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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames Test: The test substance was not mutagenic in a bacterial reverse mutation assay. Chromosome aberration test: The test substance was not clastogenic in an in vitro chromosome aberration assay. Mouse lymphoma gene mutation assay: The test substance was not mutagenic in an in vitro mammalian cell gene mutation assay.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted according to OECD Guideline and in compliance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, Phenobarbital/ß-naphthoflavone induced
Test concentrations with justification for top dose:
Cytotoxicity test concentrations:
1.22, 4.88, 19.53, 78.13, 312.5, 1250 and 5000 µg/mL (3-hour treatment period with and without S9)
0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL (24-hour treatment period without S9)

Genotoxicity test concentrations:
1st experiment: 0, 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL (3-hour treatment period, with and without S9)
2nd experiment: 0, 0.313, 0.625, 0.938, 1.25, 2.5 and 5 µg/mL (24-hour treatment period, without S9),
0, 2.5, 5, 10, 15, 20 and 30 µg/mL (3-hour treatment period, with S9)
Vehicle / solvent:
- Vehicle/solvent used: R0 medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9-mix: Cyclophosphamide, without S9-mix: Ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours and 3 hours

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Treated cultures were examined for a significant increase in mutant frequency.
Statistics:
UKEMS statistical package
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
treatment period / test concentration / decrease in relative suspension growth: without S9-mix: 3 h / 20 µg/mL / 98 %; with S9-mix: 3 h / 20 µg/mL / 70 and 73 %; without S9-mix: 24 h / 5 µg/mL / 91 %
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed at 20 µg/mL with and without S9-mix (3 hour treatment period) and at 5 µg/mL without S9 (24-hour treatment period)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test:

In the bacterial reverse mutation assay according to OECD Guideline 471 (1983) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 the test material (33 % w/w active substance in aqueous solution) was tested at concentrations of 0, 1, 3, 10, 32, 100 µg/plate with and without metabolic activation (S9-mix). In the presence of the metabolic activation system, 2 -aminoanthracene (strain TA 1535) and benzo[a]pyrene (all other strains) were used as positive controls. Positive controls in the absence of S9-mix were sodium azide (strains TA1535 and TA100), 2-aminoanthracene (strain TA1535), 9-aminoacridine (strain TA1537), 2-nitrofluorene (strain TA98) and benzo[a]pyrene (strains TA1537, TA100 and TA98). Distilled water served as negative and as vehicle control. Cytotoxicity (absence or thinning of background lawn and reduction in colony numbers) was observed at 100 µg/plate with and without S9-mix. As a result, no increase in the reverse mutation rate was observed when tested up to cytotoxic concentrations. Therefore, the test material was considered to be not mutagenic in the bacterial reverse mutation assay.

 

Chromosome aberration test:

A study was conducted according to OECD Guideline 473 to assess the clastogenic potential of the test substance in vitro in mammalian cells. Therefore, cultured human lymphocytes were used. Test concentrations were 0, 0.4, 2, 10 µg/mL (24-hour treatment period without S9-mix) and 0, 2, 10 and 50 µg/mL (3-hour treatment period with S9-mix). Chlorambucil served as positive control in the absence and Cyclophosphamide in the presence of a metabolic activation system. The cells were evaluated for genotoxicity by evaluating the mean aberrant cell frequencies. The cells were also evaluated for polyploidy. Cytotoxicity was evaluated by determination of the mitotic index. As a result, cytotoxicity was observed at 10 µg/mL both with and without metabolic activation. There was no increase in the aberrant cell frequencies, therefore the test item was concluded to be not clastogenic in this assay.

 

Mouse lymphoma gene mutation assay:

To assess the potential of the test material to induce gene mutations in vitro in mammalian cells, a study (according to OECD 476 (1997) “In vitro mammalian cell gene mutation test”) was conducted. Two independent tests were performed with duplicate cultures. The following test concentrations were used:

1st Experiment: 0, 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL (3-hour treatment period, with and without S9)

2nd Experiment: 0, 0.313, 0.625, 0.938, 1.25, 2.5 and 5 µg/mL (24-hour treatment period, without S9),

                             0, 2.5, 5, 10, 15, 20 and 30 µg/mL (3-hour treatment period, with S9)

As positive controls, Cyclophosphamide (with S9-mix) and Ethylmethanesulphonate (without S9-mix) were used.

Cytotoxicity was observed at 20 µg/mL with and without metabolic activation (3 hour treatment period) and at 5 µg/mL without metabolic activation (24-hour treatment period). No increase in mutant frequencies were observed, therefore the test substance was considered to be not mutagenic.


Justification for selection of genetic toxicity endpoint
Genetic toxicity was evaluated on the basis of three studies, adressing gene mutation in bacteria, gene mutation in mammalian cells and clastogenicity in mammalian cells, respectively. All available studies were considered reliable and suitable for classification.

Justification for classification or non-classification

In the available in vitro studies the test substance did not induce gene mutations in bacteria and in mammalian cells and was also not clastogenic in mammalian cells. Therefore the substance is considered not to be genotoxic under Regulation (EC) No 1272/2008 (CLP).