Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (equivalent to OECD TG 471): negative

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:
28 July, 2006 - 10 August, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Rat liver S9-mix induced by a combination of phenobarbital and 5, 6-benzoflavone.
Test concentrations with justification for top dose:
- Dose range finding test 1:
All 5 strains without and with S9-mix: 4.88, 19.5, 78.1, 313, 1250 and 5000 μg/plate

- Dose range finding test 2:
Strain TA98, TA100, TA1535 and TA1537 without and with S9-mix and WP2uvrA without S9-mix: 9.77, 19.5, 39.1, 78.1, 156 and 313 μg/plate

- Main test:
Strain TA98, TA100, TA1535 and TA1537 without and with S9-mix and WP2uvrA without S9-mix: 9.77, 19.5, 39.1, 78.1, 156 and 313 μg/plate
Strain WP2uvrA with S9-mix: 39.1, 78.1, 156, 313, 625 and 1250 μg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was insoluble in distilled water at 50 mg/mL but it was soluble in DMSO at 50 mg/mL.
Negative solvent / vehicle controls:
yes
Remarks:
(100 µL/plate DMSO)
Positive controls:
yes
Positive control substance:
sodium azide
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF CELLS EVALUATED: 2.5 - 4.1x10^9 cells/mL

NUMBER OF REPLICATIONS:
- Doses of the test substance and positive control were tested in duplicate in each strain, the negative control was tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibition under a stereomicroscope.
Evaluation criteria:
The test substance was judged to be positive when the number of revertant colonies increased to twice or more that in the negative control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all other cases, it was judged negative.
Statistics:
Not performed.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- Dose range finding test 1: The bacterial growth inhibition was observed at and above 313 µg/plate in all test strains without S9-mix and at and above 313 µg/plate in TA100, TA1535, TA98 and TA1537 with S9-mix and at and above 1250 µg/plate in WP2uvrA with S9-mix.
- Dose range finding test 2: The bacterial growth inhibition was observed at and above 156 µg/plate in all test strains without S9-mix and at 313 µg/plate in TA100, TA1535, TA98 and TA1537 with S9-mix.

HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Main test: The bacterial growth inhibition was observed at and above 156 µg/plate in all test strains without S9-mix and at 313 µg/plate in TA100, TA1535, TA98 and TA1537 with S9-mix and at and above 625 µg/plate in WP2uvrA with S9-mix.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed equivalent to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated equivalent to OECD 471 guideline and according to GLP principles. The test was performed in three independent pre-incubation experiments, both in the absence and presence of S9-mix up to and including 5000 μg/plate in the first dose range finding test. In the second dose range finding test and the main test, the test substance was tested up to and including the cytotoxic concentration of 313 μg/plate in the absence and presence of S9 –mix. Except WP2uvrA with S9-mix which was not tested in the second dose range finding test and was tested in the main test up to and including 1250 μg/plate. No precipitation was observed up to and including the top dose of 5000 µg/plate.

Adequate negative and positive controls were included in all three experiments. In the main test, the bacterial growth inhibition was observed at and above 156 µg/plate in all test strains without S9-mix and at 313 µg/plate in TA100, TA1535, TA98 and TA1537 with S9-mix and at and above 625 µg/plate in WP2uvrA with S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E. coli strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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

Additional information

Ames test:

The mutagenic activity of the substance was evaluated equivalent to OECD 471 guideline and according to GLP principles. The test was performed in three independent pre-incubation experiments, both in the absence and presence of S9-mix up to and including 5000 μg/plate in the first dose range finding test. In the second dose range finding test and the main test, the test substance was tested up to and including the cytotoxic concentration of 313 μg/plate in the absence and presence of S9 –mix. Except WP2uvrA with S9-mix which was not tested in the second dose range finding test and was tested in the main test up to and including 1250 μg/plate. No precipitation was observed up to and including the top dose of 5000 µg/plate.

Adequate negative and positive controls were included in all three experiments. In the main test, the bacterial growth inhibition was observed at and above 156 µg/plate in all test strains without S9-mix and at 313 µg/plate in TA100, TA1535, TA98 and TA1537 with S9-mix and at and above 625 µg/plate in WP2uvrA with S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E. coli strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP Regulation (EC) No. 1272/2008 and its amendments.