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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): not mutagenic

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:
13 December 2013 - 13 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with GLP
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
For the confirmatory assay only two, in stead of three, non-toxic dose levels were available for tester strains TA100 with and without S9, and TA1537 without S9. The deviation did not affect the integrity or interpretability of the results of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine (S. typhimurium) and tryptophan (E. coli)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9-mix) induced by Aroclor 1254
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9-mix) induced by Aroclor 1254
Test concentrations with justification for top dose:
- Initial mutagenicity assay: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate (all strains with and without S9-mix)
- Independent confirmatory assay: 157, 313, 625, 1250 and 2500 µg/plate (all strains with S9-mix) and 50, 100, 200, 400 and 800 (all strains without S9-mix)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: ICR-191: TA1537 (without S9) / 2-aminoanthracene: TA100, TA1535, TA1537 & WP2uvrA (with S9)
Remarks:
All positive control articles were dissolved in dimethyl sulfoxide (DMSO) with the exception of sodium azide which was dissolved in deionized water.
Details on test system and conditions:
METHOD OF APPLICATION: Plate incorporation methodology

DURATION
- Exposure duration: 52 ± 4 hours at ca. 37 ± 2°C

NUMBER OF REPLICATIONS: All determinations were made in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Macroscopic and microscopic (using a dissecting microscope) evaluation of the condition of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity: Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Evaluation criteria:
The test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a dose-related manner, that is =>2-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or =>3-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible. A test substance was considered to be negative in the bacterial gene mutation test if no dose-dependent, =>2-fold or =>3-fold increases was observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
other: S. Typhimurium TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
With S9-mix: toxicity was seen at ≥1250 μg/plate in all strains except TA100 and TA1537 (≥625 μg/plate and at 2500 μg/plate, resp.). Without S9-mix: toxicity was seen at ≥400 μg/plate in all strains except TA100 and TA1537 (toxicity at ≥200 μg/plate).
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: Results are for confirmatory mutagenicity assay
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Toxicity was observed at => 1250 μg/plate in the presence of S9, and at => 400 μg/plate in the absence of S9.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: Results are for confirmatory mutagenicity assay
Species / strain:
other: S. Typhimurium TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
In the presence of S9-mix, cytotoxicity was observed at a concentration of =>1600 μg/plate in all Salmonella tester strains. In the absence of S9, toxicity was observed at =>500 μg/plate in all Salmonella tester strains.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: Results are for initial mutagenicity assay
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: Results are for initial mutagenicity assay
Additional information on results:
The study comprised two studies; an initial mutagenicity test and a confirmatory mutagenicity assay.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in both experiments

HISTORICAL CONTROL DATA
- Positive historical control data: All positive controls were within the acceptance limits as specified in the study report.
- Negative (vehicle) historical control data: All negative controls were within the acceptance limits as specified in the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
During the initial mutagenicity test none of the concentrations tested showed an increase in the number of revertants for the Salmonella typhimurium strains and Escherichia coli strain. However, reduced background lawn growth was observed in S. typhimurium strains in cultures without S9 at 500 µg/plate and in cultures with or without S9 at 1600 µg/plate and 5000 µg/plate. No reduced background was observed in E. coli strain, with or without S9. Based on the results of the initial test, a confirmatory assay (also plate incorporation methodology) was performed. Again no increase in the number of revertants in any strain at any concentration. Reduced background lawn growth was observed with S9 in strain TA100 at 625 µg/plate, at 1250 µg/plate in TA98, TA100, and TA1535 and in all salmonella typhimurium strains at 2500µg/plate. Without S9 reduced background lawn growth was observed at 200 µg/plate in strains TA100 and TA1537 and in all Salmonella typhimurium strains at 400 µg/plate and 800 µg/plate. Reduced background lawn growth was also observed in the E. coli strain with S9 from 1250 µg/plate and without S9 from 400 µg/plate. Per protocol, a minimum of three non-toxic dose levels will be required for a valid assay; however, for the confirmatory assay only two non-toxic dose levels were available for tester strains TA100 with and without S9, and TA1537 without S9. The study deviation, as was stated in the report, neither affected the overall interpretation of study findings nor compromised the integrity of the study.
Conclusions:
Under the conditions of this test, the test substance was considered to be non-mutagenic and therefore the substance does not need to be classified as mutagenic in accordance with the criteria outlined in Annex 1 of the CLP Regulation (1272/2008/EC) and its amendments.
Executive summary:

