5-methyl-2-(isopropyl)cyclohexyl nicotinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 3rd to 5th, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted July 26th, 2013

GLP compliance:

no

Remarks:

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

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

Test system:

human skin model

Source species:

human

Cell type:

other: normal epidermall keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed artificial human skin model comprising normal human epidermal keratinocytes, growing as an integrated three-dimensional cell culture model, perfectly mimicking the human skin in vitro. The model exhibits normal barrier functions (presence of a differentiated stratum corneum). At the surface, a well-differentiated stratum corneum is present, over which the product to be tested is placed. Below the epidermis there is a semi-permeable membrane that communicate with the inferior well where the culture medium is placed (undernatant).
- Tissue batch number: 14-RHE-005.
- Supplier: Skinethic (Lyon).
- Mainenance medium batch: 14MA046.
- Growth medium batch: 14MPE56.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation: 37 °C (and 5 % CO2).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml MTT solution.
- Incubation time: 3 hours.
- Incubation temperature: 37 °C.
- Removal of MTT solution: after the incubation period of 3 hours, the solution was removed and replaced with 1500 μl/well of isopropanol, with further 2 hours incubation at room temperature under medium speed shaking.
- Measurement of absorbance: at 550 nm with a microplate reader (Tecan modello Sunrise remote).
- Calculation of cell viability: % cell viability = [OD(550nm) test product/meanOD(550nm)negative control] x 100.

ACCPTANCE CRITERIA
- for negative control (CN): the mean OD value of the 3 tissue has to be ≥ 1.2, the standard deviation has to be ≤ 18 %.
- for positive control (CP): the viability mean (expressed as % of the NC) has to be < 40 % the standard deviation has to be ≤ 18 %.
- for the sample : the standard deviation has to be ≤ 18 %.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viablity is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 μl.

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 117.21

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The cell viability of the test, negative and positive control are presented in the table below.

Table

Sample	%mean cell viability (SD) 42 hrs incubation	Comment
Test material	117.21 (5.16)	Non irritant
SLS 5 %	1.81 (0.11)	Irritant
Negative control (CN)	100.00 (6.51)	Non irritant

Applicant's summary and conclusion

Interpretation of results:

other: not classified as a skin irritant according to the CLP Regulation (EC) No.1272/2008

Conclusions:

The substance is not a skin irritant.

Executive summary:

The skin irritation of the substance was evaluated in-vitro in the SkinEthic 3D reconstructed artificial human skin model, according to the OECD Guideline 439. The test material was applied on each tissue in three replicates. After 42 minutes of exposure, the substance was removed and the tissue was incubated at 37 °C, 5 % CO2 for 42 hours. At the end of the exposure the MTT assay was performed to evaluate the tissue cell viability. Negative controls (phosphate buffer) and positive (5 % SLS) controls run in parallel.

The tissue cell viability was 117.21 % for the substance, 1.81 % for positive control and 100.00 for negative control.

The tissue viability is more than 50 % therefore the substance is considered as a non-skin irritant.