5-methyl-2-(1-methylvinyl)cyclohexan-1-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Remarks:

Test substance represents a main component (stereoisomer) of the registered substance

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals No. 431, April 13, 2004 (“In vitro Skin Corrosion: Human Skin Model Test”) and OECD Guideline for Testing of Chemicals No. 439, July 22, 2010 (“In vitro Skin Irritation: Reconstructed Human Epidermis Test Model”)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Test material

Test material information

Constituent 1

Reference substance name:

Isopulegol

EC Number:

201-940-6

EC Name:

Isopulegol

Cas Number:

89-79-2

Molecular formula:

C10H18O

IUPAC Name:

(1R,2S,5R)-2-Isopropenyl-5-methylcylohexanol

Details on test material:

- Test substance : Cyclohexanol, 5-methyl-2-(1-methylethenyl)-, (1R,2S,5R)-
- Name of test material (as cited in study report): L-Isopulegol
- Physical state: liquid, colorless clear
- Analytical purity: 99.4 corr.area-%
- Lot/batch No.: Muster 7 CH
- Expiration date of the lot/batch: 01 Jun 2012
- Storage condition of test material: room temperature, under N2
- Other: pH-value approx. 5 (undiluted test substance)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. The EpiDermTM tissues (surface 0.6 cm²) are commercially available as kits (EpiDerm™ 200).

Direct MTT reduction by the test substance was assessed. In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control, in the same way as described in section “Basic procedure”, additionally.

Corrosion test:
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. Fifty microliter (50 µL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 50 uL of highly de-ionized water (negative control, NC) or with 50 μL of 8-n potassium hydroxide (positive control, PC). A nylon mesh was placed carefully onto the tissue surface of the NC afterwards. The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
Three tissues were treated with the test substance, the PC (5% SDS) and NC (sterile PBS), respectively. Thirty microliter (30 µL) of the undiluted liquid test substance, PC and NC was applied using a pipette. A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol for at least 2 hours at room temperature on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µl (corrosion test)
30 µl (irritation test)

Duration of treatment / exposure:

3 min and 1 h (corrosion test)
1h (irritation test)

Duration of post-treatment incubation (if applicable):

24+/-2h and additional 18 +/-2h (irritation test only)

Number of replicates:

2 tissues assessed

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

Corrosion test - 3 min exposure

Value:

120

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

Corrosion test - 1h exposure

Value:

28

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

Irritation test - 1h exposure

Value:

8

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 120%, and it was 28% after an exposure period of 1 hour.

Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 8%.

The test substance is not able to reduce MTT directly.