2-(2-(4-methyl-3-cyclohexen-1-yl)propyl)cyclopentanone

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:

18 December 2014 to 08 January 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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

There were no deviations from standard operating procedures that affected the integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Molecular formula:

C14H24O1

IUPAC Name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Constituent 2

Reference substance name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Molecular formula:

C15H24O

IUPAC Name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Constituent 3

Reference substance name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Molecular formula:

C15H24O

IUPAC Name:

Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-

Constituent 4

Reference substance name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Molecular formula:

C15H24O

IUPAC Name:

Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-

Test material form:

liquid

Specific details on test material used for the study:

Name as stated in the report: NECTARYL
Batch No.: VE00318744
Appearance: Clear colourless liquid (determined at WIL Research Europe B.V.)
Expiration date: 14 March 2015

Method

Target gene:

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 coli (WP2uvrA).

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 induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment I : 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence and presence of 5% (v/v) S9-mix.
Experiment II : based on the results of the first mutation assay, six doses (increasing with approximately half-log steps) of the test substance were selected and tested in triplicate in each strain in the second experiment in the absence and presence of 10% (v/v) S9-mix.. The highest concentration of NECTARYL used in the second mutation assay was 5 mg/plate.

Vehicle / solvent:

NECTARYL was dissolved in dimethyl sulfoxide. Test substance concentrations were used within 3 hours after preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle of the test article, being DMSO.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 at 2.5 μg/plate (DMSO) and 2-aminoanthracene at 2.5 μg in DMSO

Details on test system and experimental conditions:

The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. To avoid indistinctness about interference of precipitation of the test substance at the colony counting, the solubility of the test substance was assessed at the beginning and end of the treatment period. In parallel selection plates with the highest dose level were used during the incubation period in the second mutation experiment.
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.

Rationale for test conditions:

according to guideline

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537,
TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle 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 is greater than two (2) times the concurrent
control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is
greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at
least one independently repeated experiment.

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) 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.
d) No more than 5% of the plates are lost through contamination or some unforeseen event.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A

Remarks:

first mutation assay (5% (v/v) S9-mix)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA, and test item precipitated (oily droplets) at 5000µg/plate in all experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A

Remarks:

Second mutation assay (10% (v/v) S9-mix)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed in all tester strains+/- S9-mix, except in tester strain WP2uvrA +/- S9-mix and TA1535 +S9-mix, and test item precipitated (oily droplets) at 5000µg/plate in all experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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.

Any other information on results incl. tables

In the first mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. NECTARYL precipitated on the plates as oily droplets at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA.

In the second mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. NECTARYL precipitated (observed as droplets) on the plates at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix and TA1535 in the presence of S9-mix. NECTARYL did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that NECTARYL is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of NECTARYL in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

NECTARYL 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 coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD, EC and Japanese guidelines.

Batch VE00318744 of NECTARYL was a clear colourless liquid with a purity of 97.1%. The test substance was dissolved in dimethyl sulfoxide.

In the first mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. NECTARYL precipitated on the plates as oily droplets at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA.

In the second mutation assay, NECTARYL was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. NECTARYL precipitated (observed as droplets) on the plates at the dose level of 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix and TA1535 in the presence of S9-mix.

NECTARYL did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that NECTARYL is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.