Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 14th of February to the 2nd of April, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKIN™
Commercial Name EPISKIN™ - 0.38 cm2
Supplier SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch 17-EKIN-014 (alive tissues) and 15-EKIN-050 (killed tissues)
Arrived at RTC on 04 April 2017
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.
– 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. Tissueswereincubatedforapproximately48hours, then transferred into a new plate and stored at -20°C. The day of the experiment, tissues were thawed at room temperature with 2mL of maintenance medium.
Vehicle:
other: maintenance medium, assay medium
Details on test system:
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 mg of test item at 37°C, 5% CO2 and 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 mg of the test item was added to 180 µL of distilled water (Baxter; batch no. 15I0211) 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
Alive tissues were treated with the test item, positive and negative controls.

Washing
At the end of the exposure, each negative and positive control tissue was rinsed with approximately 25mL of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and sample tranferred in new wells pre-filled with 2 mL/well of manteinance medium.

Post-exposure period
A 42 ± 1 °C hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

MTT staining
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 ensure the spectrophotometer linear range, an MTT formazan calibration curve was performed.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Duration of treatment / exposure:
An exposure time of 15±0.5 minutes was allowed in a ventilated cabinet at room temperature.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
101
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

PRELIMINARY TEST

In a first step, the test item was assayed for the ability of reducing MTT per se. In a first step, the test item was assayed for the ability of reducing MTT per se. A dark brown suspension, with a dark brown precipitate, was observed at the end of the incubation period when the test item was added to the MTT solution, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A dark brown suspension was observed; 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 3.153, indicating that the test item has a potential interfering ability. Based on the results, additional controls were added in the Main Assay.

MAIN TEST

The mean Optical Density of Blank Controls was 0.037, lower than the maximum acceptable value (0.1).

The negative control gave the expected baseline value (Optical Density values means > 0.6 < 1.5) 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 = 0.5). 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 colouring interference (NSC) was 7%, while non specific MTT reduction (NSMTT) was 2%. Thus the correction for colouring interference was performed.

The test item did not induce cell death in any replicate, the mean cell viability, after appropriate subtraction, was 101% when compared to the negative control.

Intra-replicate variability was acceptable with a SD of % viability value lower than 18 (4.6%) as stated in the Study Protocol.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Remarks:
Classification criteria according to the CLP Regulation 1272/2008 and its amendments
Conclusions:
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 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 101.8%.
Based on the results obtained, the test item is classified as not irritant to the skin.
Executive summary:

Method

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 experimental procedures are based on the OECD Guideline for testing of chemicals no. 439.

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. In a second step, the test item was assayed for the ability of colouring water per se.

Based on the results, additional controls were added in the Main Assay.

A dark brown suspension was observed, and additional controls were added in the main assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit (12.1 mg of active constituent/epidermis unit), with positive and negative controls.

In order to verify if the test item results had to be corrected, the non specific colour( (NSC) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colouring killed tissue (NSC killed) was performed.

Observations

The test item did not induce cell death in any replicate, the mean cell viability after appropriate subtraction was 101 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 4.6 (lower than 18, as stated in the Study Protocol).

Conclusions

The substance is classified as not irritant to the skin.