ethyl 2-acetyl-4-methyltridec-2-enoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2016 to 27 Feruary 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-acetyl-4-methyltridec-2-enoate

EC Number:

816-455-0

Cas Number:

960253-23-0

Molecular formula:

C18H32O3

IUPAC Name:

ethyl 2-acetyl-4-methyltridec-2-enoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2 , Batch no.: 17-EKIN-008, See APPENDIX 4).

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 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).

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-047).
This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied undiluted (25 μl) directly on top of the tissue

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-86-6599 compared to the negative control tissues was 99%. Since the mean relative tissue viability for GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.
The positive control had a mean cell viability of 22% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since the mean relative tissue viability for GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.
Finally, it is concluded that this test is valid and that GR-86-6599 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

In vitro skin irritation test with GR-86-6599 using a human skin model.

This report describes the ability of GR-86-6599 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of GR-86-6599 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 43 of GR-86-6599 was a yellow liquid. GR-86-6599 was applied undiluted (25 μl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-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 MTT at the end of the treatment.

GR-86-6599 did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific

reduction of MTT (NSMTT) by GR-86-6599 was -3.1% of the negative control tissues. Since the %NSMTT was ≤ 0.0, there was no correction applied on the ODs of the test item treated viable 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 GR-86-6599 compared to the negative control tissues was 99%. Since the mean relative tissue viability for

GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.

The positive control had a mean cell viability of 22% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage

viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that GR-86-6599 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.