Benzeneacetic acid, .alpha.,.alpha.-dimethyl-, ethyl ester

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:

Start of study: August 26 - End of study: September 12, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a : GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Remarks:

Grundsätze der Guten Laborpraxis (GLP), Anhang 1zu Paragraph 19a, des Chemikaliengesetzes vom Juli 1994

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α,α-Dimethyl-ethyl ester benzeneacetic acid

EC Number:

608-307-7

Cas Number:

2901-13-5

Molecular formula:

C12 H16 O2

IUPAC Name:

α,α-Dimethyl-ethyl ester benzeneacetic acid

Test material form:

solid - liquid: suspension

Details on test material:

Name: Ethyl dimethylphenylacetate
Synonyms: EDMPA
Chemical name: Benzeneacetic acid, α,α-dimethyl-, ethyl ester
Certificate of analysis: March 18, 1997
Purity: 99.8%
Appearance: clear, colorless liquid
Solubility: 0.3 g/L in water, miscible with alcohol, acetone and toluene
Batch number: B 0006
Date of production: March 12, 1997
Date of expiry: March 18, 2000
Storage conditions: darkness at approximately 20°C in a fume cupboard
Stability in the solvant: confirmed over 4h in DMSO
Concentration of stock solution: 5% (w/v)

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa uvrB for all strains. All 4 Salmonella strains are deficient in the structure of their LPS layer and in DNA excision repair system. TA 98 and TA 100 also possess a modified postreplication DNA repair system.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9 mix)

Test concentrations with justification for top dose:

First experiment with and without metabolic activation: 4, 20, 100, 500, 2500, 5000 µg/plate
Second experiment with and without metabolic activation: 0.8, 4, 20, 100, 500, 2500 µg/plate

Vehicle / solvent:

Vehicle/solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation for strain TA 100 and TA 1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation for strain TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation for strain TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Preincubation period: 48h at approx. 37°C
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawns

OTHER: A toxicity test using histidine-enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D) was performed in parallel with the second mutation experiment.

Evaluation criteria:

Criteria for a valid assay:
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive contrals induced increases in the mutation frequency which were bath statistically significant and within the laboratory's normal range

Criteria for a positive response:
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

The test results must be reproducible.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 2500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in a dose range of 500 to 2500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.