Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 21st March 2018 to 23rd April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Acid Orange 061 (a)
IUPAC Name:
Acid Orange 061 (a)
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)
- Batches: 18-EKIN-016 (alive tissues) and 17-EKIN-46 (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: 17 April 2018 and 14 November 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 before the experiment, tissues were placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthic MaintenanceMedium. Culture plates were placed in the incubator at 37 °C, 5 % CO2 and saturated humidity.
- Maintenance Medium: SkinEthic; batch: 18-MAIN3-018
- AssayMedium SkinEthic; batches: 18-ESSC-010 and 18-ESSC-015

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 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 (13300 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. A MTT formazan calibration curve was performed in order to ensure that OD values obtained in the main experiment were within the spectrophotometer linear range.

PREDICTION MODEL / DECISION CRITERIA
- Mean relative viability ≤ 50% UN GHS Category 2 or 1
- Mean relative viability > 50% UN GHS No Category
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- 20 ± 2 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:
negative control (Live tissue): 3
positive control (Live tissue): 3
test item (Live tissue): 3
test item without MTT (Live tissue): 2
test item (Killed tissue): 2
negative control (Killed tissue): 2
test item without MTT (Killed tissue): 2

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: mean cell viability %
Value:
83
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS (Preliminary test)
- Direct-MTT reduction: yes
- Colour interference with MTT: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
The assay was considered valid as the following criteria were met:
– Blank controls: the mean Optical Density of Blank Controls was 0.044, lower than the maximum acceptable value (0.1).
– Negative controls: 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 value is considered the baseline value of the experiment and thus represents 100 % of cell viability).
– Positive controls: 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 = 1.3). 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.
– Test item: acceptable intra-replicate variability was obtained (SD of % viability = 9.5 lower than 18).

Any other information on results incl. tables

PRELIMINARY TEST

Direct MTT reduction test (Step 1)

A dark orange colour was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT.

Colouring potential test (Step 2)

A dark orange solution was observed; spectrophotometric 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 2.851, indicating that the test item has a potential interfering ability.

MAIN ASSAY

The NSMTT value was −3 %, while the NSCliving value was 2 %. Based on this result, only the OD-blank background subtraction was performed and the mean cell viability was 83 %, when compared to the negative control.

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 blank subtraction was 83 % 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 per se and of colouring water per se. It resulted that the test item could direct interact with MTT and has a potential interfering ability. Based on these results, 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, each measuring 0.38 cm^2 (treatment level: 53 mg/cm^2). Positive and negative controls (sodium dodecyl sulphate 5 % in water 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 (NSCkilled) was performed.

All the validity criteria were met.

The NSMTT value was −3 %, while the NSCliving value was 2 %, thus only the OD-blank background subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 83 % when compared to the negative control. Intra-replicate variability was acceptable.

Based on the results obtained, the test item is not classified as irritant to the skin.