Registration Dossier

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Specific details on test material used for the study:
Batch no.: VE00364716
Storage conditions: room temperature protected from light
Expiry date: 02 April 2017
RTC number: 14844

In vitro test system

Test system:
human skin model
Source species:
Cell source:
other: EPISKIN Reconstructed human Epidermis (RhE)
Details on animal used as source of test system:
Commercial Name: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch: 16-EKIN-019 (alive tissues) and 16-EKIN-018 (killed tissues)
Arrived at RTC on: 10May 2016 and 03May 2016

The test system was shipped onMonday 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 SkinEthicMaintenance 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.
unchanged (no vehicle)
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 µl 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 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

Treatment: In Main Assay, alive tissues were treated with the test item, positive and negative controls.

Exposure period: An exposure time of 15±0.5 minutes was allowed in a ventilated cabinet at room temperature.

Washing: At the end of the exposure, each tissue was rinsed with approximately 25mL 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 2mL/well of maintenance medium.

Post-exposure period: A 42 ± 1 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
Amount/concentration applied:
In theMain Assay, the test item was applied as supplied in three replicates at the treatment level of 20 µL/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 µL/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit.
Number of replicates:
Three replicates

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
mean cell viability
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
The mean Optical Density of Blank Controls was 0.046, lower than the maximum acceptable value (0.1). The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 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 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 (5.15% of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 3.50). 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 overall interference, calculated as [(NSMTT +NSCliving) - NSCkilled], was -4.36%. Based on this result, only the OD-blank background subtraction was performed and the mean cell viability after treatment with test item was 56.1%, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 7.19 lower than 18).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The potential of the test item BACCARTOL CRUDE 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 blank subtraction, was 56.1%. Based on the results obtained, the test item BACCARTOL CRUDE is classified as not irritant to the skin.