2-hexylcyclopentan-1-one

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:

2014-06-17 - 2014-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

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:

2-hexylcyclopentan-1-one

EC Number:

235-970-6

EC Name:

2-hexylcyclopentan-1-one

Cas Number:

13074-65-2

Molecular formula:

C11H20O

IUPAC Name:

2-hexylcyclopentan-1-one

Test material form:

other: liquid

Details on test material:

Details on Test Material
- Name of test material (as cited in study report): Jasmatone.
- Physical state: Clear colourless liquid (determined at testing laboratory).
- Analytical purity: 99.1%
- Impurities (identity and concentrations): Not specified
- Composition of test material, percentage of components: 2-hexylcyclopentanone, 99.1%.
- Isomers composition: Not applicable.
- Purity test date: Not available.
- Lot/batch No.: PE00085776.
- Expiration date of the lot/batch: 18 November 2014.
- Stability under test conditions: Stable.
- Storage condition of test material: In refrigerator (2-8 deg C) protected from light.

Method

Target gene:

Histidine [S. typhimurium] and tryptophan [E. coli]

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9, prepared from male Sprague-Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg)

Test concentrations with justification for top dose:

Dose range finding study:
S. typhimurium TA100 and E. coli WP2 uvrA (with and without S9-mix (5% v/v)): 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 ug/plate (i.e. tested up to a limit concentration).

First mutation experiment:
S. typhimurium TA1535, TA1537 and TA98 (without S9-mix (5% v/v)): 1.7, 5.4, 17, 52, 164 and 512 ug/plate (tested up to a cytotoxic concentration).
S. typhimurium TA1535, TA1537 and TA98 (with S9-mix (5% v/v)): 5.4, 17, 52, 164, 512 and 1600 ug/plate (tested up to a cytotoxic concentration).

Second mutation experiment:
S. typhimurium TA1535, TA1537, TA100 and TA98 (without S9-mix (10% v/v)): 1.7, 5.4, 17, 52, 164 and 512 ug/plate (tested up to a cytotoxic concentration).
S. typhimurium TA1535, TA1537, TA100 and TA98 (with S9-mix (10% v/v)): 5.4, 17, 52, 164, 512 and 1600 ug/plate (tested up to a cytotoxic concentration).
E. coli WP2 uvrA (with (10% v/v) and without S9-mix): 52, 164, 512, 1600 and 5000 ug/plate (tested up to a limit concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is known to be compatible with the survival of the bacteria and the activity of the S9-mix. It is not suspected of chemical reaction with the test substance, Jasmatone.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

Each S9 batch was characterised with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA98 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Details on test system and experimental conditions:

MEDIUM: Purified agar in Vogel-Bonner Medium E (18 g/L), containing glucose (20 g/L), biotin (12.5 ug/plate) and either [S. typhimurium tests] histidine (15 ug/plate) or [E. coli tests] tryptophan (15 ug/plate).

METHOD OF APPLICATION: plate incorporation.

DURATION
- Preincubation period: not applicable.
- Exposure duration: 48 hours.
- Environmental conditions: all incubations were carried out in a controlled environment at a temperature of 37.0 +/- 1.0 deg C (actual range 34.2 - 39.0 deg C).
- Expression time (cells in growth medium): not applicable.
- Selection time (if incubation with a selection agent): not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable.

SELECTION AGENT (mutation assays): not applicable.
SPINDLE INHIBITOR (cytogenetic assays): not applicable.
STAIN (for cytogenetic assays): not applicable.

NUMBER OF REPLICATIONS: in the dose range finding test, S. typhimurium strain TA100 and E. coli WP2 uvrA were tested in triplicate. In the full mutation assay, S. typhimurium strains TA1535, TA1537 and TA98 were tested in triplicate with 5% S9-mix (in the activated conditions). An independent repeat of the assay evaluated - in triplicate - all five tester strains using 10% S9-mix for the activated conditions.

NUMBER OF CELLS EVALUATED: not evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: examination of the reduction of the bacterial background lawn, the increase in the size if the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS: precipitation of the test substance was evaluated by eye.

Evaluation criteria:

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 WP2 uvrA is greater than three (3) times the concurrent vehicle control.
b) the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Not specified.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to limit concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium, other: TA1537, TA1535 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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: not applicable - solvent vehicle was DMSO.
- Precipitation: no precipitation was observed at the start or end of the incubation period in any strains of S. typhimurium in any experiment. In the repeat experiment, precipitation of Jasmatone was observed at the start of incubation (E. coli WP2 uvrA, 5000 ug/plate), although this was apparently not seen in the dose range finding study.
- Other confounding effects: in strain TA1535, a fluctuation in the number of revertant colonies above the laboratory historical control was observed in the absence of S9-mix at a concentration of 1.7 ug/plate. As the increase was only 1.1-fold (below the criteria of 3-fold for a positive result), it was not considered to be relevant.

RANGE-FINDING/SCREENING STUDIES: dose range finding was carried out on TA100 and WP2 uvrA; both strains were included in the independent repeat experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: both 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY: a reduction of the background bacterial lawn (reported as ranging from "slight" in TA98 to "extreme" in TA100) was observed in all S. typhimurium strains, with and without S9-mix, in both the dose ranging/first experiment and repeat experiment. In the dose range finding study, there was a reduction in the background bacterial lawn in E. coli WP2 uvrA at doses above 512 ug/plate without S9-mix. This was not observed with S9-mix, nor in the repeat experiment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In a guideline study, conducted to GLP, Jasmatone was found to be not mutagenic in the bacterial reverse mutation assay, tested in four strains of Salmonella typhimurium (at up to cytotoxic concentrations) and one strain of Escherichia coli (at up to a limit concentration), in the presence and absence of metabolic activation.

Executive summary:

The mutagenicity of Jasmatone was evaluated in the bacterial reverse mutation assay, employing four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and one of Escherichia coli (WP2 uvrA). In a dose range finding study using TA100 and WP2 uvrA, tests were carried out (in triplicate) up to a limit concentration of 5000 ug/plate. Due to cytotoxicity observed in TA100 (by a reduction in the background bacterial lawn), tests (in triplicate) on TA1535, TA1537 and TA98 were carried out up to a maximum (cytotoxic) concentration of 512 ug/plate without a metabolic activation fraction (5% v/v S9-mix) and up to 1600 ug/plate with S9. An independent repeat of the assay of all tester strains (with or without 10% v/v S9-mix) was conducted up to the same maximum concentrations.

All bacterial strains showed negative responses over the entire dose range. Both negative and positive controls were within the historical laboratory control data, indicating that the test system functioned correctly. It was concluded that Jasmatone is not mutagenic in the S. typhimurium or E. coli bacterial reverse mutation assays.