1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-21 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Method B.46 of Commission Regulation 440/2008/EC (In vitro skin irritation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

EC Number:

251-649-3

EC Name:

1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

Cas Number:

33704-61-9

Molecular formula:

C14H22O

IUPAC Name:

1,1,2,3,3-pentamethyl-2,3,4,5,6,7-hexahydro-1H-inden-4-one

In vitro test system

Test system:

artificial membrane barrier model

Remarks:

In vitro skin model (EPISKIN standard model)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM
- Tissue batch number(s): not provided, source: (SkinEthic Laboratories, Nice, France)
- Production, Shipping & Delivery date: not provided
- Date of initiation of testing: 09 November 2011

TEMPERATURE USED FOR TEST SYSTEM
-Temperature used during treatment / exposure/ post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: Anthos 2001
- Wavelength: 450 nm (without a reference filter)
- Linear OD range of spectrophotometer: no information

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): Undiluted substance

POSITIVE CONTROL: Sodium Dodecyl Sulphate (SDS), prepared as a 5% w/v aqueous dilution
To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip. After 7 minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period.

NEGATIVE CONTROL: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++, used as supplied

Duration of treatment / exposure:

Exposure: 15 minutes
Incubation: 42 hours

Number of replicates:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control respectively.

Test system

Details on study design:

TEST SITE: EXPOSURE OF THE TISSUES
After receiving the EPISKIN, the tissue was pre-incubated overnight in maintenance medium at 37°C, 5% CO2 in air.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++.
- Time after start of exposure: 15 minutes.

SCORING SYSTEM: The principle of the assay was based on the measurement of cytotoxicity (irritancy) in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissue relative to the negative controls.

TOOL USED TO ASSESS SCORE: The amount of extracted formazan was determined spectrophotometrically at 540 nm in duplicate (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

max score 100

Run / experiment:

Mean

Value:

10.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

other: other: Optical density

Run / experiment:

Mean

Value:

0.101

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Max. score: 0.934, SD: ± 0.027.

Other effects / acceptance of results:

The test item was considered to be irritant.
No other effects observed

Any other information on results incl. tables

The positive control had a mean cell viability of 5.5% after 15 minutes exposure. The negative control was set at 100%.

The standard deviation of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

other: Skin irritant in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance causes skin irritation in the in vitro skin irritation test (OECD guideline 439).

Executive summary:

The ability of the test substance to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-TM)) (OECD TG 349, GLP). The possible skin irritation potential of the test substance was tested through topical application of 10 µL for 15 minutes in an in vitro test. After a 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Cashmeran compared to the negative control tissue was 10.8%. Since the mean relative tissue viability for Cashmeran was below 50% after 15 minutes treatment the test substance is considered to be irritant. The positive control had a mean cell viability of 5.5% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that the substance is irritant in the in vitro skin irritation test under the experimental conditions described in this report.