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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria:

-S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without metabolic activation, OECD 471: negative (Shin-Etsu, 2015 )

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2015 to 22 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
yes
Remarks:
only pre-incubation method
Qualifier:
according to guideline
Guideline:
other: JAPAN: The Notification stipulating the standard provided by the Minister of Health, Labour and Welfare of the Industrial Safety and Health Act Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results
Version / remarks:
01 September 1988
08 February 1999
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JAPAN: Reverse Mutagenicity Test on Bacteria" of "Mutagenicity Test" stipulated in the "Testing Methods for New Chemical Substances
Version / remarks:
31 March 2011
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Lot number: 311001
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 (Oriental Yeast) prepared from the liver of 7-week-old male SD rats administered with phenobarbital (one time at 30 mg/kg and three times at 60 mg/kg) and 5,6-benzoflavone (one time at 80 mg/kg) was used.
- method of preparation of S9 mix : One milliliter of S9 mix consisted of 8 µmol MgCl2, 33 µmol KCl, 5 µmol Glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.
- quality controls of S9: enzymatic activity
Test concentrations with justification for top dose:
Main test-1:
Without S9: 5000, 1250, 313, 78.1, 19.5, 4.88 µg/plate
With S9: 5000, 1250, 313, 78.1, 19.5, 4.88 µg/plate
Main test-2:
Without S9: 5000, 1250, 625, 313 µg/plate
With S9: 5000, 1250, 625, 313 µg/plate
Vehicle / solvent:
- Vehicle used: dehydrated DMSO
- Justification for choice of vehicle: The test substance solution of 50.0 mg/mL prepared with dehydrated DMSO was considered to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 hours after preparation. Therefore, dehydrated DMSO was selected as a vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative. No statistical methods were used.
Statistics:
None
Key result
Species / strain:
other: S.typhimurium TA98, TA100, TA1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
STUDY RESULTS
Ames test:
- Signs of toxicity : none
- Individual plate counts : Please refer to the attached background material (Table # 1 and 2)
- Mean number of revertant colonies per plate and standard deviation : Please refer to the attached background material (Table # 1 and 2)

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to the attached background material (Appendix 1)
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material (Appendix 1)
Conclusions:
The test item had no ability to induce mutations in a pre-incubation method with Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9 mix) under the present test conditions.
Executive summary:

The ability of the test item to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). The mutagenicity of the test item was judged to be negative because the number of revertant colonies in the test substance treatment groups was less than twice that in the negative control for all tester strains regardless of the presence or absence of S9 mix. The numbers of the revertant colonies in the positive controls were above twice that in the negative controls. The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility. It was also confirmed that the test system was free from bacterial contamination, which declares the test results to be valid. It was concluded that the test item had no ability to induce mutations under the present test conditions.

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

Additional information

Genetic toxicity in vitro

 

Ames test

The ability of the test item to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix) (Shin-Etsu, 2015). The mutagenicity of the test item was judged to be negative because the number of revertant colonies in the test substance treatment groups was less than twice that in the negative control for all tester strains regardless of the presence or absence of S9 mix. The numbers of the revertant colonies in the positive controls were above twice that in the negative controls. The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility. It was also confirmed that the test system was free from bacterial contamination, which declares the test results to be valid. It was concluded that the test item had no ability to induce mutations under the present test conditions.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data for genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.