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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In a bacterial reverse mutataion assay (Ames test) employing four strains of Salmonella typhimurium with and without metabolic activation no relevant increase of revertant colonies was recorded.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline Study, GLP
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
4 strains tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9
Test concentrations with justification for top dose:
0,8; 4; 20; 100; 500; 2500; 5000 µg/plate
Vehicle:
DMSO
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2 aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: microscopical evaluation of thining of bacterial lawn
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency.
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range.

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn.
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
The test results must be reproducible.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 500 µg/plate and above
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item is not mutagenic in these bacterial test systems either in the absence or in the presence o,f an exogenous metabolizing system.
Executive summary:

The test compound was suspended in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 2500, 500, 100, 20 and 4 µg/plate were used in the first experiment.

Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above. Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 2500 µg/plate and lower doses.

The test compound proved to be toxic to most of the bacterial strains at concentrations of 500 µg/plate and above. Thinning of bacterial lawns and in most cases also a reduction in the number of colonies were observed at these doses.

Because of heavy precipitation in the first experiment dose ranges from 0.8 to 2500 µg/plate were chosen for the second experiment. The toxic effects were in most cases reproduced with the Salmonella typhimurium strains in this experiment.

A toxicity test using histidine-enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D) was performed in parallel with the second experiment. Toxicity was found at concentrations of 500 µg/plate and above in the absence of metabolic activation. In the presence of metabolic activation the test compound proved to be not toxic to the bacterial strain.

Mutagenicity

In all independent mutation tests the test item was tested for mutagenicity with the same concentrations as described above. The number of colonies per plate with each strain as well as mean values of 3 plates are given. The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S9-mix in either mutation test. No dose-dependent effect was obtained.

All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

CONCLUSION

The results lead to the conclusion that the test item is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.

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

Additional information

Additional information from genetic toxicity in vitro:

In a bacterial reverse mutataion assay (Ames test) employing four strains of Salmonella typhimurium with and without metabolic activation no relevant increase of revertant colonies was recorded.

The test material is considered not mutagenic.

This finding was also confirmed in an in vivo mouse micronucleus assay.


Justification for selection of genetic toxicity endpoint
only required study

Justification for classification or non-classification

no classification

In a bacterial reverse mutataion assay (Ames test) employing four strains of Salmonella typhimurium with and without metabolic activation no relevant increase of revertant colonies was recorded.

The test material is considered not mutagenic.