DNA, only at the first 2 concentrations tested With Fpg a higher increase of % tail DNA was observed at all concentrations, but not concentration related |
Reliability: 3
Inadequate response of positive control; the use of hydrogen peroxide as positive control is not adequate for the comet + Fpg Number of cells scored in not specified |
Low |
Durovcova et al., 2018 |
|
Comet assay in human sperm cells
Cell viability measured with a Nucleocounter NC 3000 In vivo comet assay (Table 8)** |
BPA 0, 1, 1.5, 2 and 3 μmol/L for 1 h
Without metabolic activation Each concentration was scored in 3 independent experiments and 2 replicates of each experiment 600 cells were scored/concentration Cell viability: BPA from 0 to 5 μmol/L |
BPA (purity >99%, Sigma-Aldrich) |
Negative No differences in % tail DNA between control samples and BPA-treated cells at all concentrations tested Cell viability assay: concentrationdependent decrease in cell viability from 3 μmol/L (reduced cell viability to 60%) |
Reliability: 3
Test not validated and not adequate for cryopreserved samples |
Low |
Sharma et al., 2018** |
|
Comet assay in human bronchial epithelial BEAS-2B cells Cytotoxicity: MTS assay after 24 h treatment γ-H2AX foci using immunofluorescence Intracellular ROS: DCF proteins involved in the DNA damage response (p-ATM, p-ATR, p-Chk1, p-p53) using western blot |
BEAS-2B cells were exposed to BPA 200 μM for 24 h MTS assay: 12.5 to 200 μM; tests performed in triplicates and for at least 3 independent times Without metabolic activation |
BPA (Sigma-Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Increase of DNA damage, but no quantitative data are reported MTS assay: - concentrationdependent cytotoxic effect; - cytotoxicity at 200 μM: 84.7 ± 2.1%; γ-H2AX: BPAinduced phosphorylation BPA-induced also phosphorylation of ATM/ATR complex and triggered Chk1 and p53 proteins Statistically significant increase of ROS |
Reliability: 3
Only one concentration tested, which resulted in high cytotoxicity Comet assay results not reported in detail, (no quantitative data)
No positive control |
Low |
George and Rupasinghe, 2018 |
|
Comet assay in TM3 murine Leydig cells Cell viability: MTT assay Real-time cell growth kinetics [cellular index (CI)] Cell-cycle analysis (PI, FACS analysis) Morphological analysis of cell death: chromatin staining with the Hoechst 33342 dye |
BPA 0, 1, 10 and 100 μM for 3 h; cell viability analysed with trypan blue exclusion method; Positive control: doxorubicin; 250 nucleoids were analysed in each repetition (3 experiments) Without metabolic activation BPA concentrations for MTT assay and real-time cell growth kinetics: 0, 0.5, 1, 5, 10, 50, 100, 250, 500 μM MTT assay exposure: 24 or 48 h Real-time cell growth kinetics: measurement every 30 min for 96 h Cell-cycle analysis, chromatin staining: BPA 0, 1, 10 and 100 μM for 24 or 48 h |
BPA (Sigma-Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Negative
No increase in damage index (DI) Cell viability was evaluated using trypan blue exclusion method, and only treatments with an index greater than 80% were considered (results not shown) Cell viability: statistically significant and concentrationrelated decrease from 5 and from 50 μM after 24 and 48 h exposure, respectively CI: TM3 cells exhibited a decrease in their CI after 34 h of exposure at concentrations from 10 μM BPA 100, 250 and 500 μM decreased CI within a few hours of exposure Cell-cycle analysis: BPA 100 μM induced an increase in the sub-G1 phase cell population
No other effects induced in the distribution of TM3 cells in the G0 + G1, S, and G2 + M phases Morphological analysis of cell death: increase in chromatin staining upon exposure to BPA 100 μM for 24 or 48 h |
Reliability: 3
Results are reported as damage index (not a standard parameter) |
Low |
Gonçalves et al., 2018 |
|
Alkaline comet assay with repair enzymes [with DNA glycosylases, i.e. endonuclease III (Nth) and human 8- oxoguanine DNA glycosylase (hOGG1)] Oxidised purines and pyrimidines Human PBMC 300 comets from 2 independent experiments Cell viability: flow cytometry |
BPA 0, 0.01, 0.1 and 1 μg/mL for 4 h and 0, 0.001, 0.01 and 0.1 μg/mL for 48 h Positive control: H2O2 (2 blood donors) Without metabolic activation |
BPA, 99–99.5% purity (Sigma-Aldrich) |
Positive
After 4 h incubation: - statistically significant and concentrationdependent oxidative damage to purines (from 0.01 μg/mL) and to pyrimidines (from 0.1 μg/mL) After 48 h incubation: - concentrationdependent oxidative DNA damage to purines (from 0.001 μg/mL) and to pyrimidines from (0.01 μg/mL) Statistically significant differences for DNA damage between 4 h and 48 h exposure
at the highest concentrations tested (0.01 and 0.1 μg/mL) Cell viability: no significant changes |
Reliability: 2
No appropriate positive control unusual software for comet analysis |
Limited |
Mokra et al., 2018 |
|
Alkaline comet assay (CometChip platform) in mouse embryonic fibroblasts (MEF) Analysis of γH2AX (immunofluorescence) In vitro chromosomal aberrations test (Table 3)* |
BPA 150 μM for 24 and 48 h (24 h for γH2AX), or co-exposure with camptothecin (CPT) Data of 4 replicates, each with 1500 ± 300 comets Without metabolic activation |
BPA (Sigma-Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Negative
