Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Mutagenic activity of the substance was investigated in one bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A), in an in vitro gene mutation study in mammalian cells (Chinese Hamster V79 cells), in one in vitro chromosome aberration study in Chinese Hamster V79 cells and in an in vivo micronucleus test in mice which was performed to be compliant with Chinese Regulation. Negative results were obtained in all studies with and without metabolic activation.
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
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 476; GLP conform
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (only without metabolic activation):
0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10 mM
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mM
Experiment I
without metabolic activation: 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
and with metabolic activation: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 6.25, 7.5, 8.75 and 10 mM
Experiment II
without metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 1.4, 1.6, 1.8 and 2.0 mM
and with metabolic activation: 0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 mM
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was suspended in cell culture medium and processed by ultrasound for 15 min.
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: 300 µg/mL
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation

Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 2.5 mM; Experiment II without S9: ≥ 0.25 mM
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Under the test conditions described above, the test item is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

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

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

The substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The selection of the concentrations was based on data from the pre-experiments. In experiment I 10 mM (with and without metabolic activation) was selected as the highest concentration. In experiment II 2.0 mM (witout metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations. Experiments I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

without metabolic activation:

0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 5.0, 7.5 and 10 mM

and with metabolic activation:

0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 6.25, 7.5, 8.75 and 10 mM

Experiment II

without metabolic activation:

0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 1.4, 1.6, 1.8 and 2.0 mM

and with metabolic activation:

0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 mM

Precipitation of the test item was noted in experiment I and II.

Biologically rlevant growth inhibition was observed in experimen I and II only without metabolic activation. In experiment I without metabolic activation the relative growth was 62.8%, with metabolic activation 130.3% for the highest concentration (10 mM) evaluated. In experiment II without metabolic activation the relative growth was 19.0%, with metabolic activation 80.4% for the highest concentration (10 mM) evaluated.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

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

Additional information

Additional information from genetic toxicity in vitro:

The substance showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

The substance also yielded negative results in an in vitro gene mutation study in mammalian cells in concentration up to 10 mM.

The substance was assessed for its potential to induce chromosome aberrations in Chinese Hamster V79 cells in vitro. The test item did not induce chromosome aberrations. Therefore, the substance is considered to be non-mutagenic in this in vitro chromosome aberration test when tested up to 5 mM.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in murine peripheral blood cells when the substance was applied up to 2000 mg/kg bw.


Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalian cells and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 476 and GLP.

Justification for classification or non-classification

In conclusion, the substance is not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study in Chinese V79, the in vitro chromosome aberration in the presence and absence of metabolic activation and in the in vivo micronucleus test in mice up to the tested concentrations.

The substance does not have to be not classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assay (up to 10 mM) in the in vitro chromosome aberration study in concentrations up to 5 mM or in the in vivo micronucleus test in mice up to 2000 mg/kg bw.