Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-05-2008 to 02-06-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

Version / remarks:

Reference: Altern Lab Anim. 2007 Dec; 35 (6): 559-601

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2006 ; signature: January 2007

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

460-490-0

EC Name:

-

Cas Number:

477218-42-1

Molecular formula:

C18H32O3

IUPAC Name:

2-[(1S)-1-[(1R)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate; 2-[(1S)-1-[(1S)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate

Test material form:

liquid

Details on test material:

Physical state: Liquid
- Storage condition of test material: In the refrigerator at + 2 to + 8 °C, protected from light

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN TM Small Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin RHE Model (Lot no.: 08-EKIN-020). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt. to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The EPISKIN 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. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
After approx. 24 hours incubation of the EpiSkin-SM tissues, they were treated with the test item. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1 °C) maintenance medium.

Application of test item and rinsing:
The negative and positive control, and the test item were added into the insert atop the concerning EPISKIN-SMTM triplicate tissues. Solid: Additionally, the test item tissues were wetted with 15 μL of deionised water. The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1°C, 5 ± 0.5% CO2. After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. Tissues were incubated for 42 ± 1 hours at 37 ± 1°C, 5 ± 0.5% CO2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

Tissues were treated with the test material for 15 minutes and then washed with PBS to remove residual test material.
Positive control: SLS (5%) solution, 15 μL for 15 ± 1 min.
Negative control: Deionised water, 15 μL for 15 ± 1 min.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Test system

Details on study design:

TEST SITE
- Area of exposure: 15 μl of the undiluted test substance was added topically into 12-well plates 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 potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean tissue viability

Value:

92.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: n=3 ; SD = 8% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Not screened.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Any other information on results incl. tables

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be irritating to skin.

Executive summary:

The study was performed according to the method described in the ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Aitern Lab Anim. 2007 Dec; 35 (6): 559-601) in accordance with GLP and/or methodology equivalent to later OECD TG 439 and EU Method B.46 guidelines, to assess the skin irritation potential of the test substance using a human three dimensional epidermal model (EPISKIN Small Model). Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 92.6% after the 15 -Minute exposure period and 42 -Hours post-exposure incubation period. All assay acceptability criteria were considered to be met. Under the conditions of this study, the test item is considered to be not irritating to the skin.