3,7-dimethyloct-1-en-3-yl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2015 to 22 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloct-1-en-3-yl acetate

EC Number:

269-847-3

EC Name:

3,7-dimethyloct-1-en-3-yl acetate

Cas Number:

68345-17-5

Molecular formula:

C12H22O2

IUPAC Name:

3,7-dimethyloct-1-en-3-yl acetate

Test material form:

liquid

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair substitution (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537).

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed, the second experiment was performed with at least eight concentrations. 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
Strain TA 100 with and without S9 mix: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Strains TA 1535, TA 1537, TA 98 and WP2 uvrA with and without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD (without metabolic activation) 2-aminoanthracene, 2-AA (with metabolic activation)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- in agar (plate incorporation, experiment I); preincubation (experiment II)


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min
- Exposure duration/duration of treatment: 48 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h
- Method used: agar or microwell plates for the mouse lymphoma assay.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduction in the number of revertants (100-5000 µg /plate) and reduced background lawn (1000-5000 µg/plate) was observed in experiment I and II with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

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

Remarks:

A reduction in the number of revertants was not observed. However, in Experiment I at 5000 µg/plate with S9-mix and in experiment II at 1000-5000 µg/plate with and without S9-mix reduced background lawn was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

A reduction in the number of revertants was not observed. However, in Experiment I at 2500 -5000 µg/plate with S9-mix and in experiment II at 1000-5000 µg/plate with and without S9-mix reduced background lawn was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate only in the presence of S9 mix in both experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in the presence of S9 mix in both experiments. No precipitation of the test item in the overlay agar on the incubated agar plates occurred up to the highest investigated dose in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Dimethyl Octenyl Acetate (DMOE-Ac) at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations within the biologically relevant range.

Any other information on results incl. tables

Please refer to the attached document.

Applicant's summary and conclusion

Conclusions:

The test substance induced no genetic damage (considered not mutagenic) in the Ames test under the described experimental conditions.

Executive summary:

This study was performed to investigate the potential of Dimethyl Octenyl Acetate (DMOE-Ac) to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: Strain TA 100 with and without S9 mix: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Strain TA 1535, TA 1537, TA 98 and WP2 uvrA with and without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in the presence of S9 mix in both experiments. No precipitation of the test item in the overlay agar on the incubated agar plates occurred up to the highest investigated dose in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Dimethyl Octenyl Acetate (DMOE-Ac) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations within the biologically relevant range. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Dimethyl Octenyl Acetate (DMOE-Ac) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.