No significant increase in the % tail DNA No significant increase in the percentage of γH2AX-positive nuclei |
Reliability: 3
No positive controls, no sufficient details on the methods applied; single concentration; cytotoxicity not evaluated |
Low |
Sonavane et al., 2018* |
|
Comet assay in murine macrophage RAW264.7 cells Cell viability: MTT assay Intracellular ROS level: semiquantitative DCFHDA fluorescence assay Assessment of the antioxidative enzymes activities: CAT, SOD, and GPx In vitro micronucleus assay (Table 4)* |
BPA 0, 3, 10, 30, or 50 μM for 24 h; no positive control; a minimum of 50 cells/slide were analysed MTT assay: BPA 0, 3, 10, 30, or 50 μM for 12 or 24 h DCFH-DA assay and assessment of antioxidative enzymes activities: - BPA 0, 3, 10, 30, or 50 μM for 24 h Without metabolic activation |
BPA (Sigma-Aldrich) purity ≥97% not reported in the study but available on the website of the company |
Positive
Increase in tail moment and tail length in a concentrationdependent manner starting from 10 μM of BPA Cytotoxicity: concentration- and time-dependent decrease of cell viability BPA-induced ROS generation and reduced antioxidative enzyme activities from 10 μM |
Reliability: 2
No positive control |
Limited |
Huang et al., 2018* |
|
Comet assay and comet modified with FpG In cryopreserved: - Hep-2 cells (human epithelial cells from laryngeal carcinoma); - MRC-5 cells (DNA damage responsive cell line, human lung fibroblasts) Cell viability: CellTiter- Blue assay In vitro micronucleus assay (Table 4)* |
BPA 0.44 nM, 4.4 nM, 4.4 μM for 48 h; Hep-2 cells: 300 cells analysed for each treatment MRC-5 cells: 100 cells analysed for each treatment Cell viability: BPA 0.44 nM, 4.4 nM, 4.4 μM, 48 h exposure in both Hep-2 and MRC-5 cells |
BPA (Sigma) purity ≥97% not reported in the study but available on the website of the company |
Inconclusive |
Reliability: 3
Comet assay is not validated and recommended for testing cryopreserved cell samples No positive control |
Low |
Ramos et al., 2019* |
|
Comet assay in sperm cells from Sprague Dawley rats Analysis: ROS, LPO, SOD In vivo comet assay (Table 8)** |
BPA 0, 1, 10, and 100 μg/L for 2 h No positive control Without metabolic activation |
BPA (99% purity) Santa Cruz Biotechnology |
Positive Increase of tail DNA% only at 100 μg/L BPA increased SOD, ROS, TBARS [thiobarbituric acid reactive substances (TBARS) as an index of LPO] only at 100 μg/L |
Reliability: 3
The study was performed following a nonstandard, neutral protocol and unusual evaluation of comets based on the analysis of microphotographs. No positive control |
Low |
Ullah et al., 2019** |
|
Comet assay in Marc- 145 cells (rhesus monkey embryo renal epithelial cells) Cytotoxicity: MTT and LDH assays Intracellular ROS levels: DCFH-DA Lipid peroxidation: - TBARS; - SOD activity and GSH content |
BPA 10–6 to 10–3 M for 24 h; 50 cells from each of 6 independent experiments were analysed MTT assay: BPA 10–6 to 10–1 M for 24 h; DCFH-DA and TBARS assays: BPA 10–6 to 10–3 M for 24 h; SOD activity and GSH content: BPA 10–6 to 10–3 M for 24 h Without metabolic activation |
BPA (purity > 99%) Sigma-Aldrich |
Positive
Increase in % tail DNA, tail length and tail moment (10–6 - 10–3 M); Cytotoxicity: concentrationrelated increase; excess of toxicity at 10–3 and 10–4 M BPADCFH-DA, TBARS assays: - concentrationrelated increase of ROS and lipid peroxidation; - SOD activity and GSH content: concentrationrelated decrease |
Reliability: 2
No positive control |
Limited |
Yuan et al., 2019 |
|
Alkaline comet assay and Fpg modified comet assay RWPE-1 cells [human papilloma virus 18 (HPV18) immortalised, non-tumorigenic prostatic cell line] Cell viability: modified MTT assay and trypan blue exclusion Enzymatic and nonenzymatic antioxidants: analysis of GPx, GR, SOD, GSH and TAOC levels |
BPA 0, 45 μM (IC20) for 24 h 450 comets analysed/treatment; experiments in triplicates Cell viability: 0, 50, 100, 200, 300, 600 μM for 24 h Enzymatic and nonenzymatic antioxidants: BPA 0, 45 μM (IC20) for 24 h Without metabolic activation |
BPA (>99% pure) |
Positive Comet assay: increase (2.5-fold) in tail intensity (at IC20 BPA) Fpg modified comet: increase in tail intensity Cell viability: decrease in cell viability (IC20 45 μM) Enzymatic and nonenzymatic antioxidants: decrease in: - GPx1 and SOD activity (29% and 24% respectively); - TAOC levels (20%); increase in: - GR activity (4.5- fold); - total GSH level (30%) |
Reliability: 2
One concentration tested No positive control No metabolic activation |
Limited |
Kose et al., 2020 |
|
Comet assay in HepG2 cells (human hepatocellular carcinoma cell line) Cell viability: MTT test SOS/umuC assay (Table 1)* |
BPA 0, 1, 10, 100 and 1000 μg/L, for 4 and 24 h; 3 independent experiments; 50 nuclei analysed/treatment MTT test: BPA 0, 1, 10, 100 and 1000 μg/L, for 24 h |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Positive
increase of % tail DNA from 10 μg/L at both 4 h and 24 h exposure MTT test: no effects on cell viability |
Reliability: 2
Low number of nuclei analysed |
Limited |
Balabanič et al., 2021* |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vivo chromosomal aberrations assay
Table 6: In vivo chromosomal aberrations assay (OECD TG 475 was considered for the evaluation of the reliability).