The test substance was examined for its possible mutagenic activity in the bacterial reverse mutation assay, performed in accordance with OECD TG 471 and GLP. In this assay S. typhimurium strains TA1535, TA1537, TA98, TA100 and the E. coli strain WP2uvrA were used, in the absence and presence of metabolic activation (S9-mix). The test article was evaluated in an initial mutagenicity assay (plate incorporation method) in all strains at doses of 5, 16, 50, 160, 500, 1600, and 5000 µg/plate with and without S9. An independent confirmatory assay (plate incorporation method) was subsequently performed in all strains at dose levels of 157, 313, 625, 1250 and 2500 µg/plate with S9 and at dose levels of 50.0, 100, 200, 400, and 800 µg/plate without S9. Positive and vehicle controls were evaluated concurrently, and all test and control articles were evaluated in triplicate plates. In all strains, the positive and negative controls gave the expected increase in the mean numbers of revertant colonies. Therefore, both experiments were considered valid. In the initial mutagenicity assay no cytotoxicity was observed in WP2uvrA, at any tested dose level in the presence or absence of S9. However, cytotoxicity was observed in all Salmonella strains at ≥1600μg/plate in the presence of S9. In the absence of S9, toxicity was observed at ≥500μg/plate in all Salmonella tester strains. Based on the results of the initial assay, a confirmatory test was performed. In the presence of S9, toxicity was observed at ≥1250μg/plate in all tester strains except TA100 and TA1537. In TA100, toxicity was observed at ≥625μg/plate, whereas in TA1537, toxicity was observed only at 2500μg/plate. In the absence of S9, toxicity was observed at ≥400μg/plate in all tester strains except TA100 and TA1537, where toxicity was observed at ≥200μg/plate. This resulted in only two non-toxic dose levels for TA100 (with and without S9) and TA1537 (without S9), in the confirmatory assay. This is however considered not to influence the integrity and interpretability of the study. In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose-related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed of the negative control. It is therefore concluded that the test substance is not mutagenic under the conditions of this test.

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

Additional information

Genotoxicity in Ames: The test substance was examined for its possible mutagenic activity in the bacterial reverse mutation assay, performed in accordance with OECD TG 471 and GLP. In this assay S. typhimurium strains TA1535, TA1537, TA98, TA100 and the E. coli strain WP2uvrA were used, in the absence and presence of metabolic activation (S9-mix). The test article was evaluated in an initial mutagenicity assay (plate incorporation method) in all strains at doses of 5, 16, 50, 160, 500, 1600, and 5000 µg/plate with and without S9. An independent confirmatory assay (plate incorporation method) was subsequently performed in all strains at dose levels of 157, 313, 625, 1250 and 2500 µg/plate with S9 and at dose levels of 50.0, 100, 200, 400, and 800 µg/plate without S9. Positive and vehicle controls were evaluated concurrently, and all test and control articles were evaluated in triplicate plates. In all strains, the positive and negative controls gave the expected increase in the mean numbers of revertant colonies. Therefore, both experiments were considered valid. In the initial mutagenicity assay no cytotoxicity was observed in WP2uvrA, at any tested dose level in the presence or absence of S9. However, cytotoxicity was observed in all Salmonella strains at ≥1600μg/plate in the presence of S9. In the absence of S9, toxicity was observed at ≥500μg/plate in all Salmonella tester strains. Based on the results of the initial assay, a confirmatory test was performed. In the presence of S9, toxicity was observed at ≥1250μg/plate in all tester strains except TA100 and TA1537. In TA100, toxicity was observed at ≥625μg/plate, whereas in TA1537, toxicity was observed only at 2500μg/plate. In the absence of S9, toxicity was observed at ≥400μg/plate in all tester strains except TA100 and TA1537, where toxicity was observed at ≥200μg/plate. This resulted in only two non-toxic dose levels for TA100 (with and without S9) and TA1537 (without S9), in the confirmatory assay. This is however considered not to influence the integrity and interpretability of the study. In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose-related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed of the negative control. It is therefore concluded that the test substance is not mutagenic under the conditions of this test.

Genotoxicity in vitro micronucleus test (OECD TG 487): A negative result from an in vitro micronucleus test is available. This negative result further supports the absence of genotoxicity strategy and therefore the results are not summarised.

Justification for classification or non-classification

Based on the results of the Ames test, the test substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments.