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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (similar 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:
17 October 2014 - 04 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Species / strain:
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:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 4.8, 19.5, 78.1, 313, 1250 and 5000 µg/plate

- Main test:
Due to cytotoxicity observed in the dose range finding test at 1250 µg/plate in all tester strains with and without S9 mix, the following dose levels were used: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
Vehicle:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate
Solvent controls:
yes
Remarks:
(100 µL/plate DMSO)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and conditions:
METHOD OF APPLICATION:
- in agar (pre-incubation method)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Triplicate plates were used for the negative control group, and duplicate plates were used for the positive control and the test substance treatment groups.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibition
Evaluation criteria:
The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible.
Statistics:
No statistical methods were used.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
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:
- In all strains toxicity was observed at 1250 µg/plate and above, both in the absence and presence of S9 in the dose range finder.

COMPARISON WITH 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:
- Bacterial growth inhibition was observed at 625 µg/plate and above in TA100, T A1535, TA98 and TA1537, and at 1250 µg/plate in WP2uvrA with and without S9 mix.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed similar to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated similar to OECD TG 471 (1997) and according to GLP principles. A dose range finding test was performed followed by a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in the dose range dfinding test (>=1250 µg/pl). Adequate negative and positive controls were included. 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 (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic 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 similar to OECD TG 471 (1997) and according to GLP principles. A dose range finding test was performed followed by a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in the dose range finding test (>=1250µg/pl). Adequate negative and positive controls were included.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 (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic 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 Regulation (EC) No. 1272/2008.