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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 8-June 18, 2020
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability :
The test system can determine the potential of the substance to cause eye irritation without the need for animal testing.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell line:
The cell system was a reconstructed human cornea-line epithelium model with at least three layers of cells. The surface was non-keratinized with a cornea like structure. No biological contaminants were detected.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
30 minutes
Number of animals or in vitro replicates:
Details on study design:
- Details of the test procedure used :
- RhCE tissue construct used, including batch number : MaTek, batch no.: 30663
- Doses of test chemical and control substances used : 50 uL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 37 degrees C
- Justification for the use of a different negative control than ultrapure H2O (if applicable) : Distilled water was used.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Methyl acetate was used.
- Description of any modifications to the test procedure : During exposure, the well plates with test substance were incubated in a safety hood.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Controls for MTT-reducers and coloring test substances were not used since the tissue viability was less than 60%, and the ODnet was less than 0.08.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 +/- 30 nm.
- Description of the method used to quantify MTT formazan: Spectrophotometry
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) :
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
Mean tissue viability <=60% irritant (no prediction can be made
Mean tissue viability >60% non-irritant
Based on UN GHS classification.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria :
- Complete supporting information for the specific RhCE tissue construct used : Yes
- 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
- Positive and negative control means and acceptance ranges based on historical data : Postive control - mean relative viability 27.1%, range 2.1-52.1%; postive and negative controls - standard deviation viability mean 6.4%, range 0.0-19.8%
- Acceptable variability between tissue replicates for positive and negative controls :
Mean tissue viability for the positive control <50%
Mean absolute optical density of the negative control is >0.8 and < 2.8.
- Acceptable variability between tissue replicates for the test chemical:
Relative tissue viabillity difference < 20%.

Results and discussion

In vitro

Irritation parameter:
other: mean tissue viability %
Run / experiment:
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
The mean tissue viability was 16.4% of negative controls. The ocular irritation potential could not be predicted by the study. Therefore, further testing is needed.
Executive summary:

The potential of the test substance to cause irritation to eyes was studied in an OECD Guideline 492 study. The test substance was applied undiluted to reconstructed human cornea-like epithelium. Negative controls (distilled water), and positive controls (isopropanol) were also tested. The test was done in duplicate. The tissues were exposed for 30 minutes. The mean tissue viability of the test substance was 16.4% of negative controls, which shows potential eye irritant effects. Since this study alone cannot classify a substance as an eye irritant according to UN GHS guidelines, further testing will be needed to determine the eye irritancy potential of the test substance.