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Description of key information

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™ (Klimisch 1 study). Furthermore, the absence of skin effects in an acute dermal toxicity study and an in vivo LLNA study are considered.

A BCOP study is available to address eye effects of Sakura Salicylate (Klimisch 1 study).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 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
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment no. 160
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test system: isolated corneas from the eyes of freshly slaughtered bovine.
Age of animals: 6-12 months
Transport solution: Hanks balanced salt solution (HBSS) Modified supplemented with Penicillin sulphate and Streptomycin
sulphate.
Transport conditions: in transport solution at approximately 4°C.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.750 mL of the test item was tested as supplied (being a liquid non surfactant) on the epithelial surface of three idoneous bovine corneas.
Duration of treatment / exposure:
Corneas were exposed in horizontal position for an exposure period of 10 minutes.
Duration of post- treatment incubation (in vitro):
2 hours
Details on study design:
Positive control item was 100% (v/v) dimethylformamide (Labelled as N,NDimethylformamide, Sigma, batch no. SZBD0800V).
Negative control item was Physiological saline (0.9% NaCl) (Baxter, batch no. 13D0406).
Volume of treatment: 0,75 mL
Number of replicates: 3
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
95.5
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
82.3
Irritation parameter:
other: calculated permeability
Run / experiment:
mean
Value:
0.019
Negative controls validity:
valid
Remarks:
0.0067
Positive controls validity:
valid
Remarks:
0.8823
Other effects / acceptance of results:
At the macroscopic observation, no visible change of the treated corneas was noted.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of an in vitro eye irritancy test performed according to OECD/EC guidelines and GLP principles, it is concluded that Sakura Salicylate is non corrosive or irritant to the eye.
Executive summary:

The potential of Sakura Salicylate to cause corrosion/ severe irritation was tested by using the Bovine Corneal Opacity and Permeability (BCOP) assay, according to OECD/EC guidelines and GLP principles. The test item was tested as supplied (being a liquid non surfactant) on the epithelial surface of three idoneous bovine corneas. Positive and negative controls (100% (v/v) dimethylformamide and physiological saline alone, respectively) were concurrently tested in the same number of replicates. The mean opacity of the corneas treated with the test item, detected with an opacitometer at the end of the 2-hour post-exposure period, was 0.0. At the macroscopic observation, no visible change of the treated corneas was noted. After the determination of opacity, the epithelial surface was treated with a 0.4% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 0.0190. The calculated in vitro irritancy score (IVIS) for the test item was 0.3. Negative and positive controls gave the expected results. Based on these results it is concluded that the test item is non corrosive or irritant to the eye, and is not classified for eye irritancy according to Regulation (EC) 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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. It is furthermore considered that dermal exposure to Sakura Salicylate (undiluted) in an acute dermal toxicity test and in the in vivo LLNA study (undiluted) did not result in any adverse skin effects.

The potential of Sakura Salicylate to cause corrosion/ severe irritation was tested by using the Bovine Corneal Opacity and Permeability (BCOP) assay, according to OECD/EC guidelines and GLP principles. The test item was tested as supplied (being a liquid non surfactant) on the epithelial surface of three idoneous bovine corneas. Positive and negative controls (100% (v/v) dimethylformamide and physiological saline alone, respectively) were concurrently tested in the same number of replicates. The mean opacity of the corneas treated with the test item, detected with an opacitometer at the end of the 2-hour post-exposure period, was 0.0. At the macroscopic observation, no visible change of the treated corneas was noted. After the determination of opacity, the epithelial surface was treated with a 0.4% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 0.0190. The calculated in vitro irritancy score (IVIS) for the test item was 0.3. Negative and positive controls gave the expected results. Based on these results it is concluded that the test item is non corrosive or irritant to the eye

Justification for classification or non-classification

Taken all data together, Sakura Salicylate is classified as not corrosive and not irritant to the skin and eye according to Regulation (EC) 1272/2008.