Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June, 1999 - 16 February, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

EC Number:

225-193-0

EC Name:

Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Cas Number:

4707-47-5

Molecular formula:

C10H12O4

IUPAC Name:

methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Test material form:

other: solid

Method

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9.

Test concentrations with justification for top dose:

Direct plate:
- Dose range finding test (all 5 strains, without and with S9): 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg/plate
Based on the results of the preliminary test, the following dose levels were used:
- Experiment 1:
TA 98, TA 1537 and WP2uvrA (without and with S9), TA 100 and TA 1535 (without S9): 25, 75, 200, 600, 1800 and 5000 μg/plate
TA 100 and TA 1535 (with S9): 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent of choice based on solubility of the test substance and compatibility with the target cells. The test substance was soluble in acetone at approximately 500 mg/L, the maximum concentration tested.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; all Salmonella strains with S9: 1 μg/plate; WP2uvrA with S9: 10 μg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance and controls were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

OTHER EXAMINATIONS:
- Precipitate was evaluated by visual examination without magnification.

Evaluation criteria:

Evaluation of results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
For the test article to evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article.
Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value.
Data sets for strains TA98, TA100 and WP2uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Criteria for a valid test:
The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehivle control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met:
(1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count.
(2) A reduction in the background lawn.

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:

cytotoxicity

Remarks:

at concentrations of 600, 1800 and 5000 μg/plate.

Vehicle 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:

cytotoxicity

Remarks:

at concentrations of 600, 1800 and 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 600, 1800 and 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1800 and 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1800 and 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was generally observed at 5000 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and 50 μL platinf aliquot. Precipitate was observed at ≥ 3333 μg per plate and toxicity was observed at ≥ 667 to 5000 μg per plate. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 μg per plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached illustration
- Negative historical control data: see attached illustration

ADDITIONAL INFORMATION:
The study was concluded to be nagative without conducting an independent repeat assay because no unique metabolism requirements were known about the article and because no equivocal responses were observed in the assay that would suggest further testing is warranted.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed equivalent to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated equivalent to OECD 471 guideline and according to GLP principles. The test was performed in one direct plate experiment up to and including 5000 μg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding test (≥ 667 to 5000μg). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. The study was concluded to be negative without conducting an independent repeat assay because no unique metabolism requirements were known about the article and because no equivocal responses were observed in the assay that would suggest further testing is warranted. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.