Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0011440677

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal
epithelium . The EpiOcularTM tissues are cultured on cell culture inserts and are commercially available as kits containing 24 tissues on shipping agarose. The keratinocytes are tested free of bacteria, yeast, and other fungi.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
2
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ OCL-200 kit (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia)
- Doses of test chemical and control substances used: 50 µL undiluted test substance, NC and PC
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 30 min at 37°C, 2 hours post-incubation in medium
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled): 2
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device: SunriseTM Absorbance Reader, for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter.
- Description of the method used to quantify MTT formazan:
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours.Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
- Reference to historical data of the RhCE tissue construct: yes
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes
- Positive and negative control means and acceptance ranges based on historical data: yes
- Acceptance criteria for the NC:
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is >0.8. The mean OD570 of the NC should not exceed 2.5.
-Acceptance criteria for the PC:
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptance criteria for tissue variability:
Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20%

-Evaluation of results EPiOcular: The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.

Results and discussion

In vitro

Results
Irritation parameter:
other: % viability
Run / experiment:
30 min exposure/2 h post-incubation
Value:
89.1
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
23.4%
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met