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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Description: Colourless liquid
- Storage conditions: Room temperature in the dark
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Due to cytotoxicity in an initial toxicity test, the following dose levels were used in experiment 1 and 2:
Experiment 1: 5, 15, 50, 150, 500 and 1500 μg/plate
Experiment 2: 0.5, 1.5, 5, 15, 150 and 500 μg/plate (without S9)
Experiment 2: 5, 15, 50, 150, 500 and 1500 μg/plate (with S9)
Vehicle:
DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene (2AA) and 1,8-dihydroxyanthraquinone (DAN)
Details on test system and conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies, determination of background lawn
Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.9E9 bacteria per mL.
- Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.
The test material may be considered positive in this test system if the test material has induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
Preliminary Toxicity Test
The test material exhibited toxicity to TA100 from 500 µg/plate employing the plate incorporation method. A clear toxic response was also noted employing the pre-incubation method with weakened lawns initially observed at 50 and 500 µg/plate in the absence and presence of S9, respectively. The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile.

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9-mix used in both experiments was shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains employing the direct plate incorporation method, initially at 500 and 1500 µg/plate without and with S9, respectively. In Experiment 2 (pre-incubation method), the test material exhibited toxicity from 150 and 500 µg/plate without and with S9, respectively. The test material was, therefore, tested up to the toxic limit. An oily precipitate was observed from 1500 µg/plate under an inverted microscope, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a bacterial reverse mutation assay, performed according to OECD Guideline 471 and following GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA102 were exposed to the test material in DMSO (vehicle) using the plate incorporation and pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). Test concentrations used ranged from 0.5 to 1500 µg/plate. These concentrations are based on the results of a preliminary test. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains employing the direct plate incorporation method, initially at 500 and 1500 µg/plate without and with S9, respectively. In Experiment 2 (pre-incubation method), the test material exhibited toxicity from 150 and 500 µg/plate without and with S9, respectively. The test material was, therefore, tested up to the toxic limit. An oily precipitate was observed from 1500 µg/plate under an inverted microscope, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. The results of the vehicle and positive controls show the validity of the test. It was concluded that the test substance did not increase the number of revertants in the presence and absence of S9 -mix.

Justification for classification or non-classification

The test substance does not have to be classified for mutagenicity according to Regulation (EC) No 1272/2008.