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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Nov 2017-10 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
24, 76, 240, 760, 2400 ug/ml. Justification for top dose: No toxicity observed at 1600 ug/ml. At and above 3200 ug/ml plate the intensity of the bacterial background lawn was severely reduced associated with severe reduction in the mean number of revertant clones.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Evaluation criteria:
To determine a positive result, there should be a dose related increrase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the testi item either in the presence or absence of metabolic activation system. The test will be judged postive if the increase in mean revertants at the peak of the dose respomse is equal to or greater than two times the mean vehicle control values for strains TA98, TA100 or WP2uvrA(pKM101) or equal to or greater than three times the mean vehicle control values for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respecitive threshold cited above or a non-dose responsive increase that is equal to or greated than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Test item was found to be stable in DMSO for 48 hours at room temperature at the concentrations of 25 and 50000 ug/ml.
The test item did not precipitate on the basal agar plates at any of the tested doses
The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for all tester strains when conpared to the respective vehicle control plates, either in the presence or absence of the metabolic activation.
Positive control substances tested simultaneously produced more than a 3-fold increase in the mean nembers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as intensity of the bacterial background lawn of all the tester strains was compared to that of the respective vehicle control plates.

Applicant's summary and conclusion

Conclusions:
The test item 4-allyveratrole (Methyl Eugenol) was not mutagenic in the bacterial reverse mutation test.
Executive summary:

The objective of this study was to determine the potential of 4 -allylveratrole (Methyl Eugenol) 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 were based on the OECD 471 guideline.

Methyl eugenol was soluble in DMSO at 50mg/mL on sonication, and was found to be stable in DMSO for 48 hours at room temperature at the fortification levels of 25 an 50 000 ug/mL.

In a preliminart toxicity test to selct test doses for the mutaton assay, the test item did not show toxicity to the tester strain up to 1600ug/plate as the intensity of the bacterial background lawn was comparable to the vehicle control plates. Similarly, the mean number of revertant colonies was more or less comparable to the vehicle constrol plates at these test doses. However, at and above 3200ug/plate the intensity of the bacterial background lawn was severily reduced associated with a severe reduction in the mean number of revertant colonies. Based o these observations, a highest dose of 2400ug/plate was tested in the mutation assay.

At the concentrations used, 24, 76, 240, 760, 2400 ug/ml, 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. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously proving the proper functionality of the test.

In conclusion, based on the results of this study it is concluded that 4 -allylveratrole (Methyl Eugenol) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.