Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]-1-butanol and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]-1-butanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3R)-3-[(1R)-4-methylcyclohex-3-en-1-yl]butan-1-ol; (3S)-3-[(1R)-4-methylcyclohex-3-en-1-yl]butan-1-ol

EC Number:

940-702-2

Molecular formula:

C11H20O

IUPAC Name:

(3R)-3-[(1R)-4-methylcyclohex-3-en-1-yl]butan-1-ol; (3S)-3-[(1R)-4-methylcyclohex-3-en-1-yl]butan-1-ol

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Storage condition of test material: room temperature in the dark under nitrogen

Test animals

Species:

other: EPISKIN MODEL KIT

Strain:

not specified

Details on test animals or test system and environmental conditions:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Incubations were at 37°C and 5% CO2.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative group treated with DPBS; Positive group treated with SDS (5% w/v).

Amount / concentration applied:

10 micolitre of test material was applied unchanged (no vehicle).

Duration of treatment / exposure:

15 minutes

Number of animals:

For each test group, triplicate tissues were used.

Details on study design:

TEST SITE
- Area of exposure: 10 μl of the undiluted test substance was added on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes.

SCORING SYSTEM: Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Mean tissue viability

Value:

9.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Reversibility: no data. Remarks: Score in terms of percentage of negative control group. (migrated information)

In vivo

Other effects:

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Any other information on results incl. tables

Table 1. Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control	0.748	0.751	0.027	99.6	100	3.6
	0.725			96.5
	0.779			103.7
Positive control	0.092	0.066	0.023	12.3	8.8	3.1
	0.048			6.4
	0.057			7.6
Test material	0.07	0.072	0.002	9.3	9.6	0.3
	0.073			9.7
	0.074			9.9

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material is considered to be irritating to skin.

Executive summary:

The study was performed to assess the skin irritation potential of the test material to a human three dimensional epidermal model. The study was performed under GLP and followed the OECD guideline 439. Tissues were treated with the test material for 15 minutes and then washed with phosphate buffered saline to remove residual test material. Subsequently the skin tissues were incubated for 42 hours at 37°C in maintenance medium. After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. The amount of extracted formazan was determined spectrophotometrically at 540 nm. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 9.6%. Since the mean relative tissue viability for the test substance was below or equal to 50% after 15 minutes treatment it is considered to be a skin irritant.