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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The submission substance was not mutagenic in a guidline conform Salmonella typhimurium reverse mutation assay (Ames test) using the five tester strains TA 100, TA 1535, TA 1537, TA 1538, TA 98, and Escherichia coli WP2uvrA with and without metabolic activation (S9-mix from induced rat liver). Likewise, the submission substance was also not mutagenic in a guideline compliant mammalian cell gene (HPRT) mutation assay in V79 Chinese hamster cells and did not induce chromosome aberrations or clastogenic effects in a guideline conform cytogenetic study in V79 cells in vitro with and without metabolic activation.
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:
1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform study according to GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine and tryptophan reversion
Species / strain / cell type:
other: TA 100, TA 1535, TA 1537, TA 1538, TA 98, WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S 9 from rat liver
Test concentrations with justification for top dose:
0; 4; 20; 100; 500; 2500; 5000 µg/plate / first experiment with and without metaboloc activation0; 0.16; 0.8; 4; 20; 100; 500 µg/plate / second experiment with and without metabolic activation
Vehicle / solvent:
Deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: N-Methyl-N'-nitro-N~nitrosoguanidine (MNNG):
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with metabolic activation
Evaluation criteria:
In the Ames test with Salmonella typhimurium strains the effect of the test compound upon the number of back mutations to histidine prototrophy using histidine auxotrophic mutants is investigated. Using Escherichia coli WP2uvrA, a tryptophan dependent auxotroph strain, mutagenicity is based on -reversion to tryptophan independence. The strains TA 100 and TA 1535 were originally derived by a substitution mutation, the strains TA 1537, TA 1538 and TA 98 by frame shift mutations from histidine prototrophic bacteria. All five Salmonella strains are deficient in the complete structure of their lipopolysaccharide layer and in DNA excision repair system. TA 98 and TA 100 possess a modified postreplication DNA repair system which frequently causes an increase in the rate of mutations. Strain WP2uvrA carries a defect in one of the genes for tryptophan biosynthesis and is deficient in the uvrA system of DNA repair. The reversion can beinduced by a base change (substitution) .
Species / strain:
other: TA 100, TA 1535, TA 1537, TA 1538, TA 98, WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activationThe diluted test item (concentration 78 %; details see confidential details on test material) was tested for mutagenicity with Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 and E. coli WP2uvrA in the absence and presence of a metabolic activation system.The diluted test item did not cause a significant increase in the number of revertant colonies with any of the tester strains neither in the absence nor presenceof 5-9 Mix. No dose dependent effect was obtained. It is concluded that the test substance is toxic but not mutagenic in these bacteria1 test systems neither in the absence nor in the presence of an exogenous metabolizing system.
Executive summary:

The diluted test item (concentration 78 %; details see confidential details on test material) was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4-10 000 (1. experiment) and 0.16 µg/plate to 500 µg/plate

(2. experiment) was used. Control plates without mutagen showed that the number. of spontaneous revertant colonies was similar to that described in the literature. All the positiv control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be very toxic to most of the bacterial strains at 20 or 100 µg/plate. For this reason 500 µg/plate was chosen as top dose level for the mutagenicity study in the repeat experiment.

Mutagenicity: In the absence of the metabolic activation system -the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the diluted did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that the diluted test item is toxic but not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.

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

Additional information

Additional information from genetic toxicity in vitro:

The registration substance was tested for potential point mutation in a guideline conform bacterial reverse mutation assay (Ames test) according to OECD TG 471 with and without metabolic activation. Independent experiments using several test concentrations up to the limit dose of 5000 µg/plate did not cause gene mutations by base pair changes or frameshifts in the genome of any of the tester strains used. Therefore, the submission substance is considered to be non-mutagenic in this bacterial reverse mutation assay. This study was selected as key study.

The submission substance was tested for potential gene mutation in a guideline conform mammalian cell gene mutation assay (HPRT locus) according to OECD TG 476 in V79cells of the Chinese hamster. Independent experiments were performed using several test concentrations up to the limit of5000 µg/mL (with and without metabolic activation). No biologically relevant increase of mutants was found after treatment with the test item, neither with nor without metabolic activation. No dose-response relationship was obeserved. Therefore, the submission substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster. This study was selected as key study.

The submission substance was tested for the potential to induce structural chromosome aberrations in an guideline conform in vitro cytogenetic assay in Chinese hamster V79 cells according to OECD TG 473. Two independent experiments with and without metabolic activation were carried out using test item concentrations up to the limit of 5000µg/mL. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative controls. No biologically relevant increase of the aberration rates and no biologically relevant increase in the frequencies of polyploid cells were noted after treatment with the test item with as compared to the controls. EMS and CPA were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The test item did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line and is thus considered to be non-clastogenic in this test. This study was selected as key study.

In conclusion, the submission substance is found to be not mutagenic in the bacterial reverse mutation assay, the mammalian (HPRT) mutation test in V79 cells and in the in vitro chromosome aberration test in V 79 cells.


Justification for selection of genetic toxicity endpoint
There are several key studies covering bacterial point mutation testing, and testing for gen mutations and chromosome mutations in mammalian cell systems.

Justification for classification or non-classification

Based on the available data from three independent mutagenicity assays, a respective mutagenic potential of the test item can most probably be excluded. Thus, the registration substance does not have to be classified for mutagenicity in accordance with the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) as well as in the EU Classification, Labellling and Packaging Regulation (1272/2008/EC).