[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings (Deutschland) GmbH, BU Additives

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Details on test system:

SKIN DISC PREPARATION
- Procedure used: EpiSkin is an in vitro reconstructed human epidermis (RHE) from normal human keratinocytes cultured on a collagen matrix at the air liquid interface. This is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Differentiated and stratified epidermis model comprises the main basal, supra basal, spinous and granular layers and a functional stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
- Quality control for skin discs: The human keratinocytes come from mammary samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, B and C hepatitis tests were carried out on the donor bloods as well as verification of the bacteriological and fungal sterility of the cells and absence of mycoplasma.
The technical data, safety sheet and certificate of analysis of the EpiSkin kit used in this study

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The epidermis units were transferred to 12-well plates containing 2 mL of pre-warmed maintenance medium and incubated in a CO2 incubator for approximately 3 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the 15 minutes exposure at room temperature, the epidermis units were removed from the wells and thoroughly rinsed with PBS to remove residual matters, if any, from the epidermal surface.

After rinsing each of these epidermal units were gently tapped on an absorbent paper to remove the remaining PBS. The surface gently swabbed with a cotton swab and then placed in a 12-well plate pre-filled with 2 mL pre-warmed assay medium to rinse. After rinsing, all the epidermis units were placed on an absorbent paper to remove excess assay medium and then placed back to the same 12-plate pre-filled with pre-warmed maintenance medium.

DYE BINDING METHOD
- Dye used in the dye-binding assay: [MTT:] After incubation, the epidermis units were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL) and the plates were incubated for 3-hr at 37°C in a carbon dioxide incubator with 5% CO2
- Spectrophotometer: 200 μL sample from each tube was transferred into the wells of a labeled 96-well flat bottom plate (2 wells/epidermis unit) and the amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Microplate Reader, using acidified isopropanol solution as the blank.
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Ten microliters (10 μL) of Phosphate Buffered Saline (PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Ten milligram (10 mg) of 98.5% pure Sodium Dodecyl Sulfate (SDS) in powder form

Duration of treatment / exposure:

The epidermis units were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL) and the plates were incubated for 3-hr at 37°C in a carbon dioxide incubator with 5% CO2.

Number of replicates:

Three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 145.54 %.

Value:

> 145.54

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction by exposing it in direct contact with the MTT solution.
A quantity of 10 mg of the test item was mixed in a glass tube with 2 mL of MTT solution (0.3 mg/mL) along with water as control and mixed. The resulting mixture was incubated for 3 hours in a carbon dioxide incubator at 37°C protected from light. After the incubation period, the color of the MTT solution was checked.

- Colour interference with MTT: The test item was evaluated for its intrinsic color or ability to become colored in contact with water (simulating a tissue humid environment).
A quantity of 10 mg of the test item was mixed with 90 µL water in a glass tube, kept for 15 minutes at room temperature and then visually observed for color development, if any.


DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: It is considered that the NC meets the acceptance, if the mean OD value of the 3 tissues is > 0.6 and the Standard Deviation value (SD) of the % viability is < 18.
- Acceptance criteria met for positive control: It is considered that the Positive Control (SDS) meets the acceptance if the mean viability expressed as % of the NC, is < 40% and the SD is < 18.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for the test item, Licocare RBW 300 FL TP was 145.54 % and hence, in according with UN GHS it is predicted to be non-irritant under the experimental conditions described in this report.

Executive summary:

The test item, Licocare RBW 300 FL TP was tested for its possible skin irritation potential using a three dimensionalReconstructed Human Epidermis model,EpiSkin, through topical application for 15 minutes.

The test item is in solid form and was applied directly on the top of the skin tissues at 10 mg/tissue, followed by moistening with 5 µL of the negative control, and exposed for 15 minutes.  Ten microliters (10 µL) of PBS and 10 mg of SDS were used as the negative and positive controls, respectively.

After a 42 hour post-incubation period, irritation potential of Licocare RBW 300 FL TP was evaluated by assessing the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues.

The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was 0.557287. The test item had a mean cell viability of 145.54 % after 15 minutes exposure. The positive control had a mean cell viability of 14.09567 % after 15 minutes exposure, indicating that the test system functioned properly.

 

The study indicated that the test item Licocare RBW 300 FL TP is in accordance with UN GHS predicted to be non-irritant in this In Vitro Skin Irritation Test using Reconstructed Human Epidermis under the conditions of testing employed.