Registration Dossier

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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
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 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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).