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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
7 October 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 4,4'-methylenediphenol and 2,2'-methylenediphenol and o-[(4-hydroxyphenyl)methyl]phenol
EC Number:
908-912-9
Cas Number:
1333-16-0
Molecular formula:
C39H36O6
IUPAC Name:
Reaction mass of 4,4'-methylenediphenol and 2,2'-methylenediphenol and o-[(4-hydroxyphenyl)methyl]phenol
Test material form:
solid

Method

Target gene:
Histiene locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.5, 5, 50, 250, 500 µg.
All compounds were tested at the limit of solubility.
Vehicle / solvent:
DMSO
BPF was dissolved in DMSO and then added to the culture medium such as the final concentration of DMSO did not exceed 0.1% (v/v). All compounds were
tested at the limit of solubility.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate fo TA98
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate for TA100 and TA1535
Positive control substance:
sodium azide
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA1537
Positive control substance:
other: Acridine Mutagen ICR 191
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.5 µg/plate for WP2 uvrA
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate fpt TA100, TA1535, TA1537, WP2 uvrA
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
The Ames test was carried out using the plate incorporation method (with preincubation) with or without metabolic activation, with four histidine-dependent auxotrophic mutants of Salmonella typhimurium strains, TA 98, TA 100, TA 1535, TA 1537, essentially as described by Maron and Ames (1983). A fifth strain was used, a tryptophan-dependent auxotrophic mutant Escherichia coli WP2 uvrA pKM101. The test strains were cultured in the liquid broth medium for 10 h at 37 °C under agitation. After incubation, 0.5 ml of 0.1M sodium phosphate buffer (pH 7.4) (absence of metabolic activation) or 0.5 ml of S9 mix (presence of metabolic activation), 0.1ml of bacterial culture and 50 ml of BPF solutions (0.01mg/ml up to 10mg/ml) were added to a test tube and preincubated 1 h at 37 °C. Two millilitre of semi-liquid superficial agar was added to the mixture and poured onto a minimal glucose agar plate. For experiments with S. typhimurium, the top agar was supplemented with 10 ml of 0.5mM histidine/biotin solution per 100ml agar, and mutations to histidine independence were scored on minimal glucose agar plates. For experiments with E. Coli strain, mutations to tryptophan independence were scored on minimal glucose agar plates supplemented with 10 ml of 0.5mM tryptophan per 100ml agar. The plates were incubated 48 h at 37 °C, and then the number of revertant colonies was counted. All experiments were carried out in triplicate using five concentrations of BPF. Mutagenic activities were expressed as induction factors, i.e. as multiples of the background levels.
Evaluation criteria:
According to the historical values of the laboratory, a compound tested with the Ames test was considered mutagenic if the number of His+ revertant
colonies was at least twice the value of the corresponding solvent control (induction factor > 2). A dose-effect relationship is an additional indication for the mutagenic potency of a molecule. A possible mutagenic potential is assumed if the quotient ranges between 1.7 and 1.9 in combination with an observed dose-effect
relationship. No mutagenic potential is assumed if all quotients range between 1.0 (or lower) and 1.6. The latter conclusions can be strengthend by the lack of
observation of a dose-effect relationship.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
BPF was cytotoxic for the bacteria at the highest concentration tested (500 µg/plate), especially without exogenous activation system. In this study, none of the results of the Ames test (+S9 or −S9) exceeded the critical value of 2.0 and all quotients ranged below 1.6, therefore the Ames test did not show any genotoxic potential of BPF, compared to the respective positive controls.

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

Bisphenol F was cytotoxic for the bacteria at the highest concentration tested (500 µg/plate), especially without exogenous activation system. In this study, none of the results of the Ames test (+S9 or −S9) exceeded the critical value of 2.0 and all quotients ranged below 1.6, therefore the Ames test did not show any genotoxic potential of Bisphenol F, compared to the respective positive controls.