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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-07-2013 to 29-07-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised November, 2006)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one
EC Number:
939-400-3
Molecular formula:
C13H22O
IUPAC Name:
Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: clear colourless liquid

Method

Target gene:
histidine or tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Range finding test: 3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate
Experiment 1: 1, 3, 10, 33, 100, 200, 333 μg/plate
Experiment 2 * : 1, 3, 10, 33, 100, 333, 666, 1000, 3330, 5000 μg/plate
* certain dose levels depending on strain; all strains tested up to 333 μg/plate
Experiment 3 (TA98 strain only) : 100, 333, 1000, 3330, 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide was used in the assay. The test formulations were used within 4 hours for the assay.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
methylmethanesulfonate
other: ICR-191; 2-nitrofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: The plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (bacterial background lawn) and reduction in the number of revertants
Evaluation criteria:
See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.
Statistics:
No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test resultsopen allclose all
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 applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at > 333 or 666 or 1000 μg/plate in all strains/cell types tested ; depending on strain/cell type, where necessary the test item was tested up to the 5000 μg/plate limit dose.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was observed at the start of the incubation period at 1000 µg/plate and above in the range finding test/first mutation experiment, at the start and end of the incubation period at 5000 µg/plate in the second and third mutation experiment.

RANGE-FINDING/SCREENING STUDIES: A range-finding test was conducted on TA100 and WP2uvrA strains as part of the current assay

COMPARISON WITH HISTORICAL CONTROL DATA: The negative control values and strain specific positive controls were within the laboratory historical control data ranges, with the following exceptions:

The mean plate counts of the positive control of TA1535 in the absence of S9-mix (first experiment), WP2uvrA in the presence of S9-mix (first experiment) and TA100 in the presence of S9-mix (second experiment) were not within the laboratory historical range. However, these values were more than 3 times greater than the concurrent solvent control values, therefore this deviation in the mean plate count of the positive controls had no effect on the results of the study. Additionally, in the second experiment, the mean plate count of the solvent control of TA1535 in the absence of S9-mix was not within the laboratory historical range. Historical control data from experiments was presented within the study report.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat).In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 μg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants or a reduction of the bacterial background lawn, was observed in all tester strains, except in the tester strains TA98 (presence of S9-mix) and WP2uvrA. Since in tester strain TA98 in the presence of S9-mix (first experiment) no toxicity or precipitate on the plates was observed and the recommended dose level of 5000 μg/plate was not selected, an additional third mutation experiment was performed utilising this strain only. The test item precipitated on the plates at the top dose of 5000 μg/plate in the second and third experiment. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. The negative control values were within the laboratory historical control data ranges, except the response for TA1535 in the absence of S9-mix (second experiment). However, since this value was just without the limit of the range, the validity of the test was considered to be not affected. The 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, except the response for TA1535 in the absence of S9-mix (first experiment), WP2uvrA in the presence of S9-mix (first experiment) and TA100 in the presence of S9-mix (second experiment). The values were above the limit of the range. However since more than a three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.