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Chromosomal aberration assay in bone marrow Swiss albino mice Six animals (3 females and 3 males)/group (control and BPAtreated animals) 100 metaphases were scored per animal Mitotic effects In vivo micronucleus assay (Table 7)* |
BPA 0, 10, 50 and 100 mg/kg bw; 2% gum acacia was used as the suspending medium for BPA Single oral dose administered by gavage Sampling of bone marrow at 6, 24, 48 and 72 h Cumulative dose level: 10 mg/kg bw for 5 consecutive days Sampling of the bone marrow 24 h after the last administration of BPA |
BPA, purity 98% (Loba Chemie, Mumbai, India) |
Negative No significant increase of structural chromosomal aberrations Significant increases in the frequencies of gaps at all doses at 48 and 72 h sampling time and at 50 and 100 mg/kg bw at the 24 h sampling time C-mitotic effects through increases of mitotic indices and decrease in anaphase for both higher dose level at 24, 48 and 72 h sampling times |
Reliability: 2
Low number of animals/sex, but in total 6 animals/group Low number of metaphases scored, treatment with colchicine shorter (1.5 h) than recommended (5–6 h) |
Limited |
Naik and Vijayalaxmi, 20091* |
|
Chromosomal aberration in bone marrow
Holtzman rats Ten animals (5 females and 5 males)/group (control and BPA-treated animals) Analysis of 100 metaphases per animal In vivo micronucleus assay (Table 7)* and comet assay (Table 8)* Bacterial reverse mutation assay (Table 1)**
|
BPA 0, 2.4 μg, 10 μg, 5 mg and 50 mg/kg bw administered orally once a day for 6 consecutive days; BPA dissolved in distilled ethyl alcohol and diluted with sesame oil Sampling of the bone marrow 24 h after the last administration of BPA |
BPA, ∼99% purity (Sigma Chemical Company) |
Positive
Dose-related increase of structural chromosomal aberrations starting from 10 μg |
Reliability: 2
Mitotic index as a measure of cytotoxicity not determined |
Limited |
Tiwari et al., 20121*,** |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vivo micronucleus assay
Table 7: In vivo micronucleus assay
(OECD TG 474 was considered for the evaluation of the reliability).
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Micronucleus assay Male ICR mice Peripheral blood reticulocytes (1000/animal analysed, 5 mice per group) Bacterial reverse mutation assay (Table 1)** |
228 mg/kg bw of BPA dissolved in DMSO, once by gavage; controls received vehicle alone Peripheral blood collected at 24, 48 and 72 h after administration |
BPA purity >99% (Tokyo Kasei Kogyo Co., Ltd) |
Inconclusive
(negative with no demonstration of bone marrow exposure) No increase of micronucleated reticulocytes at any sampling time Cytotoxicity was not evaluated |
Reliability: 2
Single dose tested, although relatively high; 1000 scored reticulocytes/animal instead of 2000 as in OECD TG 474 (1997) No positive control |
Low |
Masuda et al., 20051** |
|
Micronucleus assay in bone marrow Male mice (102/ElxC3H/El)F1 (5 animals per group) |
BPA 0, 0.002, 0.02 and 0.2 mg/kg bw oral gavage on 2 days Cells collected 24 h after last administration 2000 polychromatic erythrocytes (PCE) were scored per animal |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Inconclusive
(negative with no demonstration of bone marrow exposure) No induction of micronuclei in the bone marrow polychromatic erythrocytes |
Reliability: 2
No positive control; very low doses applied |
Low |
Pacchierotti et al., 20081 |
|
Cytogenetic analyses of oocytes and zygotes in female C57Bl/6 mice Assessment of meiotic delay in spermatocytes by BrdU incorporation and aneuploidy in epididymal sperm by multicolor FISH in male 102/ElxC3H/El)F1 mice (5 mice per dose) |
Acute exposure: 0.2 or 20 mg/kg Sub-acute exposure: 0.04 mg/kg for 7 days by gavage Sub-chronic exposure: 0.5 mg/L for 7 weeks in drinking water 0.2 mg/kg bw starting on day 8 after BrdU, for 6 consecutive days BPA 0, 0.002, 0.02 and 0.2 mg/kg for 6 consecutive days |
BPA (Sigma-Aldrich) |
Negative
No significant induction of hyperploidy or polyploidy in oocytes and zygotes in any treatment condition
No delay of meiotic divisions No induction of hyperploidy or polyploidy in epididymal sperms |
Reliability: 2
This study was adequately planned, performed and reported, even though specific guidelines for the effects in germ cells are not available No positive control
Very low doses for the analysis of sperm aneuploidy |
Limited |
|
|
Micronucleus in bone marrow Swiss albino mice Six animals (3 females and 3 males)/group (control and BPAtreated animals); 2000 PCE/animal In vivo chromosomal aberration (Table 6)* |
