Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from March 29th, 2017 to April 14th, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The complete read-across justification is detailed in section 13; source study has reliability 1.
Justification for type of information:
The complete read-across justification is detailed in section 13.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
GLP compliance:
yes

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:
other: commercial reconstructed human epidermis (RhE)
Cell source:
other: human derived epidermis keratinocytes
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Commercial Name: EPISKIN™ - 0.38 cm^2
- Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
- Batch: 17-EKIN-015 (alive tissues), 17-EKIN-012 (killed tissues)
- 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.
- Delivery date: 11st April 2017
- Examination at arrival:
Temperature indicator: pale grey (suitable for use)
pH indicator: orange (suitable for use)
- Preparation and pre-treatment incubation period:
i) 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.
ii) 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.
- Maintenance Medium: SkinEthic; batch: 17-MAIN3-014
- AssayMedium SkinEthic; batches: 17 ESSC 011 and 17-ESSC-014

REMOVAL OF TEST MATERIAL AND 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
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 4 hours at room temperature 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, a MTT formazan calibration curve was performed.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- 20 mg/epidermis unit (treatment level: 53 mg/cm^2).

NEGATIVE CONTROL
- At the treatment level of 20 µL/epidermis unit

POSITIVE CONTROL
- At the treatment level of 20 µL/epidermis unit

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):
A 42 ± 1 hour recovery period was allowed by incubation at 37 °C, 5 % CO2 and saturated humidity.
Number of replicates:
three for negative control, positive control, test item and two for test item without MMT

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: mean cell viability %
Value:
104
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

PRELIMINARY TEST

Direct MTT reduction test (Step 1)

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.

Colouring potential test (Step 2)

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 2.872, indicating that the test item has a potential interfering ability. Based on these results, additional controls were added in the Main Assay.

MAIN ASSAY

The mean Optical Density of Blank Controls was 0.039, lower than the maximum acceptable value (0.1). The negative control gave the expected baseline value (mean Optical Density value ≥ 0.6 and ≤ 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 (2 % of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 0.1). 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 1 %, while the non specific MTT reduction (NSMTT) was -1 %, thus only the blank subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 104 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.6 (lower than 18).

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 104 % when compared to the negative control.
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™, according to the the OECD Guideline 439 (2015). 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 in the test system being this reaction an index of cell viability. A preliminary test was carried out to evaluate the compatibility of the test item with the test system: for the abilities of reducing MTT and of colouring water per se. In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 mg/cm^2). Positive and negative controls (sodium dodecyl sulphate and D-PBS, respectively) were concurrently tested at the treatment level of 20 µL/epidermis unit. 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 Colour in killed tissue (NSC killed) was performed.

All the validity criteria were met. The colouring interference (NSC) was 1 %, while the non specific MTT reduction (NSMTT) was -1 %; thus only the blank subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 104 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.6.