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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isopulegol

EC Number:

201-940-6

EC Name:

Isopulegol

Cas Number:

89-79-2

IUPAC Name:

2-isopropenyl-5-methylcyclohexanol

Constituent 2

Reference substance name:

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

EC Number:

232-102-8

EC Name:

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

Cas Number:

7786-67-6

Molecular formula:

C10H18O

IUPAC Name:

5-methyl-2-(prop-1-en-2-yl)cyclohexan-1-ol

Details on test material:

Name of test material: L-Isopulegol

Test system

Type of coverage:

open

Amount / concentration applied:

50 μL of the undiluted liquid test substance (corrosion test)
30 μL of the undiluted liquid test substance (irritation test)

Details on study design:

- Corrosion test condition
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance
application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation
medium was replaced with fresh medium immediately before application. 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 μL 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. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until
all tissues per application time were dosed and rinsed. 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 condition
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). 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.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: OD570

Basis:

mean

Time point:

other: 3 minutes

Score:

ca. 1.91

Reversibility:

no data

Remarks on result:

other: Corrosion test

Results 2

Irritation parameter:

other: OD570

Basis:

mean

Time point:

other: 1 hour

Score:

ca. 0.453

Reversibility:

no data

Remarks on result:

other: Corrosion test

Results 3

Irritation parameter:

other: OD570

Basis:

mean

Time point:

other: 1 hour

Score:

ca. 0.146

Reversibility:

no data

Remarks on result:

other: Irritation test

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Isopulegol shows a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.