BPA 0, 10, 50 and 100 mg/kg bw; 2% gum acacia was used as the suspending medium for BPA Single oral dose administered by gavage sampling of bone marrow at 6, 24, 48 and 72 h Cumulative dose level: 10 mg/kg bw for 5 consecutive days Sampling of the bone marrow 24 h after the last administration of BPA |
BPA purity 98% (Loba Chemie, Mumbai, India) |
Negative No significant decrease of PCE/NCE ratio Significant increase of gaps and C-mitoses |
Reliability: 2
Low number of animals/sex in each group, but in total 6 animals/group |
Limited |
Naik and Vijayalaxmi, 20091* |
|
Micronucleus in bone marrow Male Sprague Dawley rats 8 rats/group (control and BPA-treated animals) In vivo comet assay (Table 8)* |
BPA 0, 200 mg/kg bw per day for 10 days Orally via drinking water Bone marrow processed at the end of treatment |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Inconclusive (negative with no demonstration of bone marrow exposure) No data on bone marrow toxicity are reported |
Reliability: 2
Exposure of the bone marrow not demonstrated Single dose tested No positive control |
Low |
De Flora et al., 20111* |
|
Micronucleus in bone marrow Holtzman rats Ten animals (5 females and 5 males)/group (control and BPA-treated animals) In vivo chromosomal aberration (Table 6)* Comet assay (Table 8)* Bacterial reverse mutation assay (Table 1)** |
BPA 0, 2.4 μg, 10 μg, 5 mg and 50 mg/kg bw per day administered orally for 6 consecutive days Sampling of the bone marrow 24 h after the last administration of BPA Analysis of 2000 PCE |
BPA, ∼99% purity (Sigma Chemical Company) |
Positive
Dose-related increase of MN-PCE starting from 10 μg/kg bw per day |
Reliability: 2
Inappropriate staining |
Limited |
Tiwari et al., 20121*,** |
|
Micronucleus test in peripheral blood reticulocytes and in bone marrow of Pzh:Sfis female mice No. of animals/group: 9 in control, 6 in BPA 5 mg/kg bw, 8 in BPA 10 mg/kg bw, 6 in BPA 20 mg/kg bw; 1000 reticulocytes or PCE were scored In vivo comet assay (Table 8)* |
BPA 5, 10, or 20 mg/kg bw per day for 2 weeks in drinking water Animals were sacrificed 24 h after the end of treatment Blood was collected at 1 and 2 weeks of exposure |
BPA, no information on purity or the supplier company |
Positive in reticulocytes at 10 and 20 mg/kg bw after 2 weeks of exposure
Negative in reticulocytes after 1 week of treatment
Negative in bone marrow |
Reliability: 2
No criteria for scoring micronuclei were described No positive control |
Low
No information on source and purity of BPA |
Gajowik et al., 2013* |
|
Micronucleus test in bone marrow cells Adult male Wistar albino rats Ten animals per group |
Oral administration of 5 μg, 50 μg and 100 μg BPA/100 g bw once a day for 90 days, sacrifice and sampling of bone marrow on the 91th day |
BPA (<99% pure) purchased from Sigma-Aldrich, diluted in olive oil |
Positive
Increases (2–3-fold at the highest dose) in the frequency of micronuclei in polychromatic erythrocytes and normochromatic erythrocytes Statistical significance of the difference with negative controls not determined No decrease in PCE/NCE ratio |
Reliability: 3
Major limitation in data presentation and analysis: low number of scored cells per animal lack of historical control data |
Low |
Srivastava and Gupta, 2016 [ |
|
Micronucleus test in bone marrow Male Swiss albino mice, 10 animals/group; analysis of 2000 PCE/animal In vivo comet assay (Table 8)* |
50 mg/kg bw, orally once a day for 28 days Sampling of the bone marrow at the end of treatment |
BPA, purity ≥ 99%, (Sigma-Aldrich) |
Positive
Increase in the mean values of MNPCEs (66.40 ± 9.94 vs 10.40 ± 2.96) Cytotoxic (reduction in the ratio of PCE/NCE compared to control) |
Reliability: 2
No positive control only one dose |
Limited |
Fawzy et al., 2018* |
|
Micronucleus test in bone marrow Male Wistar rats; 6 animals/group Analysis of 2000 PCE for MN scoring and of 200 cells for PCE/NCE Ratio
Lipid peroxidation: serum level of malondialdehyde (MDA) (8-OHdG) in urine In vivo comet assay (Table 8)* |
0, 50 and 100 μg/kg bw per day, 4 weeks, by gavage Sampling at the end of treatment |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Positive Significant dose-related increase (up to 3-fold) in the mean values of MNPCEs compared with control Cytotoxic (a weak statistically significant decrease in PCE/NCE ratio); dose-related increase of MDA in blood and of urinary 8- OHdG levels |
Reliability: 2
No positive control only 2 doses |
Limited |
Panpatil et al., 2020* |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vivo DNA damage
Table 8: In vivo DNA damage (comet assay, OECD TG 489 was considered for the evaluation of the reliability).
