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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): Negative with and without metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 18 August 2014 and 20 September 2014
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)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine
- E. coli: Tryptophan
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 Aroclor 1254
Test concentrations with justification for top dose:
INITIAL TEST:
- TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 5000, 1500, 500, 150, 50, 15, 5.0, 1.5 µg/plate

CONFIRMATORY TEST:
- TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 5000, 1500, 500, 150, 50, 15, 5.0 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Dimethyl sulfoxide (DMSO) was selected based on the solubility of the test substance and compatibility with the target cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
A detailed overview of positive control substances per strain is included in "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

NUMBER OF REPLICATIONS:
- Initial test: Vehicle control, positive controls and dose levels of the test substance were plated, two plates per dose.
- Confirmatory test: All dose levels of test substance, vehicle control and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Thinning of the background lawn or a microcolony formation
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.
- Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value.
- Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
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
Untreated negative 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative 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
Untreated negative 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.
- Precipitation: No precipitate was observed in any of the experiments.
- Other confounding effects: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based on these findings, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

HISTORICAL CONTROL DATA
The negative and strain-specific positive control, mean revertants per plate were within the laboratory historical control data ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Cytotoxicity results: Toxicity was observed beginning at 1500 or at 5000 μg per plate in both tests.
Conclusions:
A bacterial reverse mutation assay was conducted according to OECD 471 guideline and GLP principles. It is concluded that the substance is not mutagenic under the conditions of the test.
Executive summary:

The mutagenic activity of the substance was evaluated according to OECD TG 471 and according to GLP principles. The test was performed with the plate incorporation method, both in the absence and presence of S9-mix. The dose levels were selected based on an initial test with doses up to 5000 µg/plate. As a result, S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) and E.coli WP2 uvrA were exposed to test substance concentration of 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate in the confirmatory test. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation (both experiments). Negative and positive controls were included and were found to be adequate. Based on the results of this study, it is concluded that the substance is not mutagenic with and without metabolic activation.

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

Additional information

Ames test

The mutagenic activity of the substance was evaluated according to OECD TG 471 and according to GLP principles. The test was performed with the plate incorporation method, both in the absence and presence of S9-mix. The dose levels were selected based on an initial test with doses up to 5000 µg/plate. As a result, S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) and E.coli WP2 uvrA were exposed to test substance concentration of 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate in the confirmatory test. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation (both experiments). Negative and positive controls were included and were found to be adequate. Based on the results of this study, it is concluded that the substance is not mutagenic with and without metabolic activation.

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in EU CLP (EC no 1272/2008 and its amendments).