Vanillic acid

Genetic toxicity: in vitro

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:

10/03/02 - 10/14/02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No certificate of analysis; only two strains of bacteria were used.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Simplified study.

Deviations:

yes

Remarks:

Only two strains of bacteria were used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vanillic acid

EC Number:

204-466-8

EC Name:

Vanillic acid

Cas Number:

121-34-6

Molecular formula:

C8H8O4

IUPAC Name:

4-hydroxy-3-methoxybenzoic acid

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vanillic acid
- Expiration date of the lot/batch: no data
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature
- Stability under test conditions: yes

Method

Target gene:

Histidine locus in two strains of Salmonella typhimurium (S. typhimurium; TA98, TA100),

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

61.7, 185, 556, 1667 and 5000 µg/plate with and without metabolic activation.

Vehicle / solvent:

DMSO.

Controls

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide.

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide.

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene.

Remarks:

with and without metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: 2 plates/dose/strain
DETERMINATION OF CYTOTOXICITY
- Method: background lawn of microcolonies observation

Evaluation criteria:

The test article was considered to be positive for mutagenicity if the mean number of revertant colonies in the test article group was no less than 2 times that of the negative control group, and if the number of revertant colonies increased with increasing dose of the test article.

Statistics:

Statistical methods were not used in the determination of test results.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data.
RANGE-FINDING: none

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Any other information on results incl. tables

Table 1: Strain TA98 (+/- S9 mix) results:

Strain TA98
Dose level (µg/plate)	Without metabolic activation			With metabolic activation
	Plate counts		Mean	Plate counts		Mean
Positive control	83	90	86.5	82	84	83
Negative control	17	15	16	24	19	21.5
Untreated	21	22	21.5	24	34	29
61.7	19	15	17	20	33	26.5
185	20	14	17	29	27	28
556	23	16	19.5	31	28	29.5
1667	16	14	15	16	21	18.5
5000	13	20	16.5	24	22	23

Table 2: Strain TA100 (+/- S9 mix) results:

Strain TA100
Dose level (µg/plate)	Without metabolic activation			With metabolic activation
	Plate counts		Mean	Plate counts		Mean
Positive control	1602	1425	1513.5	1590	1441	1515.5
Negative control	559	562	560.5	544	584	564
Untreated	677	606	641.5	534	574	554
61.7	588	563	575.5	554	576	565
185	614	568	591	596	520	558
556	483	587	535	589	603	596
1667	468	586	527	592	615	603.5
5000	578	524	551	574	549	561.5

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, Vanillic acid is not mutagenic in the Salmonella typhimurium reverse mutation assay (simplified study).

Executive summary:

In a reverse gene mutation assay in bacteria (Reliability 2, GLP), Vanillic acid dissolved in dimethylsulfoxide (DMSO) was tested in S. typhimurium TA100 and TA98 in the presence and the absence of mammalian metabolic activation (S9) prepared from the livers, using the plate incorporation method. The highest dose-level used, unless limited by the solubility of the substance, was 5000 µg/plate.Three known mutagens, dissolved in DMSO, were used to check the sensitivity of the test system.

The results of experiment showed that for both test systems (with and without metabolic activation), and each bacterial strain, the mean number of revertant colonies in the test substance group was less than 2 times that of the negative control group and no increase in revertant colonies accompanied an increase in dose. No cytotoxicity was observed at all tested concentrations with and without metabolic activation. This showed that the test substance was negative for mutagenicity (simplified study). The mean number of revertant colonies in the negative control group for each bacterial strain was within the control range based on historical data. There was also a clear increase (2 times or higher) in the mean number of revertant colonies in the positive control group of each bacterial strain compared to the negative control group.

Based on these results, each bacterial strain was confirmed to be appropriately sensitive to mutagenic substances. Under the test conditions, Vanillic acid did not show any mutagenic activity in the bacterial reverse mutation test using S. typhimurium in two strains (simplified study).