|
object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Comet assay in peripheral blood lymphocytes Sprague Dawley rats 8 rats/group (control and BPA-treated animals); 100 nuclei were scored In vivo micronucleus assay (Table 7)* |
200 mg/kg bw for 10 consecutive days, orally via drinking water Sampling at the end of treatment |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Negative |
Reliability: 2
Tail moment, used as only parameter to report the results for the comet assay, is not recommended by the Comet international Committee; single dose tested; no positive control |
Limited |
De Flora et al., 20111* |
|
Comet assay in peripheral whole blood cells of Wistar rats (6 animals/group BPAtreated animals; 5 animals in the control group; 3 animals in the positive control group) |
0, 125 and 250 mg/kg bw; oral administration (gavage) for 4 weeks Positive control: MMS (i.p., sampling after 24 h); 50 cells were analysed on each replicated slide |
BPA purity > 99% (Merkolab Chemistry) |
Positive
Increase of both tail length and tail moment at 250 mg/kg bw |
Reliability: 3
Inappropriate presentation and evaluation of results Group mean tail length and tail moment values, rather than the means of animal median values (OECD TG 489) Sampling time, and frequency of administrations not stated |
Low |
Ulutaş et al., 20111 |
|
Comet assay in blood lymphocytes Holtzman rats Ten animals (5 females and 5 males)/group (control and BPAtreated animals); analysis of 50 nucleoids/animal Plasma concentrations of 8- hydroxydeoxyguanosine (8-OHdG), lipid peroxidation (MDA) and glutathione activity In vivo micronucleus assay (Table 7)* chromosomal aberrations assay (Table 6)* Bacterial reverse mutation assay (Table 1)** |
2.4 μg, 10 μg, 5 mg and 50 mg/kg bw per day administered once a day for 6 consecutive days Sampling 24 h after the last administration of BPA |
BPA, ∼99% purity (Sigma Chemical Co.) |
Positive
Dose-related increase starting from 10 μg/kg bw per day Significant increase in plasma concentration of 8- OHdG only at 50 mg/kg bw per day Dose-related increase of MDA and decrease of glutathione in liver Inconsistent results of 8- OHdG with comet assay |
Reliability: 2
Inappropriate sampling time Low number of nucleoids scored |
Limited |
Tiwari et al., 20121*,** |
|
Comet assay in bone marrow, spleen, liver and kidney and germ cells Male Pzh:SFIS mice; 5 animals/group; 100 cells were analysed |
0, 5, 10, 20 or 40 mg/kg bw Orally in drinking water Daily for 2 weeks Animals were sacrificed 24 h after the last treatment |
BPA, no information on purity or the supplier company |
Positive
Increases of DNA tail moment in bone marrow, spleen, kidney and lung cells at any dose level without a clear dose response No increase of tail moment was detected in liver cells In sperm cells increase of tail moment: at all doses 24 h after the end of exposure; at the 2 highest doses 5 weeks after the end of treatment |
Reliability: 3
No information on purity; drinking water consumption (containing BPA) not measured, inadequate sampling time, poor study report; tail moment, used as only parameter to report the results for the comet assay, is not recommended by the Comet International Committees |
Low |
Dobrzyńska and Radzikowska, 20131 |
|
Alkaline comet assay in epididymal sperm of Holtzman rats In vivo dominant lethal mutations in male rats (Table 9)* |
Oral gavage of 10 μg/kg bw and 5 mg/kg bw BPA dissolved in ethyl alcohol and diluted in sesame oil, for 6 consecutive day |
BPA ∼99% purity (Sigma Chemical Co.) |
Positive
Significant increase in the sperm DNA damage at 5 mg/kg bw |
Reliability: 3
Comet assay is not considered appropriate to measure DNA strand breaks in mature germ cells due to the high and variable background levels in DNA damage in this cell type (OECD TG 489); moreover, the sampling time, i.e. 8 weeks after last treatment, is inappropriate for in vivo comet assay |
Low |
Tiwari and Vanage, 20131* |
|
Comet assay in lung, spleen, kidneys, liver and bone marrow of Pzh:Sfis female mice No. of animals/group 9 in control, 6 in BPA 5 mg/kg bw, 8 in BPA 10 mg/kg bw; 6 in BPA 20 mg/kg bw 100 nucleoids scored/animal In vivo micronucleus assay (Table 7)* |
BPA 5, 10, or 20 mg/kg bw/day for 2 weeks in drinking water Sampling 24 h after the end of treatment |
BPA, no information on purity or the supplier company |
Positive in lung at 5 and 10 mg/kg Negative in spleen, kidneys, liver and bone marrow |
Reliability: 2
Inappropriate sampling time, tail moment, used as only parameter to report the results for the comet assay, is not recommended by the Comet International Committees |
Low
No information on source and purity of BPA |
Gajowik et al., 2013* |
|
Alkaline comet assay in brain cells of KM male mice; (11 animals/group); 200 cells for each group analysed |
BPA 0.5, 50 and 5000 μg/kg bw (daily dose, diluted in tea oil, by gavage) for 8 weeks After 8 weeks of exposure, mice were sacrificed and the brain samples were immediately removed The tail DNA%, tail length and tail moment were measured using CASP comet analysis software Based on the DNA percentage of the tail intensity, the damage level was divided into 5 grades Arbitrary units computed with the score of DNA damage in analysed cells were used to express the DNA damage |
