Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl 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:

6 September 2001- 17 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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:

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:

-

EC Number:

435-360-1

EC Name:

-

Cas Number:

144447-11-0

Molecular formula:

C20H22O5

IUPAC Name:

4-methoxyphenyl 4-[4-(oxiran-2-yl)butoxy]benzoate

Details on test material:

Identification: Epoxyhexmes
Trade Name: Epoxyhexmes
Chemical name: 4-methoxyphenyl-4(5,6-epoxyhexyloxy)benzoate
CAS-No: 144447-11-0
Description: White crystalline solid
Batch: RTC9811501003
Purity: 97.3%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 31 December 2001

Method

Target gene:

Histidine/tryptophan operon

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV.

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: See below

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0, 3, 10, 33, 100, 333, 666, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: none

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan

NUMBER OF REPLICATIONS: triplicate plates per concentration

NUMBER OF CELLS EVALUATED: all colonies were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, background lawn

Evaluation criteria:

- The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
- The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
- The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
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) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.

However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

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 1535

Metabolic activation:

with and without

Genotoxicity:

positive

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:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

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 5

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: precipitated > 666 µg/plate in the main study
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
In the range finding test the substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10,33, 100,333, 1000,3330 and 5000 µg/plate in the absence and presence of S9-mix.
Based on the results of the dose range finding test, it was tested up to concentrations of 3330 µg/plate in the absence and presence of S9-mix in a mutation assay. The test substance was tested beyond the limit of the solubility to obtain adequate mutagenicity data. The mutation experiment was performed with the strains TA1535, TA1537 and TA98.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The substance is mutagenic in the bacterial reverse mutation assay.

Executive summary:

The substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and.TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coliWP2uvrA.

The test was performed in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). In the dose range finding test, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The substance precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the mutation assay, the substance was tested up to concentrations of 3330 µg/plate in the absence and presence of S9-mix. the substance precipitated on the plates at dose levels of 1000 and 3330 µg/plate. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

In tester strain TA100, the substance induced up to 2.0- and 4.8-fold, dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA1535, the substance induced an up to 2.0- fold, dose related increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix and an up to 4.9-fold, not dose-related increase in the presence of S9-mix. In the other three tester strains (TA1537, TA98 and WP2uvrA) in the absence and presence of S9-mix, the substance did not induce a dose-related increase in the number of revertant colonies.

Based on the results of this study it is concluded that the substance is mutagenic in the bacterial reverse mutation assay.