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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from May 17th to July 4th, 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Justification for test system used:
The test system 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.
Details on test system:
Commercial Name: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch: 16-EKIN-026

Functional controls
Quality controls: histology scoring, magnitude of viability and barrier function (IC50 determination).
Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.

Preparation of the Test System
- Examination at arrival
Temperature indicator: pale grey (suitable for use)
pH indicator: orange (suitable for use)
- Preparation and pre-treatment incubation period
The test system was shipped on Monday and received on Tuesday. According to the supplier procedure, tissues were prepared as follows: 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.
Maintenance Medium: SkinEthic batch: 16-MAIN3-041
Assay Medium: SkinEthic batches: 16 ESSC 019 and 16-ESSC-026

PRELIMINARY TEST DIRECT MTT REDUCTION TEST (Step 1)
Non-specific reduction of MTT was evaluated as follows: two ml of MTT ready-to-use solution (0.3 mg/ml) was incubated with 20 µl of test item at 37 °C, 5 % CO2and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

COLOURING POTENTIAL TEST (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 20 µl of the test item was added to 180 µl of distilled water (Baxter; batch no. 15D2902) in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Colouring of the solution/suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm.

MAIN ASSAY
In the Main Assay, alive tissues were treated with the test item, positive and negative controls. At the end of the exposure, each tissue was rinsed with approximately 25 ml of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 ml/well of maintenance medium.
Each tissue insert was incubated with 2 ml/well of MTT ready-to-use solution. Plates were incubated for approximately 3 hours at 37 °C, 5 % CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µl of acidic isopropanol. Tubes were preserved for approximately 3 days at 4 °C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µl from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200 µl) of acidic isopropanol were analysed and used as blank. In order to verify the linearity range of the spectrophotometer, ensuring that the MTT formazan calibration curve was still appropriate, on the day of the experiment two quality controls were assayed. These quality controls were prepared diluting MTT formazan in acidic propanol at concentrations of 0.3 and 0.05 mg/ml.

CONTROL ITEMS
Positive control item was 5 % (w/v) sodium dodecyl sulphate (SDS) solution, obtained by 1:1 dilution in sterile water (Baxter, batch no. 15D2902) of a sterile commercial 10 % (w/v) SDS solution in water (SIGMA, batch no. BCBP9757V). Negative control item was D-PBS (GIBCO, batch no.1734656). Positive and negative control items were obtained commercially and characterised by labelling. Determination of the stability and concentration of solutions of the positive control were not undertaken since it is sufficient to provide evidence for the correct expected response of the test system to it.

EVALUATION CRITERIA
Mean relative viability ≤ 50%: Irritant
Mean relative viability > 50%: Not irritant
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 µl per well for negative and positive controls and for test item
Duration of treatment / exposure:
An exposure time of 15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature
Duration of post-treatment incubation (if applicable):
42 ± 1 hour recovery period was allowed by incubation at 37 °C, 5 % CO2 and saturated humidity
Number of replicates:
3 for negative control, 3 for positive crontrol and 3 for test item

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
60
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
63.4
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
61.4
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

In a first step, the test item was assayed for the ability of reducing MTT per se. No colour change and absence of particle in suspension were noticed in the MTT solution at the end of the incubation period, indicating that the test item could not direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. No colour change was noticed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 0.069, indicating that the test item does not have a potential interfering ability. Based on these results, no additional controls were added in the Main Assay.

In the main assay, the mean Optical Density of Blank Controls was 0.0, lower than the maximum acceptable value (0.1). The negative control gave the expected baseline value (Optical Density values of the three replicates higher or equal to 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean 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% of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 1.9). 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 mean cell viability of the test itme was 62 %, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 1.7 lower than 18).

Applicant's summary and conclusion

Interpretation of results:
other: not irritant to the skin according to the CLP regulation (EC) No 1272/2008)
Conclusions:
Not irritant to the skin
Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The study was performed according to the OECD Guideline 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. Before the main assay, the substance was tested for its direct MTT reduction potential and for its ability of colouring water per se.

The blank, negative and positive controls gave acceptable results and the study was accepted as valid. After appropriate subtraction, the mean cell viability of the test item treated tissues was 62 %. Based on the tissue viability obtained, the substance is not considered to be a skin irritant.