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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
2. Histidine for Salmonella typhimurium and tryptophan Escherichia coli
3. Histidine for Salmonella typhimurium and tryptophan Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
2 and 3
Details on mammalian cell type (if applicable):
Not specified
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2. Type and composition of metabolic activation system: NADH 4 μmol, Magnesium chloride 8 μmol, NADPH 4 μmol, Potassium chloride 33 μmol, 0.2 M phosphate buffer (pH 7.4) 1000 μmol, Glucose · 6-phosphate 5 μmol, FMN 2 μmol
- source of S9: Kikkoman Corporation
- method of preparation of S9 mix : S9 was prepared from non-induced liver of 8-week-old Syrian Hamster (Std: Syrian) males used.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.3 ml
3. Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system.
Test concentrations with justification for top dose:
2. 0, 312.5, 625, 1250, 2500, 5000 µg/plate
3. 42 - 5000 µg/plate without and with S9 mix
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test chemical was stable in DMSO solution, and the content of test substance in preparation liquid was within a predetermined value range.
3. - Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: Test chemical was dissolved in deionised water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: -S9, AF-2 (TA100, WP2, TA98), +S9, 2-aminoanthracene (all strains), trypane blue (TA100, TA98, azo reduction method)
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
not specified
Positive controls:
yes
Remarks:
in accordance with the OECD guideline
Positive control substance:
other: in accordance with the OECD guideline
Remarks:
3
Details on test system and experimental conditions:
2. NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
- Number of independent experiments - 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in medium (Standard method of Ames and Azo reduction method)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: The pre-incubation method was used to test by direct method and metabolic activation method.
- Exposure duration/duration of treatment: 48 hours (Standard method of Ames); 20 min (Azo reduction method)
- Harvest time after the end of treatment (sampling/recovery times): No data
3. NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
- Number of independent experiments - 2 individual experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: pre-incubation method was used with 30 minutes pre-incubation
- Exposure duration/duration of treatment: at least 48 h incubation time both without and with S9 mix in both experiments.
- Harvest time after the end of treatment (sampling/recovery times): No data
Evaluation criteria:
2. Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control , and when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system.
3. Toxicity was evaluated on the basis of a reduction in the number of revertant colonies and/or a clearing of the bacterial background lawn.
Statistics:
2. Yes, Mean ±standard deviation was observed.
3. No data
Species / strain:
S. typhimurium, other: S. typhimurium TA100, TA1535, TA98, TA1537
Remarks:
2 and 3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
2 and 3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
2. RANGE-FINDING/SCREENING STUDIES (if applicable): When test chemical was tested at a common ratio of approximately 3 in the range of 50 to 5000 μg / plate, no antibacterial activity was observed in either direct method or metabolic activation method of all test bacteria. From the above results, it was decided that the maximum dose in this test was set to 5000 μg / plate in all the test bacteria, in the direct method and the metabolic activation method, and the dose was set to 5 in the common ratio 2.
3. TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of test chemical was not observed.

RANGE-FINDING/SCREENING STUDIES (if applicable): Test concentrations were based on the level of toxicity in a pre-experiment with all Salmonella and Escherichia strains.Since it was freely
soluble and non toxic in this preliminary toxicity test, it was tested up to the prescribed maximum concentration of 5000 μg/plate. The pre-experiment is reported as main experiment I. Both experiments were performed with the preincubation method.
Remarks on result:
other: No mutagenic effects were observed.
Conclusions:
The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Genetic toxicity in vitro study was assessed for the given test chemical according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and as per Prival modification (MJ Prival, and VD Mitchell, 1982). The test chemical was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9 (0.3 ml) prepared from non-induced liver of 8-week-old Syrian Hamster (Std: Syrian) males was used with composition of NADH 4 μmol, Magnesium chloride 8 μmol, NADPH 4 μmol, Potassium chloride 33 μmol, 0.2 M phosphate buffer (pH 7.4) 1000 μmol, Glucose · 6-phosphate 5 μmol, FMN 2 μmol at test concentrations of 0, 312.5, 625, 1250, 2500, 5000 µg/plate. Test chemical was dissolved in DMSO. The positive control substances used are as follows, AF-2 : Frill Furide, SA : Sodium azide, 9-AA : 9-aminoacridine, 2-AA : 2-aminoanthracene and TB : Trypan blue. The study was conducted by Standard method of Ames and Azo reduction method. In Azo reduction method, the pre-incubation method was used to test by direct method and metabolic activation method. 0.1 ml of the test bacterial solution, 0.1 ml of the test substance preparation solution, 0.5 ml of the phosphate buffer solution (0.5 ml of the S9 mixed solution in the metabolic activation method)) were mixed in a small test tube and preincubated at 37 ° C. for 20 minutes After it was done, 2 ml of top agar was added and mixed, and it was poured onto a synthetic medium flat plate. At the same time, a control test was conducted using a solvent or two kinds of positive control substance solutions instead of the test substance preparation solution. When test chemical was tested at a common ratio of approximately 3 in the range of 50 to 5000 μg / plate, no antibacterial activity was observed in either direct method or metabolic activation method of all test bacteria. From the above results, it was decided that the maximum dose in this test was set to 5000 μg / plate in all the test bacteria, in the direct method and the metabolic activation method, and the dose was set to 5 in the common ratio 2. Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control, and when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA by Ames test. Hence the substance cannot be classified as gene mutant in vitro.

