(Z)-hex-3-enyl benzoate

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:

Experimental phase was performed between 07 November 2012 and 25 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

(Z)-hex-3-enyl benzoate

EC Number:

246-669-4

EC Name:

(Z)-hex-3-enyl benzoate

Cas Number:

25152-85-6

Molecular formula:

C13H16O2

IUPAC Name:

(3Z)-hex-3-en-1-yl benzoate

Test material form:

liquid

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbitone and ß-naphthoflavone

Test concentrations with justification for top dose:

- Dose range finding test: Pre-incubation method
TA 100 with S9-mix, WP2uvrA with and without S9-mix: 0, 50, 150, 500, 1500 and 5000 µg/plate
TA 100 without S9-mix: 0, 5, 15, 50, 150 and 500 µg/plate

- Experiment 1: Pre-incubation method
TA 1535, TA 1537, TA 100 and TA98 (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA 1535, TA 1537, TA 100 and TA98 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate
WP2uvrA (with S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate

- Experiment 2: Pre-incubation method
The test item dose range was amended slightly, following results of Experiment 1 and was as follows:
TA 1535, TA 1537, TA 100 and TA98 (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA 1535, TA 1537, TA 100 and TA98 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5 and 15 µg/plate
WP2uvrA (with S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50 and 150 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The test item was immiscible in sterile distilled water but was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

(untreated plates, plate incorporation method)

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL/plate DMSO)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Preliminary toxicity test, Experiment 1 and 2: pre-incubation method

DURATION
- Exposure duration: pre-incubation for 20 minutes, incubation for approximately 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain, with or without S9, in experiment 1 and 2.

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a reduction in the growth of the bacterial background lawn.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test-item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
- The test-item was toxic to TA100 from 15 µg/plate in the absence of S9-mix and 500 µg/plate in the presence of S9-mix and non-toxic to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The solvent control values were within the historical control values. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 5 µg/plate in the absence of S9-mix and 150 µg/plate in the presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence of S9-mix although weakened bacterial background lawns were noted to the same strain in the absence of S9-mix at and above 50 µg/plate. The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experiment number. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.

ADDITIONAL INFORMATION:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate; all were found to be satisfactory. The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the reverse mutation assay using Salmonella typhimurium and Escherichia coli strains, performed according to OECD 471.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. The test was performed in two independent experiments using the pre-incubation method at up to seven dose levels, in triplicate, both in the absence and presence of S9-mix. The dose levels for the first experiment were selected based on observed cytotoxicity in the preliminary toxicity study and ranged between 0.05 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The second experiment was performed using a similar, slightly amended, dose range to the first experiment. The test substance was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. Adequate negative and positive controls were included. No significant increases in the frequency of revertant colonies were recorded for any of the S. typhimurium strains (TA1535, TA1537, TA98 and TA100) or E. coli strain (WP2uvrA), with any dose of the test substance, either with or without metabolic activation. Based on the results of this study the substance is considered to be non- mutagenic in the reverse mutation assay using Salmonella typhimurium and Escherichia coli strains.