3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

EC Number:

276-650-6

EC Name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

Cas Number:

72403-67-9

Molecular formula:

C15H24O2

IUPAC Name:

[3-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate; [4-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate

Test material form:

other: liquid

Details on test material:

Identification: Myraldyl Acetate
Appearance: Colourless to pale yellow liquid
Test item storage: At room temperature protected from light
Stable under storage conditions until: 28 April 2017 (expiry date)

Test animals

Species:

other: EPISKIN Small Model

Details on test animals or test system and environmental conditions:

Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-001, See APPENDIX 4).
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.

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise 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).

Source
SkinEthic Laboratories, Lyon, France.

Test system

Number of animals:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Details on study design:

6.7.1. Application/Treatment of the test item
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well 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.

6.7.2. 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-solution (0.3 mg/ml in PBS). 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 separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 71 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

Results and discussion

Any other information on results incl. tables

Myraldyl Acetate was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Myraldyl Acetate did not interact with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with Myraldyl Acetate and controls are presented in APPENDIX 1, Table 2 (see attached study).

The individual OD570 measurements are presented in APPENDIX 2 (see attached study).

Table 3 (see attached study) shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with Myraldyl Acetate compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Myraldyl Acetate compared to the negative control tissues was 40%. Since the mean relative tissue viability for Myraldyl Acetate was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See APPENDIX 3 see attached study). The standard deviation value of the percentage viability of three tissues treated identically was less than 13%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Finally, it is concluded that this test is valid and that Myraldyl Acetate is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified as Category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.