[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxic properties of a structural analogue substance were assessed with the following outcomes:

Ames Test: Negative;

HPRT Assay: Negative;

Chromosome Aberration Assay: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to read-across justification attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to read-across justification attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In both experiments, clear cytotoxicity between 54- 60% was observed at the highest concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to read-across justification attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence and presence of metabolic activation clear cytotoxicity of the test item was observed at the highest concentration applied (60 µg/mL in the absence and 40 µg/mL in the presence of S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro genotoxicity tests are available with a structural analogue substance. For detailed read-across justification please refer to attached document in IUCLID Section 13.

Chromosme Aberrration Assay:

The test item dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments five concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

Experiment Awith 3/20 h treatment/sampling time

without S9 mix: 0,12.5, 25, 50 and 100μg/mL test item

with S9 mix: 0,12.5, 25, 50 and 100μg/mL test item

Experiment Bwith 20/20 h treatment/sampling time

without S9 mix: 0,1.6, 3.2, 6.3 and 12.5μg/mL test item

Experiment Bwith 20/28 h treatment/sampling time

without S9 mix: 0,1.6, 3.2, 6.3 and 12.5μg/mL test item

Experiment Bwith 3/28 h treatment/sampling time

with S9 mix: 0,12.5, 25, 50 and 100μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts for the highest test concentrations. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

HPRT Assay:

The test item, dissolved in Ham's F12 medium, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: Mutation Assay 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50, 60 and 70 µg/mL Mutation Assay 5-hour treatment period with S9-mix: 5, 10, 20, 30 and 40 µg/mL. This concentration was tested but was very toxic and there were not enough cells to start the phenotypic expression period after the treatment.In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity.In the absence and presence of metabolic activation clear cytotoxicity (survival between 15-17 %) of the test item was observed at the highest concentration applied (60 µg/mL in the absence and 40 µg/mL in the presence of S9 mix). Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit). The mutation frequency found in the solvent controls was in the 95 % confidence interval of the historical control data. The concurrent positive controlsethyl methanesulfonate(1.0 µL/mL) and7, 12-dimethyl benzanthracene(20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.The test itemtested up to cytotoxicconcentrationswith and without metabolic activationover a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency. It is concluded that the test item was not mutagenic in thisin vitromammalian cell gene mutation test performed with Chinese hamster ovary cells.

Ames Test:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a preliminary solubility tests, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I). At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration. Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate.

In the preliminary concentration range finding test strong inhibitory effect of the test item was observed at the recommended maximum test concentration of 5000 μg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study. In the initial and confirmatory mutation tests unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn), affected colony development (pinpoint colonies) and decreased revertant colony counts (absent revertants or revertants below the historical control data ranges and/or corresponding vehicle control data ranges). In general, 320 μg/plate (noticed following the pre-incubation procedure in S. typhimurium strains) was considered as lowest concentration showing unequivocal cytotoxicity.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data with a structural analogue substance are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP).