Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: Clear colorless liquid
- Storage Conditions: Room temperature, in the dark

In vitro test system

Test system:
human skin model
Remarks:
EPISKIN™
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (Supplier: SkinEthic Laboratories, 4, A. Fleming – 69366 Lyon, France)
- Tissue batch number: 16-EKIN-004

The test system EPISKIN™ is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The principle of the RhE test method is based on the premise that chemicals are able to penetrate the stratum corneum and irritant chemicals are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS N. 298-93-1] into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 µL/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 µL/cm^2).
Duration of treatment / exposure:
15 +/- 0.5 minutes (in a ventilated cabinet at room temperature)
Duration of post-treatment incubation (if applicable):
42 +/- 1 hour
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
72.8
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
4.1%
Other effects / acceptance of results:
The test item did not induce relevant cell death in any replicate, with a mean cell viability of 72.8%, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 12.3, i.e. lower than 18).

Before the main assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A colourless solution, with a sticky transparent precipitate was observed at the end of the incubation period, indicating no interaction with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A transparent solution was observed indicating that the test item has no colour capabilities. Based on these results, no additional control was added in theMain Assay.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of a reliable in viro skin irritation test, it is concluded that Sakura Salicylate is classified as not irritant to the skin.
Executive summary:

The potential of Sakura Salicylate to be irritant to the skin was investigated through an in vitro skin irritation study, using a reconstructed human epidermis (RhE) model EPISKIN™. The experimental procedures were according to OECD Guideline 439 and GLP principles. The test item was shown not to interfere with the test system and positive and negative controls (a 5% (w/v) SDS solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively) gave responses as expected. The test item did not induce relevant cell death in any replicate, with a mean cell viability of 72.8% when compared to the negative control. Intra-replicate variability was acceptable with an SD of % viability value equal to 12.3. Based on the results obtained, Sakura Salicylate is classified as not irritant to the skin according to Regulation (EC) 1272/2008.