 

Another gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical in Salmonella typhimurium and Escherichia coli (Ames test) as per OECD Guideline 471 (Bacterial Reverse Mutation Assay) and equivalent and similar to Prival modification. Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and E. coli WP2 uvrA were used in this study at 42 - 5000 μg/plate without and with S9 mix in both experiments. Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system. Test concentrations were based on the level of toxicity in a pre-experiment with all Salmonella and Escherichia strains. Toxicity was evaluated on the basis of a reduction in the number of revertant colonies and/or a clearing of the bacterial background lawn. Since it was freely soluble and non toxic in this preliminary toxicity test, it was tested up to the prescribed maximum concentration of 5000 μg/plate. The pre-experiment is reported as main experiment I. Both experiments were performed with the preincubation method. Negative and positive controls were in accordance with the OECD guideline. Test was conducted in 3 replicates in 2 individual experiments both in the presence and absence of S9 mix. Pre-incubation method was used with 30 minutes pre-incubation (Prival, 1982) and at least 48 h incubation time both without and with S9 mix in both experiments. Precipitation of test chemical was not observed. Slight toxicity was exclusively observed in experiment II at the maximum concentration of 5000 μg/plate in TA1535 with S9 mix and TA98 without S9 mix. No substantial and biological relevant increase in revertant colonies of any of the five tester strains was observed in both experiments following treatment with chemical at any dose level neither in the absence nor in the presence of metabolic activation. Under the experimental conditions used it was not genotoxic (mutagenic) in the gene mutation tests in bacteria.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames assay:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Genetic toxicity in vitro study was assessed for the given test chemical according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and as per Prival modification (MJ Prival, and VD Mitchell, 1982). The test chemical was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9 (0.3 ml) prepared from non-induced liver of 8-week-old Syrian Hamster (Std: Syrian) males was used with composition of NADH 4 μmol, Magnesium chloride 8 μmol, NADPH 4 μmol, Potassium chloride 33 μmol, 0.2 M phosphate buffer (pH 7.4) 1000 μmol, Glucose · 6-phosphate 5 μmol, FMN 2 μmol at test concentrations of 0, 312.5, 625, 1250, 2500, 5000 µg/plate. Test chemical was dissolved in DMSO. The positive control substances used are as follows, AF-2 : Frill Furide, SA : Sodium azide, 9-AA : 9-aminoacridine, 2-AA : 2-aminoanthracene and TB : Trypan blue. The study was conducted by Standard method of Ames and Azo reduction method. In Azo reduction method, the pre-incubation method was used to test by direct method and metabolic activation method. 0.1 ml of the test bacterial solution, 0.1 ml of the test substance preparation solution, 0.5 ml of the phosphate buffer solution (0.5 ml of the S9 mixed solution in the metabolic activation method)) were mixed in a small test tube and preincubated at 37 ° C. for 20 minutes After it was done, 2 ml of top agar was added and mixed, and it was poured onto a synthetic medium flat plate. At the same time, a control test was conducted using a solvent or two kinds of positive control substance solutions instead of the test substance preparation solution. When test chemical was tested at a common ratio of approximately 3 in the range of 50 to 5000 μg / plate, no antibacterial activity was observed in either direct method or metabolic activation method of all test bacteria. From the above results, it was decided that the maximum dose in this test was set to 5000 μg / plate in all the test bacteria, in the direct method and the metabolic activation method, and the dose was set to 5 in the common ratio 2. Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control, and when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA by Ames test. Hence the substance cannot be classified as gene mutant in vitro.

 

Another gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical in Salmonella typhimurium and Escherichia coli (Ames test) as per OECD Guideline 471 (Bacterial Reverse Mutation Assay) and equivalent and similar to Prival modification. Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and E. coli WP2 uvrA were used in this study at 42 - 5000 μg/plate without and with S9 mix in both experiments. Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system. Test concentrations were based on the level of toxicity in a pre-experiment with all Salmonella and Escherichia strains. Toxicity was evaluated on the basis of a reduction in the number of revertant colonies and/or a clearing of the bacterial background lawn. Since it was freely soluble and non toxic in this preliminary toxicity test, it was tested up to the prescribed maximum concentration of 5000 μg/plate. The pre-experiment is reported as main experiment I. Both experiments were performed with the preincubation method. Negative and positive controls were in accordance with the OECD guideline. Test was conducted in 3 replicates in 2 individual experiments both in the presence and absence of S9 mix. Pre-incubation method was used with 30 minutes pre-incubation (Prival, 1982) and at least 48 h incubation time both without and with S9 mix in both experiments. Precipitation of test chemical was not observed. Slight toxicity was exclusively observed in experiment II at the maximum concentration of 5000 μg/plate in TA1535 with S9 mix and TA98 without S9 mix. No substantial and biological relevant increase in revertant colonies of any of the five tester strains was observed in both experiments following treatment with chemical at any dose level neither in the absence nor in the presence of metabolic activation. Under the experimental conditions used it was not genotoxic (mutagenic) in the gene mutation tests in bacteria.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.