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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 12nd to 15th, 2018
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)
Version / remarks:
adopted 9th Oct. 2017
Principles of method if other than guideline:
Additional information was taken from:
- MatTek Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals, for use with MatTek Corporation’s Reconstructed Human EpiOcularTM Model, 14. July 2014
- Stern M., Klausner M., Alvarado R., Renskers K., Dickens M., 1998. “Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test”. Toxicology in Vitro 12, 455-461
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 4-oxovalerate
EC Number:
EC Name:
Ethyl 4-oxovalerate
Cas Number:
Molecular formula:
ethyl 4-oxopentanoate

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Description of the cell system used: commercially available EpiOcularTM tissue kit. The EpiOcularTM tissue consists of primary 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.
- Designation of the kit: OCL-200-EIT
- Day of delivery: 13. Mar. 2018
- Batch no.: 27027

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied: 50 μl
Duration of treatment / exposure:
28 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
two replicates
Details on study design:
- MTT solution: contains 1 mg/ml 3-(4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide, which can be reduced to a blue formazan by the viable cells of the tissue. A MTT stock solution of 5 mg/ml in DPBS buffer was prepared and stored in aliquots of 2 ml at – 20 ± 5 °C. 2 ml of the stock solution were thawed and diluted with 8 ml of as-say medium (resulting in 1 mg/ml). This MTT-solution with the concentration of 1 mg/ml was used in the test. For the pre-test (testing the ability of direct MTT reduction), the MTT concentrate in DPBS is thawed and diluted with serum-free MEM directly before use. For the main test, the MTT concentrate in DPBS is thawed and diluted with assay medium directly before use. Composition of the subset procured: KCl 0.2 g, KH2PO4 0.2 g
NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 litre. Composition of the subset prepared: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 litre.
- MEM with Phenol Red for Pre-Test: serum-free MEM (Minimum Essential Medium)
- Assay Medium for Main Test: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)

- Assessment of Direct Reduction of MTT by the test Item: the test item was tested for the ability of direct MTT reduction. To test for this ability, 50 μl of the liquid test item 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 ± 1 % CO2 and 80-100 % relative humidity for 3 hours. 1 ml of MTT solution plus 50 μl of sterile demineralised water are 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 Items: 50 μl of the liquid test item were added to 2 ml isopropanol, incubated in 6-well plates on an orbital shaker for 2 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 the mean OD for isopropanol, the mean OD of the test item solution was 0.00 (≤ 0.08). Therefore, the main test was performed without colourant controls.

- Preparations: on the day of the start of the experiment, the MTT stock solution was thawed. The MTT stock solution was diluted with the MTT solvent directly before use. The assay medium was pre-warmed to 37 ± 1 °C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 ml assay medium in the appropriate wells. All 24 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 will be 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 and 30 minutes.
- Exposure and Post-Treatment: after overnight incubation, the tissues were pre-wetted with 20 μl DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes. After that, 50 μl of the controls and the test item were applied in duplicate in 1-minute intervals. This was done in such a fashion that the upper surface of the tissue is covered. 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 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of the exposure time, the inserts were removed from the plates in 1-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 ml of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 ml assay medium. For post-treatment incubation, the tissues were incubated for 120 minutes 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-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was then incubated for 180 minutes at 37 ± 1 °C, 5 ± 1.0 % 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 sealed and then shaken for 2 hours at room temperature, protected from light.
- Measurement: 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-well-plate. Eight wells with 200 µl isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

- The test substance is considered to be irritant to the eyes or cause eye damage if the viability after the exposure is less than or equal to 60 %
- The test substance is considered to be non-irritant to eyes if the viability after the exposure is greater than 60 %.

Results and discussion

In vitro

Irritation parameter:
other: tissue viability %
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Values for negative control and for positive control were within the range of historical data of the test facility
- OD of negative control: 2.0
- % mean relative viability of positive control: 31.5 %
- Variation within replicates: 5.2 % (negative control); 0.3 % (positive control); 2.3 % (test item)

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate 1 2 3 4 5 6 7 8 Mean 
Absorbance  0.037 0.038 0.036 0.038 0.037  0.038 0.038 0.038 0.038

The absorbance values of negative control, test item and positive control are given in the following table:

Table: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation Measurement Negative Control Positive Control Test substance
Tissue 1  1 2.075 0.660 0.617
2 2.074 0.673 0.644
Tissue 2  1 1.898 0.656 0.681
2 2.046 0.667 0.672

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol (= corrected values).

Table: Mean Absorbance Negative Control, Positive Control and Test Item

Designation Negative Control Positive Control Test substance
Mean – blank (Tissue 1) 2.037 0.629 0.593
Mean – blank (Tissue 2) 1.934 0.624 0.639

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control

Designation Positive Control Test substance
% Viability (Tissue 1) 31.7 % 29.8 %
% Viability (Tissue 2) 31.4 % 32.2 %
% Viability Mean 31.5 % 31.0 %

Applicant's summary and conclusion

Interpretation of results:
other: the substance should be classified according to the CLP Regulation (EC) No.1272/2008 in Category 1 or Category 2
The substance is at least an eye irritant (Cat.2).
Executive summary:

The potential of the test item to evoke eye irritation was evaluated in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study according to the OECD Guideline 492.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes. 50 µl of the liquid test was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, 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.

The controls showed the following results: after treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD>0.8 and < 2.5, OD was 2.0. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 31.5 % (< 50 %). Variation within tissue replicates of the controls and test item was acceptable (< 20 %).


After treatment with the test item, the mean value of relative tissue viability was 31.0 %. This value is well below the threshold for eye hazard potential (≤ 60 %). Under the conditions of the test, the substance is considered either eye irritant or inducing serious eye damage in the EpiOcularTMEye Irritation Test.