Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 October 2017 to ****
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS number: Not yet known
EC Number: Not yet known
Specific details on test material used for the study:
Purity: >93%
Physical state/Appearance: Dark amber viscous liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis tissues
Cell source:
other: not specified
Details on test system:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker at room temperature for 15 minutes to homogenize the released mediators in the maintenance medium. Maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator IL-1α determination.

MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled micro tubes containing acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer and then refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Control samples:
other: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Amount/concentration applied:
10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the exposure period each tissue was rinsed before incubating for 42 hours.
Number of replicates:
Three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
86.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that negligible direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results.

 

Assessment of Color Interference with the MTT Endpoint

The test item did not change the color of the solution. However the test item was an amber viscous liquid which may have adhered to the tissue culture surface during the main test and thus cause color interference. Therefore, as a precaution, color correction tissues were incorporated into the test to correct for this possibility. However, the results obtained showed that negligible color interference occurred (Appendix 3). It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 86.1% (>50%) after a 15 Minute exposure period and 42 hour post exposure incubation period. Traces of residual test item remained on the test item treated tissue culture surfaces after rinsing due to viscosity.

 

Table 1: Mean Viabilities of the EPISKIN™ Tissues

Treatment

OD570
(Mean ± SD)

Relative Tissue Viability
(Mean ± SD)

Test Item

0.701 ± 0.044

86.1% ± 5.4%

DPBS (Negative Control)

0.814± 0.049

100%± 6.0%

0.5% SDS (Positive Control)

0.302 ± 0.044

37.1% ± 5.4%

 

Quality Criteria

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 37.1% relative to the negative control treated tissues and the standard deviation value of the viability was 5.4% (≤18%). The positive control acceptance criteria were therefore satisfied.

 

The mean OD570 for the negative control (DPBS) treated tissues was 0.814, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 6.0% (≤18%). The negative control acceptance criteria were therefore satisfied.

 

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.4% (≤18%). The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).
Executive summary:

The skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model. Reconstructed human epidermal cultures were treated topically in triplicate with the test material for 15 minutes, rinsed, and then incubated post-exposure for 42 hours. Cell viability was measured by means of the colorimetric MTT reduction assay, in which viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue/purple formazan salt within the mitochondria of viable cells. 

The test item was found to directly reduce MTT; therefore additional non-viable tissues were incorporated for correction purposes. The test item was found to have the possibility to cause color interference with the MTT endpoint; therefore, as a precaution, additional tissues were incorporated to correct for this. A third set of controls was included, comprising non-viable tissues, to prevent a double correction for a colored test item that also reduces MTT.

At the end of the 42-hour post exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals. At the end of the formazan extraction period each tube was mixed thoroughly, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate, and the optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 86.1%, greater than 50%, and therefore, the test item was considered as non-irritant to skin and was not classified according to GHS. The quality criteria required for acceptance of results in the test were satisfied.