Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2017 to 19 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Principles of method if other than guideline:
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 second experiment in the presence of S9-mix. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 5 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
TEST MATERIAL
Name ( stated on the report) : TETRAHYDRO CITRAL
Appearance: Colourless to pale yellow liquid
Batch: SC00019145
Stable under storage conditions until: 18 March 2018 (expiry date)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
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:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
First mutation assay : tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.
Second mutation assay : tested up to concentrations of 1600 μg/plate in the tester strains TA1535, TA1537, TA100 and TA98, and up to 5000 μg/plate in the tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix.
Vehicle / solvent:
The vehicle of the test item was dimethyl sulfoxide (Merck, Darmstadt, Germany).
Controls
Untreated negative controls:
yes
Remarks:
The negative control was dimethyl sulfoxide, the vehicle of the test item.
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: 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:
see additional information on results fileds below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First Mutation Experiment :
- Toxicity : A decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence and presence of S9-mix at concentrations of 512 μg/plate and upwards. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
Second Mutation Experiment :
- Toxicity : A decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains at concentrations of 164 μg/plate and upwards in the absence of S9-mix and at concentrations of 512 μg/plate and upwards presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that TETRAHYDRO CITRAL is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of TETRAHYDRO CITRAL and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study procedures described in this report were based on the most recent OECD, EC and METI guidelines.

Batch SC00019145 of the test item was a colourless to pale yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

In the second mutation assay, the test item was tested up to concentrations of 1600 μg/plate in the tester strains TA1535, TA1537, TA100 and TA98, and up to 5000 μg/plate in the tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

TETRAHYDRO CITRAL 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 an independently repeated experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that TETRAHYDRO CITRAL is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.