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Genetic toxicity: in vitro

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Administrative data

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: other: 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.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1989

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- purity: ≥97 %

Method

Species / strain
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
Controls
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.

Results and discussion

Test results
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'.

Applicant's summary and conclusion

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.