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Toxicological information

Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 16, 2017 to October 19, 2017
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)
Principles of method if other than guideline:
This study was performed in order to evaluate the potential of test substancein a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study. The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of irreversible tissue damage caused by cytotoxic effects in the human cornea model. Strategic combinations of other accepted test methods with OECD Guideline 492 can be used as replacement of the in vivo Draize test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage or eye irritation potential. Eye irritant substances are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-iphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison with untreated negative controls is used to predict the eye irritation potential. The test was performed according to the protocol provided from MatTek Corporation.
GLP compliance:
yes (incl. QA statement)

Test material

Test material form:
solid: bulk

Test animals / tissue source

other: human-derived keratinocytes
Details on test animals or tissues and environmental conditions:
The EpiOcular tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
1) Tissue 51.7 mg
2) Tissue 49.8 mg
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:

Results and discussion

In vitro

Irritation parameter:
other: % relative tissue viability
Run / experiment:
6 h
Vehicle controls validity:
not valid
Negative controls validity:
Positive controls validity:
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

% Viability positive control and test substance:

Designation Positive Control Test substance
% Viability (Tissue 1) 39.90% 7.30%
% Viability (Tissue 2) 43.00% 10.30%
% Viability Mean 41.40% 8.80%

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met (Inconclusive)
Under the study conditions, based on the percentage viability of 8.8%, the test substance wasconsidered either eye irritant or inducing serious eye damage to the human eye.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the substance according to OECD Guideline 492, in compliance with GLP. Two tissues of the EpiOcular tissue model were treated with the test substance, positive control and negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50±2 mg of neat test substance. Sterile demineralised water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcular reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were performed at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 8.8%, the test substance was concluded to be irritant to the human eye. However further GHS classification Category 1 or 2 cannot be concluded based on the result. Under the study conditions, based on the percentage viability of 8.8%, the test substance wasconsidered either eye irritant or inducing serious eye damage to the human eye (Andres, 2018).