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EC number: 204-464-7
CAS number: 121-32-4
Dose-Range Finding Test: Mutagenic Response of Ethylvanillin in the
Salmonella Typhimurium Reverse Mutation Assay and in the Escherichia
Coli Reverse Mutation Assay
Direct plate assay
Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Normal bacterial background lawn
The objective of this study was to determine the potential of
Ethylvanillin 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 was performed according to international guidelines (OECD
guideline No. 471 adopted 21 July 1997 and test method No. B13/14 of EC
regulation No. 440/2008) and in compliance with the principles of Good
The test was performed in two independent experiments, at first a direct
plate assay was performed and secondly a pre-incubation assay.
The test item was dissolved in dimethyl sulfoxide (DMSO). In the dose
range finding study, the test item was initially tested up to
concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the
direct plate assay. The test item did not precipitate on the plates at
this dose level. The bacterial background lawn was not reduced at any of
the concentrations tested and no biologically relevant decrease in the
number of revertants was observed.
In the first mutation experiment, the test item was tested up to
concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98.
The test item did not precipitate on the plates at this dose level. The
bacterial background lawn was not reduced at any of the concentrations
tested and no biologically relevant decrease in the number of revertants
In the second mutation experiment, the test item was tested up to
concentrations of 5000 μg/plate in the tester strains TA1535, TA1537,
TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did
not precipitate on the plates at this dose level. Cytotoxicity, as
evidenced by a decrease in the number of revertants, was observed in the
tester strains TA98 and TA1537 in the absence and presence of S9-mix,
respectively, at the highest tested concentration.
The negative and 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
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 a follow-up
In conclusion, based on the results of this study it is concluded that
Ethylvanillin is not mutagenic in the Salmonella typhimurium reverse
mutation assay and in the Escherichia coli reverse mutation assay in the
presence or in the absence of S9-metabolic activation.
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