Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 15 - August 7, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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)
GLP compliance:
yes (incl. certificate)
Remarks:
GLP cokpliance certificate included in full study report

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Details on test material:
Identification: [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids
Appearance: Brown semi-solid gel (at 20°C)

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
EpiOcular™ tissues, Lot No. 27430 Kit E, were received from MatTek on 22 May 2018 and refrigerated at 2-8°C. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium for an additional overnight equilibration of 16 to 24 hours. After the overnight incubation, the tissues were moistened with 20 μl of phosphate-buffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 μl of the test article were applied to duplicate EpiOcular™ tissues. Each treatment with test article or control was conducted in duplicate.
Duration of treatment / exposure:
The tissues were incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.
Duration of post- treatment incubation (in vitro):
After dosing and incubation, the tissues were rinsed with phosphate-buffered saline (PBS) and soaked in 5 ml of room-temperature assay medium in a 12-well plate for 12 minutes. The soaked tissues were then incubated in fresh assay medium at 37±1°C, 5±1% CO2 for 120 minutes. At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in Dulbecco's Modified Eagle's Medium). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiOcular™ tissue was rinsed with PBS, and then treated with 2.0 ml of extractant solution (isopropanol) overnight at room temperature.

Results and discussion

In vitro

Results
Irritation parameter:
other: Tissue viability
Run / experiment:
Mean tissue viability of two treated tissues.
Value:
45.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
The test item is determined to be an irritant based on a mean tissue viability less than or equal to 60% according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”.
Other effects / acceptance of results:
The mean OD570 of the negative control tissues was 2.022, which met the acceptance criteria of greater than 0.8 and less than 2.5. The mean relative viability of the positive control tissues was 35.3%, which met the acceptance criterion of less than 50%. The differences in viability between identically treated tissues were 0.21% to 8.33%, which met the acceptance criterion of less than 20%. All controls passed the acceptance criteria for a valid study.

Any other information on results incl. tables

The summarized results and irritation classifications are as follows:

 Test and Control Article Identity

 Mean Tissue Viability

 Tissue Viability Std. Deviation

Irritancy

Classification

GHS Calssification 
 [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids  45.8% 8.3%  Irritant   Eye Irritation Category 1 or 2
 Tissue culture water (Negative Control)  100% 0.2%  Non-Irritant   Not classified
 Methyl acetate (Positive Control)  35.3% 1.3%   Irritant   Eye Irritation Category 1 or 2

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the outcome of a Reconstructed Human Cornea-like Epithelium (RhCE) Test Method performed according to OECD 492 guideline and GLP principles, it is concluded that the substance is irritanting to the eye.
Executive summary:

A Reconstructed Human Cornea-like Epithelium (RhCE) Test Method was performed with the substance according to OECD guideline 492 and GLP principles. The substance was applied undiluted (50 μL/ tissue, n=2). The mean in vitro tissue viability of the positive control was 35.3%, and the mean in vitro tissue viability of the negative control (tissue culture water) was 100%. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The mean viability of tissue exposued to the test substance was 45.8%. Since the mean tissue viability of the treated tissues was ≤60%, the substance is considered an eye irritant and should be classified as either Category 1 or 2 according to Regulation (EC) No 1272/2008.