Benzeneacetic acid, 2-(6-carboxy-2,3-dimethylphenoxy)-

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:

07-NOV-2008 to 20-NOV-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid

EC Number:

601-485-7

Cas Number:

117570-93-1

Molecular formula:

C17 H16 O5

IUPAC Name:

2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid

Details on test material:

- Name of test material (as cited in study report): ASA404 C7
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 072301
- Expiration date of the lot/batch: 18 March 2009
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Main 1
TA100: 3, 10, 33, 100, 333, 3330 and 5000 µg/plate
TA1535, TA97, TA98 and TA102: 100, 333, 3330 and 5000 µg/plate
Main 2:
All strains: 100, 333, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9

Migrated to IUCLID6: 0.1 µg/plate in DMSO for TA102

Controls 2

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Controls 5

Positive controls:

yes

Positive control substance:

other: ICR-191, 1 µg/plate in DMSO for TA97

Remarks:

without S9

Controls 6

Positive controls:

no

Positive control substance:

other: 2-aminoanthracene, TA1535, TA97, TA100, TA98

Remarks:

with S9

Controls 7

Positive controls:

yes

Positive control substance:

other: 1,8 dihydroxyanthraquinone , 30 µg/plate in DMSO for TA102

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 bacteria per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, and the reduction of the revertant colonies

OTHER EXAMINATION
- Precipitation of the test substance

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, and TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.



A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535 and, TA98 is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No statistics are performed

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: The results of solvent and positive controls were considered valid and it can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that ASA404 C7 is not mutagenic in the Salmonella typhimurium reverse mutation assay