5-methyl-2-(isopropyl)cyclohexyl nicotinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Principles of method if other than guideline:

The evaluation of pro-sensitising activity of the substance on human monocytes through FACS analysis was performed.

GLP compliance:

no

Remarks:

not-GLP study but considered as reliable based on the reporting of the study

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-2-(isopropyl)cyclohexyl nicotinate

EC Number:

254-991-1

EC Name:

5-methyl-2-(isopropyl)cyclohexyl nicotinate

Cas Number:

40594-65-8

Molecular formula:

C16H23NO2

IUPAC Name:

5-methyl-2-(isopropyl)cyclohexyl nicotinate

In vitro test system

Details on the study design:

CELL MODEL: monocyte-like human line called THP-1. Cells are cultured in RPMI containing 10 % FCS and 2 mM glutamine.

PRELIMINARY CYTOTOXICITY STUDY: after exposure of the cells to the test material, the cells are washed with PBS and exposed to the MTT-medium at 37°C. At the end of the incubation period, the MTT-medium is removed and the cells receive the MTT solubilization solution. The plate is shaken on a rotatory plate shaker for 20-30 minutes, ensuring that all the crystals have dissolved from the cells and have formed a homogeneous solution. The absorbance is measured as described with background elimination. The results are expressed in terms of viability: % viability = (OD treated cultures*100)/OD untreated control cultures.

SENSITISATION ASSAY:
-Sample preparation: the sample was dissolved in ethanol and then diluted in the cell medium at the two different dilutions in order to obtain the desired final concentrations in contact with the THP-1cells in vitro.
- Test concentrations: 3 μg/ml and 0.6 μg/ml.
- Exposure period: 48 h.
- Exposure conditions: at 37 °C with 5 % CO2.
- Staining: after the incubation with the test substance and the controls, cells were collected, checked under the microscope for their vitality by staining with Trypan Blue dye and counted in a cell counter chamber.
- Measurement of markers expression: after staining the cells were washed in PBS and then marked with a fluoresceinated anti-B7.1 or B7.2 antibody. After washing, to eliminate the excess antibody, the MFI (Mean Fluorescence Intensity) linked to the cells was evaluated by means of a flow cytofluorimeter (FACS, Fluorescence Activated Cell Sorter, Becton Dickinson, Mountain View, CA). This value is proportional to the expression of costimulatory molecules. The MFI of the non-treated THP-1 cells and of cells after reaction with a monoclonal isotype-matched antibody was used as an internal control (basal fluorescence). MFI is the geometric average of the fluorescence intensity of the cells treated with the fluoresceinated antibody and it is proportional to the number of stained molecules per cell.
- Positive control: nickel sulphate.
- Negative control: cell medium, non treated cells.

Results and discussion

Positive control results:

Test on nickel sulphate, a known sensitising substance, is characterised by a) high increase of both the markers; b) direct correlation between concentration and intensity of the response; c) relevant effects even at very low doses. The tested dose of 4 mg/ml of Nickel Sulphate (NiSO4.6H2O) corresponds to approx. 1 ppm of Nickel, concentration that is around the minimal sensitising threshold in already sensitised individuals with irritated skin (1,2). A low dose of nickel sulphate can cause a detectable increase of CD80 respect to untreated cell. The concentration that is able to cause an allergic reaction in most of the sensitive subjects in intact skin is over the 100 ppm of Nickel Sulphate.

1. Gawkrodger DJ, Nickel dermatitis: how much Nickel is safe? Contact Dermatitis 1996; 35: 267-271.
2. Tanojo H, Hostynek JJ, Mountford HS, Maibach HI. In vitro permeation of nickel salts through human stratum corneum. Acta Derm Venereol Suppl, 2001; 212: 19-23.

In vitro / in chemico

Results

Results 1

Parameter:

other: MFI (CD80)

Value:

-0.42

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: MFI values obtained for test conc. 3 and 0.6 μg/ml/.

Results 2

Parameter:

other: MFI (CD86)

Value:

-0.82

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: MFI values obtained for test conc. 3 and 0.6 μg/ml/.

Other effects / acceptance of results:

The sample did not show cytotoxic effects on the cells used for the test (IC50 = 15 μg/ml)
No signal related to apoptosis on the treated cells was observed.
The sample did not show any increase in the expression of the investigated markers.

Any other information on results incl. tables

The results expressed as co-stimulatory molecules expression after the exposure to the substance for the positive control substance and test substance are presented in the table below.

Table

Sample	CD80 (MFI)	CD86 (MFI)
Nickel sulphate 20 μg/ml	11.35	57.66
Nickel sulphate 10 μg/ml	9.63	24.88
Nickel sulphate 4 μg/ml	1.43	11.39
Test material 3 μg/ml	-0.42	-0.82
Test material 0.6 μg/ml	-0.33	-0.8

Applicant's summary and conclusion

Interpretation of results:

other: not considered to be a skin sensitiser according to the in vitro study results

Conclusions:

The substance did not show any increase in the expression of the investigated markers. It is not considered to have a sensitising potential.

Executive summary:

The sensitising potential of the substance was evaluated in-vitro by quantifying the changes of cell surface marker expression (CD80, CD86) on a human monocytic leukemia cell line, THP-1 cells, following 48 hours exposure to the two concentrations of the test substance. The changes of surface marker expression were measured by a flow cytofluorimeter following cell staining with fluoresceinated antibodies. A preliminary cytotoxicity measurement (MTT assay) was also conducted to determine the non-cytotoxic concetrations to be used in the sensitisation assay. The Mean Fluorescence Intensity (MFI) of surface markers for the test material and positive control (nickel sulphate) compared to negative control were calculated.

The results of nickel sulphate is characterised by a) high increase of both the markers; b) direct correlation between concentration and intensity of the response; c) relevant effects even at very low doses (4 μg/ml). The sample did not show any increase in the expression of the markers CD80 and CD86.

Comparing the behaviour of the positive substance and the test material it is deemed that the substance does not have sensitising potential.