BPA from Sigma- Aldrich (HPLC grade) purity >97% not reported in the study but available on the website of the company |
Positive
Significant increase of damaged cells from 23.0% in the control group to 47.3%, 66.6% and 72.5% in the low-, medium and high-exposed groups Severity of DNA damage, expressed as arbitrary units (AUs), increased with AUs of 0.28 in the control to AUs of 0.59, 0.96 and 1.28 in the low, medium and highly exposed groups, respectively |
Reliability: 2
DNA damage was evaluated using arbitrary units and considering the distribution of DNA damage in the cell population analysed (n = 440), rather than using median animals data as the statistical unit, as recommended in OECD TG 489 |
Limited |
Zhou et al., 2017 |
|
Comet assay in liver female Wistar rats; (7 animals/group) Serum biochemical analysis: ALT, ALP, TP, Alb, GGT, TC, Triglycerides, HDL; LDL Hepatic antioxidants and lipid peroxidation level: GPx, SOD, MDA CYPR450 (ELISA) Histopathology Immunohistochemical evaluation of caspase-3 |
7 animals/group: control (corn oil) BPA 10 mg/kg bw; daily administration via gavage for 30 days Sampling at the end of treatment |
BPA (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Positive: increase of tail DNA % BPA-induced: - increase of ALT, ALP, GGT, TC, LDL, MDA, caspase-3; - decrease of Alb, TP, GPx, SOD, CCYPR450 Histopathological analyses showed deleterious hepatic changes ranging from hepatocytes’ vacuolisation and eccentric nuclei to focal necrosis and fibrosis
|
Reliability: 3
Use of frozen tissues; without a positive control; a single dose applied; toxic effects in liver |
Low |
Abdel-Rahman et al., 2018 |
|
Comet assay in liver of Sprague Dawley rats of either sex; 7 animals/group Serum analysis: ALT, ALP, AST, bilirubin Analysis of antioxidant effects: CAT, POD, SOD, GSH Lipid peroxidation assay, hydrogen peroxide assay, nitrite assay Liver histopathology |
BPA, 25 mg/kg by i.p. negative control group; vehicle control group (10% DMSO in olive oil) Sampling: 4 weeks after the treatment |
BPA, no information on purity or the supplier company |
Positive increase of tail DNA % 28.35 ± 1.2 vs 0.01 ± 0.005 BPA-induced: - increase of WBC, ALT, AST, ALP, bilirubin, H2O2, nitrite - decrease of RBC, platelets, Hb, albumin, CAT, POD, SOD, GSH, ‘Histopathological examination of BPAtreated animals revealedintense hepatic cytoplasm inflammation, centrilobular necrosis, cellular hypertrophy, fatty degeneration, vacuolisation, steatosis and distortion of portal vein’ |
Reliability: 3
Limitations: - a single administration by i.p. and comet, analysis after 4 weeks; - unusual software used for the comet analysis; - the results reported using the different parameters (tail length, % of DNA in tail, tail moment) are not consistent; - the value of % of DNA in tail in controls is extremely low with respect to the data reported in the scientific literature; - high liver toxicity |
Low
A single administration by i.p. No information on source and purity of BPA |
Kazmi et al., 2018 |
|
Comet assay in liver of Male Swiss albino mice (10 animals/group); images of 50 randomly selected nuclei/ experimental group Analysis of liver toxicity markers (AST and ALT) and liver histopathology |
BPA dissolved in ethanol and diluted in corn oil by gavage at 50 mg/kg bw, once a day for 28 successive days |
BPA (≥ 99 %) Sigma- Aldrich |
Positive
Mean tail length, tail moment and % tail DNA were significantly increased (p < 0.05) in liver of BPA-treated mice Increase of AST, ALT, marked histopathological alteration in liver of BPAtreated animals ‘congestion of the hepatic blood vessels as well as marked vacuolar degeneration of the hepatocytes with many necrotic cells’ |
Reliability: 3
Major deviation from OECD TG 489: -too low number of analysed cells per animal -aggregated mean data analysed (instead of animal median) -no positive control - too high liver toxicity associated with treatment |
Low |
Elhamalawy et al., 2018 |
|
Alkaline comet assay in liver, kidney, testes, urinary bladder, colon and lungs cells CD-1 male mice (5 mice/group) In vitro comet assay (Table 5)** |
Gavage 0, 125, 250 and 500 mg/kg bw BPA (maximum tolerated dose) as suspensions in corn oil prepared by ultrasonication 2 doses (24 h apart) Animals were sacrificed 3 h after 2nd dose 200 cells analysed/mice (100 cells per gel and 2 gels per mouse) |
BPA (purity >99%, Sigma-Aldrich) |
Negative
None of the tissues showed an effect of BPA except in testicular cells, in which an increased level of DNA strand breaks (p < 0.01 compared with control group) was observed at the lowest dose only |
Reliability: 1
This study basically followed the OECD TG 489 |
High |
Sharma et al., 2018** |
|
Comet assay in liver and testes of male Swiss albino mice Male Swiss albino mice, 10 animals/group 50 nuclei/group were analysed In vivo micronucleus assay (Table 7)* |
50 mg/kg bw, orally once a day for 28 days Sampling at the end of treatment |
BPA, purity ≥ 99% (Sigma-Aldrich) |
Positive
Increase (p ≤ 0.05) in the mean values of tail length, percentage of tail DNA and Olive tail moment in liver and testes Histopathological examination hepatocyte vacuolar degeneration with many necrotic cells Defective spermatogenesis characterised by severe necrosis and loss of the spermatogonial layers with multiple spermatid giant cells formation in most of the seminiferous tubules and a congestion of the interstitial blood vessels |
