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:
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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

Constituent 1
Chemical structure
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
Controlsopen allclose all
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
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
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
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 resultsopen allclose all
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
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.