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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

no mutagenic potential

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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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2
Reason / purpose for cross-reference:
read-across source
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Experiment 1, 1b, 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment 1, 1b, 2
Vehicle controls validity:
valid
Remarks:
Experiment 1, 1b, 2
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Experiment 1, 1b, 2
Additional information on results:
Experiment 1
-Confirmation of the Criteria and Validity
All strains met the criterion of at least 10E9 bacteria/mL (correlating to 100-200 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
For TA1537 the experiment 1 was not evaluated as a high number of colonies was visually found on the control plates, what might be occur due to a contamination of the bacterial culture.
- Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
- Mutagenicity
No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment (exp. 2) was performed.
For TA1537, the experiment 1 was repeated in exp. 1b.

Experiment 1b
- Confirmation of the Criteria and Validity
The strain TA1537 met the criterion of at least 10E9 bacteria/mL (correlating to 100-200 colo-nies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
- Solubility and Toxicity
In experiment 1b, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strain TA1537 could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
- Mutagenicity
No relevant or concentration-related increase of the number of revertant colonies in the treatment with and without metabolic activation could be observed.
Therefore, the test item is stated as not mutagenic to TA1537 under the conditions of this experiment.

Experiment 2
- Confirmation of the Criteria and Validity
All strains met the criterion of at least 10E9 bacteria/mL (correlating to 100-200 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.
- Solubility and Toxicity
In experiment 2, the test item showed no precipitates on the plates in all tested concentra-tions.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
- Mutagenicity
No relevant or dose-increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment

VALIDITY
All negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly
All positive controls showed f(I) values > 2 or > 3 which demonstrated the mutagenic potential of the diagnostic mutagens.
The confirmation tests of the genotype performed by the supplier did not show any irregularities. The control of the titer within the demanded value of 1-2x10E9 bacteria/mL. In the sterility control no growth of bacteria could be detected.
Since all criteria for acceptability have been met, the study is considered valid
Conclusions:
The substance is not mutagenic with and without metabolic activatio
Executive summary:

The test item was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).

The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. The initial experiment had to be repeated for the strain TA1537 due to high colony numbers on the control plates.

Experiment 1:

In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 μL/plate (maximum test concentration for soluble substances) in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. Thus, the test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant or dose-related increase in the number of revertants in all evaluated strains, in the presence and the absence of metabolic activation.

The experimental part for TA1537 was declared invalid due to high colony numbers on the control plates and was repeated in experiment 1b.

Experiment 1b:

Experiment 1b was performed for the strain TA1537 and exactly as in experiment 1.

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed on the plates with TA1537. Thus, the test item showed no signs of toxicity towards the bacteria strain in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant or dose-related increase in the number of revertants in TA1537, in the presence and the absence of metabolic activation.

Experiment 2:

Based on the results of the first experiment, and since no cytotoxicity was observed up to the highest tested concentration, the test item was tested up to concentrations of 5 μL/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.

The test item showed no signs of toxicity in all tested concentrations (5 μL/plate to 0.08 μL/plate).

The results of this experiment showed that the test item caused no significant or dose-related increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation.

Conclusion:

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2
Reason / purpose for cross-reference:
read-across source
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- No opacity nor precipitation of the medium was observed at the beginning or end of treatment, in the absence or presence of S9 metabolism at any dose level.
- Following treatment with the test item, for all treatment series, a dose-related reduction of pH was observed at higher dose levels. However, pH values at the dose levels selected for metaphase analysis were deemed adeguate since only pH changes higher than one unit are considered culture conditions leading to artifactual positive results ( Scott et al., 1991).
- No remarkable variation of osmolality was observed at any dose level, in the absence or presence of S9 metabolism.

SOLUBILITY TEST
A preliminary solubility trial was performed using dimethylsulfoxide (DMSO).
The test item was found soluble in DMSO at the concentration of 116 mg/ml. An aliquot of this solution, added to culture medium in the ratio of 1:100, gave a clear solution. Based on these results, the final concentration of 1160 µg/ml in DMSO was selected as the highest dose level to be used in the cytogenetic assay.

SELECTION OF DOSE LEVELS FOR SCORING
For short term treatment, no toxicity was observed at any dose level in the absence or presence of S9 metabolic activation.
For continuous treatment in the absence of S9 metabolism, dose related toxicity was observed at higher dose levels reducing the mitotic index to 45 % of the concurrent negative control at 5 mM concentration.
On the basis of these results, the dose levels selected for scoring of aberrationswere as follows:
10.0, 5.00 and 2.50 with and without S9 in case of 3 h treatment
5.00, 1.25 and 0.313 without S9 in case of 24 h treatment
Conclusions:
The test substance does not induce chromosome aberrations in human lymphocytes after in vitro treatment
Executive summary:

The test item was assayed for the ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation, according to OECD guideline 473. Three treatment series were included in the study. A short term treatment was performed where the cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long term(continuous) treatment was also performed, only in the absence of S9 metabolism, until harvest at 24 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Dose levels of 1160, 580, 290, 145, 72.5, 36.3, 18.2, and 9.08 µg/ml) were used for all treatment series. Appropriate negative and positive control were included. Two replicate cell cultures were prepared at each test point. For all treatment series, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Where no toxicity was observed, the highest dose level was selected for scoring chromosomal aberrations.

