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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 98, TA 100, TA 1535, TA 1537 and TA 102 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:
2011-07-07 to 2011-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest.
Untreated negative controls:
yes
Remarks:
A. dest., BSL Lot No. 110526, 110617
Negative solvent / vehicle controls:
yes
Remarks:
A. dest., BSL Lot No. 110526, 110617
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment II
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I.

Toxic effects of the test item were noted in tester strains TA 98, TA 100, TA 1535 and TA 1537 in experiment II at a concentration of 5000 µg/plate (without metabolic activation).

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations, a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. The submission substance was tested in two independent experiments at concentrations of 31.6, 100, 316, 1000, 2500 up to the limit of 5000 µg/plate with and witout metabolic activation. All concentrations were tested in triplicate.

No toxic effects of the test item were noted in any of the tester strains up to the highest dose evaluated with and without metabolic activation in experiment I. In experiment II, cytotoxicity were noted in tester strains TA 98, TA 100, TA 1535 and TA 1537 without metabolic activation at the limit concentration of 5000 µg/plate.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed folowing teratment at any concentration level, neither in the presence nor in the absence of metabolic activation in experiment I and II. The positive controls induced distinct increases of revertant colonies indicating the validity of the experiment.

In conclusion it can be stated that the test item 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.

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

Additional information

Additional information from genetic toxicity in vitro:

The submission 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 of 5000 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 submission substance do 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).