Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2010 - May 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 SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. 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 model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC 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.
One 10-Minute treated tissue and one 60-Minute treated tissue were retained for possible tissue histology.
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

Test animals / tissue source

Species:
human
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
30 mg of the test item was topically applied to the corresponding tissues
Duration of treatment / exposure:
10 and 60-minute exposure periods
Duration of post- treatment incubation (in vitro):
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing DPBS. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 microL of maintenance medium (at room temperature) until all the tissues were rinsed (including the negative and positive control treated tissues). Following rinsing the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 microL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for three hours at 37°C, 5% CO2 in air.
At the end of the incubation period, the inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.
Number of animals or in vitro replicates:
Triplicate tissues were treated with the test item for exposure periods of 10 minutes and 60 minutes. 30 mg of the test item was topically applied to the corresponding tissues. The tissues were dosed at regular timed intervals, to allow for the period taken to rinse each insert following exposure, and to ensure each tissue received an equal exposure time. Duplicate tissues were treated with 30 μL of solution A to serve as negative controls for the 10 and 60-minute exposure periods. Duplicate tissues were treated with 30 μl of SDS 0.5% w/v to serve as positive controls for the 10 and 60-Minute exposure periods. In order to eliminate the possibility of volatile test items affecting the viability of other treated tissues, the plates were sealed with a gas permeable sealing membrane. The plates were incubated at 37°C, 5% CO2 in air for the respective exposure times.
Details on study design:
The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic Reconstructed Human Corneal model (RHC, SkinEthic Laboratories, Nice, France) following treatment periods of 10 and 60 minutes.
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. 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 model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC 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.
One 10-Minute treated tissue and one 60-Minute treated tissue were retained for possible tissue histology.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
10-Minute exposure
Value:
104
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
60-Minute exposure
Value:
104.9
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 relative mean viability of the test item treated tissues was 104.0% after a 10-Minute exposure period and 104.9% after a 60-Minute exposure period.
It was considered unnecessary to proceed with tissue histology.
Negative result, considered not likely to be a severe ocular irritant.