Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6th September 2018 to 30th November 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

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)
GLP compliance:
no
Remarks:
The protocol meets the requirements of the OECD guideline, "In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method" (TG 439).

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl {5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamate
EC Number:
943-940-5
Cas Number:
1869118-24-0
Molecular formula:
C16H16N4O4S
IUPAC Name:
ethyl {5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamate
Test material form:
solid: crystalline

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Justification for test system used:
The protocol meets the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to a test article.
The test article is a solid and was not evaluated in the mesh compatibility test.
The test articles, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period.
25 μL of sterile CMF-DPBS were added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.
The MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After a 3-hour incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2ml isopropanol per tissue and the optical density of the extracted formazan is determined with a spectrometer at 570nm. Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Since the test article is a solid, 25 μL of sterile CMF-DPBS were added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.
Duration of treatment / exposure:
The test articles, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period.
Duration of post-treatment incubation (if applicable):
42-hour post-exposure expression period
Number of replicates:
Triplicate

Test system

Details on study design:
Since the test article was a solid, it was not evaluated in the mesh compatibility test.
The test article was not observed to directly reduce MTT in the absence of viable cells.
The test article was not considered to have probable photometric MTT interference.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
94.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
94.9%±7.51
Other effects / acceptance of results:
The assay was accepted when the following criteria were met: 1) the positive control (5% SDS) resulted in a mean tissue viability ≤ 20%, 2) the mean OD570 value of the negative control tissues was ≥ 0.8 and < 2.8, and 3) the standard deviations of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were < 18%.

The mean tissue viability of the positive control was 4.03% ±0.29.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A test article was predicted to be an irritant (GHS Category 1 or 2) when the mean relative viability of the triplicate-treated tissues is ≤50% of the mean viability of the negative control. A test article was not predicted to be a skin irritant (GHS No Category) when the mean relative viability of the triplicate tissues was >50%.
The test article had a value of 94.9% ± 7.51, therefore is classed as a non-irritant.
Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to a test article1. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)2.

 

The test article, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period. Since the test material is a solid, 25 μL of sterile CMF-DPBS was added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.

 

Residual test article was suspected on the tissues treated with the test material, after the rinsing process. The test article was attempted to be removed from the exposed EpiDerm™ tissues using cotton-tipped applicators soaked in CMF-DPBS. The residual was observed to remain on the tissues. The test article prolonged the exposure to the tissues, which may have influenced the toxic effect; however, all of the tissue viabilities were >50%, therefore, there was no significant impact on the final prediction for the test articles.

 

A test article was predicted to be an irritant (GHS Category 1 or 2) when the mean relative viability of the triplicate-treated tissues is ≤50% of the mean viability of the negative control. A test article was not predicted to be a skin irritant (GHS No Category) when the mean relative viability of the triplicate tissues was >50%. The mean viability of the test articles was 94.9% ± 7.51, therefore the test material is classed as a non-irritant.