[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP complaint, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD draft proposal: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one

EC Number:

260-709-8

EC Name:

1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one

Cas Number:

57378-68-4

IUPAC Name:

1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Details on test material:

Description: clear colourless to pale yellow liquid
Batch: 2030337
Purity: 97.6%
Test substance storage: in refrigerator (2-8°C) in the dark
Stability under storage conditions: stable
Expiry date: 18 January 2011

Test animals

Species:

other: not applicable

Strain:

other: not applicable

Test system

Type of coverage:

open

Preparation of test site:

other: Human skin tissue

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

Test substance: 10 μl
Negative control: Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, The Netherlands)
Positive control: 5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) (CAS Number 151-21-3)

Duration of treatment / exposure:

15 minutes

Observation period:

After a 42 hour incubation period.

Number of animals:

Not applicable

Details on study design:

Test system: EPISKIN Standard Model (EPISKIN-SM, 0.38 cm², Lot no.: 10-EKIN-010). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Preparation and preincubation;
-Tissues: on the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. The level of Maintenance Medium was just beneath the tissue. Maintenance Medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.
- MTT medium: MTT concentrate (3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml) Sigma Aldrich, Zwijndrecht, The Netherlands)
- Environmental conditions: All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 91 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.0 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Study design:
1. Test for reduction of MTT by the test substance; Delta Damascone was checekd for possible direct MTT reduction before the study was started. To access the ability of the test substance to reduce MTT, approximatley 10 μl of test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml). The mixture was incubated for approximately 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.
1. Application/Treatment of the test substance; The test was preformed on a total of 3 tissues per test substance together with negative and positive controls. Ten μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl 5% SDS (positive control) respectively. The positive control was respread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
3. Cell viability measurement; 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. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was seperated from the collagen matrix and both parts were placed in prelabeled microtobes and extrated with 500 μl isopropanol. Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (Thermo Labsystems).
Cell viability was calculated for each tissue as a precentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Electronic data capture; Obeservations/measurements in the study were recorded electronically using the following programme(s): REES centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USE): Temperature and humiduty.
Multiskan spectrum version 1.00 (Thermo labsystems, Breda, The Netherlands) for optical density measurement.

Interpretation:
1. acceptability of the assay; The in vitro skin test is considered acceptable if it meets the following criteria:
a.) The absolute mean OD 570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b.) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
2. Data evaluation and statistical procedures; 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 postincubation is ≤ 50% of the mean viability of the negative controls.
A test substance is considered non-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 postincubation is > 50% of the mean viability of the negative controls.

Results and discussion

In vivo

Results

Irritation parameter:

other: tissue viability (% of control)

Basis:

other: mean of three tissue culture wells

Time point:

other: 42 h

Score:

5

Max. score:

100

Reversibility:

other: examination of reversibility is not part of this study

Other effects:

In a three-dimensional human epidermis model (EPISKIN Standard Model), application of delta-damascone directly to the skin tissue resulted in a mean of 5% cell viability (percentage of control) after an incubation period of 42 hours.

Any other information on results incl. tables

Delta Damscone was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Delta Damascone did not interact with MTT.

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 Delta Damascone compared to the negative control tissues was 5%. Since the mean relative tissue viability for Delta Damascone was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 minutes of exposure of 5%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the precentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.