Benzeneacetic acid, 4-(cyclopropylcarbonyl)-.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: March 08; End of evaluation March 31, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to international guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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 OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(Cyclopropylcarbonyl)-α,α-dimethyl-ethyl ester benzeneacetic acid

EC Number:

605-546-9

Cas Number:

169280-10-8

Molecular formula:

C16 H20 O3

IUPAC Name:

4-(Cyclopropylcarbonyl)-α,α-dimethyl-ethyl ester benzeneacetic acid

Test material form:

solid - liquid: suspension

Details on test material:

- Physical state: white, crystalline powder
- Content: 100%
- Lot/batch No.: H 0080 dated January 24, 2000
- Expiration date of the lot/batch: September 30, 2000
- Solubility: solution in DMSO
- Stability under test conditions: confirmed over 4h in DMSO
- Storage condition of test material: darkness at approximately 5°C in a refrigerator
- Concentration of stock solution: 50 mg/ml

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Bacterial strains were checked half-yearly with regard to their respective biotin, histidine and/or tryptophan requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate incorporation test:
- without metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
- with metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
Repeat plate incorporation test: without metabolic activation: 1.6, 5, 16, 50, 160 and 500 µg/plate (TA 100, TA 1535, TA 1537, TA 98)
Preincubation test:
- without metabolic activation: 1.6, 5, 16, 50, 160 and 500 µg/plate (TA 100, TA 1535, TA 1537, TA 98)
16, 50, 160, 500, 1600 and 5000 µg/plate (WP2uvrA)
- with metabolic activation: 16, 50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without metabolic activation for strain TA 100 and TA 1535

Positive control substance:

sodium azide

Remarks:

99% (w/w), batch n° 04786, 1.0 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without metabolic activation for strain TA 1537

Positive control substance:

9-aminoacridine

Remarks:

98% (w/w), batch n° 55731, 50.0 µg/plate

Controls 3

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without metabolic activation for strain TA 98

Positive control substance:

2-nitrofluorene

Remarks:

98% (w/w), batch n° 70202, 2.5 µg/plate

Controls 4

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without metabolic activation for strain WP2uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

99.9 % (w/w), batch n° 037H0792, 2.0 µg/plate (plate incorp.), 0.5 µg/plate (preincub.)

Controls 5

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

With metabolic activation for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

96% (w/w), batch n°70056, 0.5 µg/plate (TA 98 and TA 100), 1.0 µg/plate (TA 1535 and TA 1537), 10.0 µg/plate (WP2uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Preincubation period: 20 min at ca. 30°C
- Exposure duration: 48h at ca. 37°C
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: Thinning of the bacterial lawn

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
1. the negative control counts fell within the normal ranges for the spontaneous mutant frequency
2. the positive control chemicals induced clear increases in revertant numbers and within the laboratory's normal range

Evaluation criteria
For valid data, the test article was considered to be mutagenic if:
1. it produces at least a 2-fold increase in the mean number of revertants/plate of at least one of the tester strains over the mean number of revertants/plate of the appropriate vehicle control at complete background lawn.
2. it induces a dose-related increase in the mean number of revertants/plate of at least one of the tester strains over the mean number of revertants/plate of the appropriate vehicle control in at least 2 to 3 concentrations of the test compound at complete bacterial background lawn.

The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

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:

at 160 and 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Only in the preincubation test visible precipitation of the test compound on the plates was observed at 5000 µg/plate with metabolic activation with all tester strains and without metabolic activation with the strain E.coli WP2uvrA. Test toxicity was observed without metabolic activation at 160 µg/plate and above with all S.thyphimurium strains. In the presence of metabolic activation, the test compound proved to be toxic to the S.thyphimurium strains in a dose range of 500 to 5000 µg/plate.

Remarks on result:

other: other: First plate incorporation test

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not mutagenic with and without metabolic activation.