Reliability: 3
No positive control, low number of nucleoids analysed, toxic effects observed in liver and testes, a single dose applied The standard alkaline comet assay applied is not considered appropriate to measure DNA strand breaks in mature germ cells |
Low |
Fawzy et al., 2018* |
|
Comet assay in heart of Wistar rats; 20 animals/group |
BPA dissolved in corn oil 30 mg/kg bw per day injected subcutaneously (SC) 6 days/week for 4 weeks Sacrifice at the end of treatment |
BPA Sigma-Aldrich; purity >97% not reported in the study but available on the website of the company |
Positive
Increase tail DNA % (6.88 vs 1.67) Histopathological changes: focal disruption of cardiomyocytes with some nuclear changes, such as karyolysis and pyknosis and sarcoplasmic vacuolisation The mitochondria appeared swollen and deranged with different sizes and shapes |
Reliability: 3
Single dose; no positive control; inadequate cell preparation for comet assay; high toxicity |
Low
route of administration: subcutaneous |
Amin et al., 2019 |
|
Comet assay in testes of Sprague Dawley rats; 7 rats/group Histopathology Antioxidant enzymes: CAT, SOD, GSH, POD, NO |
BPA (50 mg/kg bw) injected intraperitoneal on alternate days for 21 days Sacrifice 24 h after the end of treatment |
BPA analytical grade (Merck KGaA); purity >97% not reported in the study but available on the website of the company |
Positive
Histopathology: ‘BPA caused significant damage and abrasions to seminiferous tubules with low cellular density’ BPA-induced: - decrease of body weight, epididymis and testes weight, testosterone, FSH, LH, CAT, SOD, GSH, POD; - decrease of sperm count, viability, motility - increase of estradiol |
Reliability: 3
Single dose; no positive controls; an unusual software for the comet analysis used; the comet presented in the microphotographs are of low quality The standard alkaline comet assay applied is not considered appropriate to measure DNA strand breaks in mature germ cells |
Low
BPA was administered by i.p. |
Majid et al., 2019 |
|
Comet assay (neutral) on spermatozoa of Sprague Dawley rats (7 per group) 100 scored cells per animal In vitro comet assay (Table 5)** |
Animals treated by gavage with 5, 25 and 50 mg BPA/kg bw per day for 28 days and sacrificed on day 29th, control received the vehicle alone (0.1% ethanol) |
BPA (99% purity) from Santa Cruz Biotechnology |
Positive
Both tail moment and % tail DNA were significantly (p < 0.05) increased in the BPA 50 mg/kg bw per day group compared to vehicle controls, while no significant difference with controls was observed in the BPA 5 and 25 mg/kg bw per day groups |
Reliability: 3
The study was performed following a non-standard, neutral protocol and unusual evaluation of comets based on the analysis of microphotographs No detailed information on data analysis is provided (e.g. the use of median vs mean as individual animal descriptor) No positive control |
Low |
Ullah et al., 2019** |
|
Comet assay in testes of offspring of BPA treated mice (pregnant Kumming mice, 20 in each group) |
Animals were randomly divided into 7 groups. One group served as controlthe others received BPA in drinking water at 0.05, 0.5, 5, 10, 20 or 50 mg/kg bw per day, for 40 days from gestation day 0 to lactation day 21. F1 male mice were sacrificed at weaning (post-natal day 21) and DNA damage in testes evaluated by comet assay |
BPA (purity 99%, Sigma) |
Positive
The results obtained showed significantly increased Olive tail moment (OTM) in testes cells of F1 animals treated with 5, 10, 20 and 50 mg/kg bw per day, compared with the control group (p < 0.05). |
Reliability: 3
The results obtained showed significantly increased Olive tail moment (OTM) in testes cells of F1 animals treated with 5, 10, 20 and 50 mg/kg bw per day, compared with the control group (p < 0.05). |
Low |
Zhang et al., 2019 |
|
Alkaline comet assay in thyroid tissue Male albino rats 20 rats/group Biochemical investigation of MPO activity, GSH, SOD activity and MDA |
BPA dissolved in corn oil 200 mg/kg bw per day (1/20 of the oral LD50) for 35 days Sacrifice 24 h after the last administration |
BPA (99.5% purity) was obtained from Sigma-Aldrich Co. |
Positive
% tail DNA 4 times increase compared with control level The histopathological examinations of thyroid gland showed severe congestion of interstitial blood capillaries, severe lymphocytic infiltration associated with variablesized follicles, most of which contain scanty colloid secretion, and some are atrophied in BPA group Significant induction of MPO activity and MDA concentration associated with significant decreases of SOD activity and GSH concentration in the thyroid gland of BPA group |
Reliability: 3
Only one dose level No positive control Comet method poorly described The microphotographs of comets are of low quality High toxicity |
Low |
Mohammed et al., 2020 |
|
Alkaline comet assay in testes Male juvenile Sprague Dawley (SD) rats (7 animals/group) Sperm DNA damage was evaluated by the comet and Halo assays using duplicate slides; apoptosis in testes cells was quantified using TUNEL assay, and testicular levels of 8- OHdG were determined by immunohistochemistry |
Gavage 8 weeks BPA (100 mg/kg bw per day) daily/5 days per week by gavage for 8 consecutive weeks Animals were sacrificed after 8 weeks |
