2-cyclohexylethyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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:

other: BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures.

GLP compliance:

yes

Type of assay:

other: BlueScreen HC Assay

Test material

Test material information

Constituent 1

Reference substance name:

2-cyclohexylethyl acetate

EC Number:

244-543-3

EC Name:

2-cyclohexylethyl acetate

Cas Number:

21722-83-8

Molecular formula:

C10H18O2

IUPAC Name:

2-cyclohexylethyl acetate

Method

Target gene:

GADD45a gene

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

A genetically modified strain of cultured human lymphoblastoid TK6 cells
is used (GLuc-T01). Incorporated into this strain is a patented Gaussia luciferase (GLuc) reporter system
that exploits the proper regulation of the GADD45a gene, which mediates the adaptive response to
genotoxic stress.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

BlueScreen HC operates in a final aqueous solvent solution of 1% (v/v), with respect to the solvent. In the final step of assay preparation, equal volumes of cells in assay medium and test compound solution are combined producing a 50% dilution of stock solution. Hence, test compound solutions are prepared at double the required top test concentration in 2% (v/v) aqueous solvent. The necessary 50-fold dilution from the solvent stock to aqueous solvent solution (20 μl solvent stock + 980 μl sterile deionised water) usually defines the highest concentration that can be tested since it is not desirable to serially dilute from a precipitating dose.
Cyclohexaneethyl acetate was supplied as a colourless liquid which was combined with 100 % DMSO to give a 1250 mM stock solution. The compound precipitated when added to water and therefore further dilution of the DMSO stock solution was required. The concentration of test compound used in the assay is shown in Table 1.

Vehicle / solvent:

DMSO

Controls

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

A genetically modified strain of cultured human lymphoblastoid TK6 cells is used (GLuc-T01). Incorporated into this strain is a patented Gaussia luciferase (GLuc) reporter system that exploits the proper regulation of the GADD45a gene, which mediates the adaptive response to genotoxic stress. Exposure to a genotoxic compound increases expression of GLuc, which is quantified at the assay endpoint by the detection of luminescence generated from the reaction of GLuc with a
coelenterazine substrate, added to the microplate wells just before measurement.
The microplates are covered with a breathable membrane and incubated at 37°C with 5% CO2 and 95% humidity for 48 hours.

Evaluation criteria:

The assay plates are then analysed using a microplate reader that provides measurements of fluorescence and flash luminescence for cells and solutions in the microplate wells. Following a recent protocol enhancement, cytotoxicity is measured by lysis of the cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence. This technique for the estimation of relative cell density replaces the previous optical absorbance measure. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminescence intensity is proportional to the activity of the cell’s DNA repair system, which is increased by genotoxic analytes. Luminescence is normalised to the fluorescence signal to correct for variation in cell yield caused by cytotoxicity.
Raw data collected from BlueScreen HC assay plates are saved to an MS Excel™ file. The luminescence and fluorescence data are automatically analysed using the BlueScreen HC software template to produce a result summary, with data presented both in tabulated and graphical formats, giving a semi-quantitative assessment of cytotoxicity and genotoxicity. The overall assay outcome presented for a compound results from the average of the duplicate test series performed for the compound.

Results and discussion

Test results

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Cyclohexaneethyl acetate was negative for genotoxicity both with and without metabolic activation in the BlueScreen HC assay.