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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames Test (OECD 471, GLP, K, rel. 2): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98 & TA 100.
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:
From October 25 to November 19, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted according to OECD 471 Guideline (1981 version where only 4 strains were tested) with deviations from current Test Guideline: 4 strains only tested (TA 102 or E.coli WP2 strain not tested); historical vehicle/positive control data not reported
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
4 strains only tested (TA 102 or E.coli WP2 strain not tested); historical vehicle/positive control data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % v/v S9 mix; S9 from liver of CD (Sprague- Dawley derived) rats injected with Aroclor 1254
Test concentrations with justification for top dose:
Dose range finding test: 5, 50, 500 and 5000 µg/plate in TA 1535, TA 1537, TA 1538, TA 98 and TA 100 strains, with or without S9-mix

Mutation test:
Test 1: 50, 150, 500, 1500 and 5000 µg/plate in TA 1535, TA 1537, TA 98 and TA 100 strains, with or without S9-mix
Test 2: 50, 150, 500, 1500 and 5000 µg/plate in TA 1535, TA 1537, TA 98 and TA 100 strains, with or without S9-mix

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of substantial reduction in revertant colony counts or absence of a complete background bacterial lawn.

OTHER:
- Revertant colonies were counted using a Biotran Colony Counter.
Evaluation criteria:
A compound is deemed to provide evidence of mutagenic potential if:
- a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments and;
- the increase in the number of revertant colonies is at least twice the concurrent solvent control value.
Statistics:
None
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: The test material was not toxic towards any of the tester strain.

OTHERS:
- Test material and S9 mix used in this experiment was shown to be sterile.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See the attached document for information on tables of results

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) according to the criteria of the Annex VI of the of the Regulation (EC) No 1272/2008 (CLP).
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test material diluted in DMSO using the plate incorporation method. The dose range for the mutation test was determined in a dose range finding test and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose levels. Vehicle (DMSO) and positive control groups were also included in mutagenicity tests. 

Test material was not toxic towards any of the tester strains in any of the experiments. The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No substantial increase in the frequency of revertant colonies was recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A key study was identified (Huntington, 1985, Rel.2). In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test material diluted in DMSO using the plate incorporation method at up to 5000 μg/plate. No substantial increase in the frequency of revertant colonies was recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. The test material does not induce gene mutations in bacteria under the test conditions whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The test material is therefore considered as non-mutagenic according to the Ames test.

Justification for selection of genetic toxicity endpoint
Only one study available, GLP-compliant and of high quality (Klimisch score = 2). No further testing is required for substances at the REACH Annex VII tonnage level.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including ATP6.

Self classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).