BPA (Sigma-Aldrich) Purity >99% not reported in the study but available on the website of the company |
Negative All comet assay parameters (tail length, Olive tail moment and % DNA in the tail) and the nuclear diffusion factor in Halo assay, were slightly but not significantly increased in testes cells of BPA-treated rats compared with controls TUNEL-positive cells and per cent of 8-OHdG positive areas in testicular tissue were also slightly but non-significantly increased in BPA-treated rats |
Reliability: 3 The standard alkaline comet assay applied (OECD TG 489) is not considered appropriate to measure DNA strand breaks in mature germ cells Other test methods (Halo and immunohistochemical determination of 8- OHdG) are not standardised and/or validated for regulatory use For all end-points, only a single dose was tested Sampling time not specified No positive control |
Low |
Sahu et al., 2020 |
|
Comet assay on whole brain cells from KM mice of F1 and F2 (8 male and 8 female) |
Pregnant mice (F0) were orally dosed with BPA dissolved in tea oil at 0.5, 50, 5000 μg/kg bw per day from gestational day 1 until weaning (post-natal day 21). Then, the first generation (F1) of mice were used to generate the F2 DNA damage in brain cells was evaluated by comet assay in mice from both F1 and F2 |
BPA (purity: 98 %) Sigma-Aldrich |
Equivocal DNA damage, expressed as arbitrary units, was slightly (less than twofold) increased in the F1male mice at the lowest dose and in females at the intermediate dose. No effect of BPA exposure was observed in the F2 mice |
Reliability: 3 The study protocol is only shortly described
The presentation and interpretation of the results is inadequate No positive control |
Low |
Zhang et al., 2020 |
|
Comet assay in blood liver and kidney Male Wistar rats (WNIN) 6 animals/group 50 nuclei/slides were scored Lipid peroxidation: serum level of malondialdehyde (MDA) 8-Hydroxy-2- deoxyguanosine (8- OHdG) in urine collected 24 h before the sacrifice In vivo micronucleus assay (Table 7)* |
0, 50, and 100 μg/kg, per oral (gavage) for a period of 4 weeks Sampling at the end of treatment |
BPA, (Sigma-Aldrich) purity >97% not reported in the study but available on the website of the company |
Positive
A weak but statistically significant and doserelated increase of tail length in liver In kidney increase of DNA damage observed only at the dose of 50 μg/kg Comet parameters are not reported for blood cells Dose-related increase of MDA in serum and of 8- OHdG levels in urine |
Reliability: 2
Low number of nucleoids analysed No positive controls |
Limited |
Panpatil et al., 2020* |
|
Evaluation of sperm DNA damage by alkaline comet and DNA ladder assays Male Sprague Dawley rats (groups of 7 animals) ROS, Catalase, POD and SOD, GSH, Lipid peroxidation, TBARS, hydrogen peroxide, nitrite assay, AOPP |
BPA diluted in 10% DMSO was injected intraperitoneally at 25 mg/kg bw on alternate days for 30 days |
BPA, no information on purity or the supplier company |
Positive
Significant (p < 0.01) increase of all comet parameters in BPA-treated animals compared with vehicle controls Electrophoresis on agarose gel showed extensive DNA fragmentation in testes of BPA-treated rats Significant increase in ROS level and decreased levels of CAT, GSH SOD and POD in the testis of BPA-treated group |
Reliability: 3
The standard alkaline comet assay applied (OECD TG 489) is not considered appropriate to measure DNA strand breaks in mature germ cells
The comet protocol is shortly described, with no information on the number of analysed sperm cells per animal; sampling time not specified; cytotoxicity not evaluated; no positive control The DNA ladder assay is a biochemical method not validated for genotoxicity assessment |
Low
For insufficient reliability and lack of information on test item purity |
Zahra et al., 2020 |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).
In vivo dominant lethal assay
Table 9: In vivo dominant lethal assay (OECD TG 478 was considered for the evaluation of the reliability).
|
Test system/Test object |
Exposure conditions (concentration/ duration/metabolic activation) |
Information on the characteristics of the test substance |
Results |
Reliability/ Comments |
Relevance of the result |
Reference |
|
Dominant lethal test with male Holtzman rats (7 per group) Each treated male was mated with 2 females per week over a period of 8 weeks; the mated females were sacrificed on 15th day of gestation and uterine content examined In vivo comet assay in rat epididymal sperm (Table 8)* |
Rats treated by oral gavage with BPA dissolved in ethyl alcohol and diluted in sesame oil, at dose levels of 10 μg/kg bw and 5 mg/kg bw once a day for 6 consecutive days Negative controls were treated with vehicle |
BPA ∼99% purity (Sigma Chemical Co.) |
Positive
Significant decrease in total implants/female and live implants/female, in females mated with males treated with 5.0 mg BPA/kg bw the fourth week and sixth week after treatment |
Reliability: 2
No positive control No negative historical control Limited study design, with less analysable total implants and resorptions than recommended (OECD TG 478) |
Limited |
Tiwari and Vanage, 20131* |
Source: Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs, EFSA, (2021).