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Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (OECD 471): Positive

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Nov 2016 to 24 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name as cited in study report: Orasol Red 335
- Batch identification: 002-141706
- Content: 96 g/100 g (100 g/100 g minus water content)
- Physical state / colour: Solid / red
- Storage conditions: Room temperature
Target gene:
S. typhimurium: his
E. coli: trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital (i.p.) and β-naphthoflavone (oral) induced Wistar rat livers
Test concentrations with justification for top dose:
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.3 mg/plate was used as top dose in all experiments.

EXPERIMENT 1 (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA)
with and without S9: 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate

EXPERIMENT 2 (TA 100, TA 1537 and TA 98)
with and without S9: 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

OTHER
- To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION
EXPERIMENT 1 and 2: Standard plate test (plate incorporation method)

DURATION
- Exposure duration: 48 to 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS
- 3 test plates per dose or per control

EVALUATION
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.
- Toxicity: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
- Solubility: If precipitation of the test material was observed, it would be recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.
Rationale for test conditions:
Mutagenicity was observed in the standard plate test. Therefore, the standard plate test was repeated instead of the prival modification.
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect was observed in the standard plate test without S9 mix at the tester strain E. coli at 2650 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:
A weak bacteriotoxic effect was observed in the standard plate test without S9 mix at the tester strain TA 1535 at 5300 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 100, TA 1537 and TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Test substance precipitation was found from about 333 μg/plate onward.

ADDITIONAL INFORMATION ON MUTAGENICITY
- TA 100 without S9 mix: 1st Experiment: Increase in the number of his+ revertants at concentrations of 2650 and 5300 μg/plate (factors 2.2 and 2.2, respectively). 2nd Experiment: Increase in the number of his+ revertants at a concentration of 2650 μg/plate (factor 2.3).
- TA 1537 without S9 mix: 1st Experiment: Increase in the number of his+ revertants at concentrations of 1000 and 2650 μg/plate (factors 3.4 and 3.3, respectively). 2nd Experiment: Increase in the number of his+ revertants at concentrations of 2650 and 5300 μg/plate (factors 3.2 and 3.7, respectively).
- TA 98 without S9: 1st Experiment: Increase in the number of his+ revertants at concentrations of 33, 100, 333, 1000, 2650 and 5300 μg/plate (factors 2.7, 4.2, 8.3, 13.0, 20.7 and 17.8, respectively). 2nd Experiment: Increase in the number of his+ revertants at concentrations of 33, 100, 333, 1000, 2650 and 5300 μg/plate (factors 2.3, 4.2, 5.5, 5.0, 4.2 and 6.7, respectively).
- TA 100 with S9 mix: 1st Experiment: Increase in the number of his+ revertants at a concentration of 2650 μg/plate (factor 2.5). 2nd Experiment: Increase in the number of his+ revertants at a concentration of 2650 μg/plate (factor 2.1).
- TA 1537 with S9 mix: 1st Experiment: Increase in the number of his+ revertants at concentrations of 333, 1000, 2650 and 5300 μg/plate (factors 3.3, 8.2, 7.0 and 7.0, respectively).
- TA 98 with S9 mix 1st Experiment: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2650 and 5300 μg/plate (factors 2.9, 9.3, 14.4, 10.6 and 8.0, respectively). 2nd Experiment: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2650 and 5300 μg/plate (factors 4.9, 8.3, 4.1, 3.2 and 3.0, respectively).

Additional information

OECD471 (Ames test)

In an Ames test performed according to OECD 471 the mutagenic potential of the test substance was evaluated in the bacterial strains S. typhimurium TA 1535, TA 100, TA 1537 and TA 98 and E. coli WP2 uvrA with and without metabolic activation (S9 mix from phenobarbital and β-naphthoflavone induced rats livers). The test substance was tested in triplicate at 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate in the standard plate test using DMSO as vehicle. In the second experiment this procedure was repeated for strains TA 100, TA 1537 and TA 98 because mutagenicity was observed in the first experiment. Therefore, the standard plate test was repeated instead of the prival modification. Test substance precipitation was found from about 333 μg/plate onward. A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was observed in the standard plate test without S9 mix at the tester strain TA 1535 at 5300 μg/plate and at the tester strain E. coli at 2650 μg/plate. An evident and partly dose dependent increase in the number of his+ revertants with the tester strains TA 100,TA 1537 and TA 98, all with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. According to the results of the present study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen (BASF 2017).

Justification for classification or non-classification