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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 September 11, 2017 to September 28, 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
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)

Test material

Test material form:
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 % (UVCB); Appearance: amber, homogeneous liquid

Test animals / tissue source

other: EpiOcularTM tissue
Details on test animals or tissues and environmental conditions:
Commercially available EpiOcularTM kit.
The EpiOcularTM 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.

EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Batch no.: 27006

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Tissue 1: 51.1 mg
Tissue 2: 51.6 mg
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
Details on study design:
Assessment of direct reduction of MTT by the test substance:
50.9 mg of the test substance was added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 μL of H2O demin was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Assessment of coloured or staining test substances:
51.9 mg of the test substance was added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 3 hours at room temperature. Then, two 200 μL aliquots of the resulting solution and two 200 μL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of OD for isopropanol, the OD of the test substance solution was 0.00 (≤ 0.08). Therefore, the main test was performed without colourant controls.

Main Test:
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1 °C. 6-well-plates were labelled with test substance, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.

Exposition and Post-Treatment:
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 μL of the controls and a defined amount of the test substance were applied in duplicate in 1- min- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.

After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

MTT Assay and Extraction:
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.

The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm

Results and discussion

In vitro

Irritation parameter:
other: tissue viability (%)
Run / experiment:
6 h
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
Validity criteria:
OD of negative control: >0.8 and <2.5, result: 1.6
% mean relative viability of positive control: < 50 % of negative control, result: 42.2 %
Variation within replicates: < 20 %,
0.2 % (negative control)
3.5 % (positive control)
0.4 % (test substance)

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Under the study conditions, the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcularTM eye irritation test.
Executive summary:

A study was conducted to determine an eye irritation potential of the tests substance according to OECD Guideline 492, in compliance with GLP. The test substance was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 h. The jellylike test substance was applied to two tissue replicates (51.1 and 51.6 mg). After treatment, the respective substance was rinsed from the tissue and cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the mean absorbance values was 1.6, being within the required acceptability criterion of mean OD >0.8 and <2.5. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 42.2% (<50%). Variation within tissue replicates was acceptable (<20%). After treatment with the test substance, the mean value of relative tissue viability was reduced to 2.4%. This value is below the threshold for eye irritation potential (≤60%). According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required. Under the study conditions, the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcularTM eye irritation test (Andres, 2017).