Registration Dossier

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: According to official guidelines

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)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: V516690
Appearance: Brown powder
Batch: V516690/AY
Purity/Composition: LC-MSpurity (Area% UV-VIS): 95.8% (Reference: PA064492)
Test item storage: At room temperature
Stable under storage conditions until 17 July 2016 (expiry date)
Molecular formula: C49H49ClN2O6S2
MW: 861.52

Test animals

Species:
other: reconstructed human rpidermis (RhE) model
Details on test animals and environmental conditions:
The test system EPISKINTM 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 pen- etrate 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.

Alive tissues:
at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium. Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

Killed tissues:
a sufficient number of epidermis units were placed in a 12-well plate in which each well had previously been filled with 2 mL/well sterile water for injection. Tissues were incubated for approximately 48 hours, then transferred into a new plate and stored at -20°C. The day of the experiment, tissues were thawed at room temperature with 2 mL of maintenance medium.

Test system

Amount / concentration applied:
Live tissues treatments: Negative control, Positive control, 20 mg test item, and 20 mg test item treated tissues without MTT
Killed tissues: Negative control, 20 mg test item

Duration of treatment / exposure:
15 +/- 0.5 minutes
Observation period:
42h +/- 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity
Number of animals:
3 replicates per treatment
Details on study design:
Commercial Name: EPISKINTM - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch number Alive tissues: 15-EKIN-048
Batch number Killed tissues: 15-EKIN-039
Arrived at: 01 December 2015 (Alive tissues) and 30 September 2015 (Killed tissues)

Maintenance Medium: SkinEthic; batch: 15-MAIN3-052
Assay Medium: SkinEthic; batches: 15-ESSC-050 & 15-ESSC-045

The epidermis surfaces were moistened with 10 μL of sterile water before application of the test item.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
> 100
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure. Max. score: 100.0. Remarks: Viability for the test item was 145%. (migrated information)

In vivo

Irritant / corrosive response data:
The mean cell viability of the test item treated tissues, after the appropriate background subtraction, was 145.1%. Based on the results obtained, the test item V516690 is classified as not irritant to the skin.

Any other information on results incl. tables

The mean Optical Density of Blank Controls was 0.0415, lower than the maximum acceptable value (0.1). Using alive tissues, the negative control gave the expected variability [Standard Deviation (SD) of % viability lower or equal to 18] though the mean Optical Density value (OD = 0,57817) was continuous but not within the acceptable range (mean OD ≥0.6 and ≤1.5). However, since this value was very close to the minimum acceptable value stated in the OECD Guideline no. 439 (0.6) and taking into account the observed OD values for the test item treated tissues (higher than 0.6), this result was considered to be attributable to a chance event, thus the assay was accepted as valid. According to the method, the mean negative control value is considered the baseline value of the experiment and thus represents 100% of cell viability.

Positive control results indicated an appropriate cell death with an acceptable relative cell viability (4.8 % of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 2.2). Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.

The Non Specific Colour (NSC) induced by the test item was 1.8%, while the non specific MTT reduction (NSMTT) was -1.9%. Based on these results, only the OD-blank background subtraction was performed.

The test item did not induce cell death in any replicate with a mean cell viability of 145.1%, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 11.3 lower than 18).

Applicant's summary and conclusion

Conclusions:
The potential of the test item V516690 to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKINTM.
The blank negative and positive controls gave acceptable results and the study was accepted as valid.
The mean cell viability of the test item treated tissues, after the appropriate background subtraction, was 145.1%. Based on the results obtained, the test item V516690 is classified as not irritant to the skin.