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:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to determine the skin irritation potential of the test item using the SkinEthic Reconstructed Human Epidermal model (RHE, SkinEthic Laboratories, Nice, France) following treatment periods of 4 and 24 hours. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
Methods. The experimental design of the study consists of a test for direct reduction of MTT by the test item, followed by the main test.
For the main test, triplicate SkinEthic tissues were treated with approximately 25 mg of test item and exposed for 4 hours and 24 hours. The tissues were incubated at 37C in a humidified atmosphere of 5% CO2 in air for the appropriate exposure times.
Duplicate untreated tissues were used for each exposure period to serve as negative controls. Duplicate tissues treated with 50 μL of Triton X-100 (0.1% w/v) were used for the 24-Hour exposure period to serve as a positive control.
At the end of the 4-Hour exposure period each SkinEthic tissue
At the end of the 4-Hour exposure period each SkinEthic tissue was rinsed using Dulbecco’s Phosphate Buffered Saline (DPBS) and placed into a ‘holding plate’ until all the tissues had been rinsed. The rinsed tissues (2 per group) were then transferred to an MTT ‘loading plate’ and incubated at 37C for 3 hours in a humidified atmosphere of 5% CO2 in air. At the end of this time, each SkinEthic tissue was blotted dry and placed into an MTT ‘extraction plate’ in order to extract all of the reduced MTT from the tissues. The remaining test item treated tissue was retained for possible histology. The same rinsing, loading, extraction and retention procedures were repeated for the 24-Hour tissues once the 24-Hour exposure period was complete. The maintenance medium in each well of the 24-Hour exposure period treatment plates were retained for possible analysis of inflammatory mediator levels of IL-1alfa and IL-8.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N-{3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzene-1-sulfonamide
Cas Number:
1195765-45-7
Molecular formula:
C23H20F3N5O2S2
IUPAC Name:
N-{3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzene-1-sulfonamide
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: airlifted, living, multi-layered epidermal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionally equivalent to human epidermis in vivo
Cell source:
other: SkinEthic Reconstructed Human Epidermal model
Source strain:
not specified
Details on animal used as source of test system:
The SkinEthic RHE model incorporates several features which make it advantageous in the study of skin irritancy potential. The model consists of an airlifted, living, multi-layered epidermal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionally equivalent to human epidermis in vivo. Test items are applied directly to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly.
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
Cytotoxicity was determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues (quantitative measurement of tissue viability) relative to the negative control.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent no treatment
yes, concurrent MTT non-specific colour control

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4-Hour exposure
Value:
105
Negative controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
24-Hour exposure
Value:
108
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a Non-Irritant.
GHS: Considered not to be a significant irritant.