Dose levels selected for scoring were as follows: 10.0, 5.00 and 2.50 with and without S9 in case of 3 h treatment and 5.00, 1.25 and 0.313 without S9 in case of 24 h treatment.

For each replicate culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level and treatment condition. It is concluded that the substance does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mutant frequencies in the negative control cultures fell within the 95 % control limits of the distribution of the laboratory’s historical control database. Treatment with the positive control items gave marked responses that were compatible with those generated in the historical control database and produced a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control, indicating the correct functioning of the test system. Adequate number of cells and concentrations was analysed.

The pH values and osmolality of the post-treatment media were determined. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism.

No relevant increases over the spontaneous mutation frequency were observed at any treatment level in the presence of S9 metabolic activation. A statistically significant increase (p<0.05) over the concurrent vehicle control was observed at the intermediate dose level of 145 µg/ml, in the absence of S9 metabolism. However, the mutant frequency at this concentration was within the historical control range and no dose-effect relationship was
indicated. Hence, the observed increase was considered of no biological relevance.
Analysis of variance indicated that replicate culture was not a significant factor in explaining the observed variation in the data, in the absence and presence of S9 metabolism. Dose level was a significant factor (p<0.05) in explaining the observed variation in the data, only in the presence of S9 metabolism. This result was probably due to the mean mutation frequency observed at the highest dose level, slightly higher than the concurrent vehicle control. However, this increase was not statistically significant and the mutation frequencies of the two replicate cultures were within the historical control range.
Conclusions:
The test item does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation
Executive summary:

The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment, according to OECD guideline 476. A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 1160 µg/ml (10 mM) and at a wide range of lower dose levels: 580, 290, 145, 72.5, 36.3, 18.1, 9.06 and 4.53 µg/ml. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. No precipitation was noted at any concentration tested. A main assay was performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO). Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 9 days to allow phenotypic expression of induced mutation. The following dose levels were used: 1160, 580, 290, 145, 72.5, 36.3 µg/ml.

No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism.

It is concluded that the substance does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genetic toxicity potential of the substance was evaluated in the in vitro gene mutation study in bacteria. Moreover, data on Similar Substance 01 were considered, obtained from the in vitro gene mutation study in mammalian cells and in the in vitro chromosome aberration study. Justification for Read Across is given in Section 13 of IUCLID.

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of S. typhimurium, according to an internal method similar to the OECD Guideline 471. The test was performing without and with metabolic activation in the range of concentration of 0.1 to 100 µg/20µl/plate, using strains of Salmonella typhimurium (TA 97, TA 98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA 1538). Each concentration and control were tested in triplicate. No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation. The test substance did not induce gene mutations in the strains of S. typhimurium used, both with and without metabolic activation.

The similar substance was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. The initial experiment had to be repeated for the strain TA1537 due to high colony numbers on the control plates. In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 μL/plate (maximum test concentration for soluble substances) in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. Thus, the test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant or dose-related increase in the number of revertants in all evaluated strains, in the presence and the absence of metabolic activation. The experimental part for TA1537 was declared invalid due to high colony numbers on the control plates and was repeated in experiment 1b.

The Experiment 1b was performed for the strain TA1537 and exactly as in experiment 1. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed on the plates with TA1537. Thus, the test item showed no signs of toxicity towards the bacteria strain in both the presence and the absence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant or dose-related increase in the number of revertants in TA1537, in the presence and the absence of metabolic activation.

Based on the results of the first experiment, and since no cytotoxicity was observed up to the highest tested concentration, the test item was tested up to concentrations of 5 μL/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method in a second experiment. The test item showed no signs of toxicity in all tested concentrations (5 μL/plate to 0.08 μL/plate).

The results of this experiment showed that the test item caused no significant or dose-related increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Similar Substance 01 was assayed for its ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation, according to OECD guideline 473. Three treatment series were included in the study. A short term treatment was performed where the cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long term(continuous) treatment was also performed, only in the absence of S9 metabolism, until harvest at 24 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). Dose levels of 1160, 580, 290, 145, 72.5, 36.3, 18.2, and 9.08 µg/ml) were used for all treatment series. Appropriate negative and positive control were included. Two replicate cell cultures were prepared at each test point. For all treatment series, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Where no toxicity was observed, the highest dose level was selected for scoring chromosomal aberrations.For each replicate culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells. No statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level and treatment condition. It is concluded that the substance does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Similar Substance 01 was further examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment, according to OECD guideline 476. A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 1160 µg/ml (10 mM) and at a wide range of lower dose levels: 580, 290, 145, 72.5, 36.3, 18.1, 9.06 and 4.53 µg/ml. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. No precipitation was noted at any concentration tested. A main assay was performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 9 days to allow phenotypic expression of induced mutation. The following dose levels were used: 1160, 580, 290, 145, 72.5, 36.3 µg/ml. No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. It is concluded that the substance does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two following categories:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances, which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The test substance did not show any reasons of concern in all in vitro tests performed.

Therefore, the substance is not classified for genetic toxicity according to the CLP Regulation (EC) No. 1272/2008.