Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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 25 Jun. 2018
GLP compliance:
yes (incl. QA statement)
Landesamt für Umwelt, Mainz, Germany

Test material

Reference substance name:
Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester
EC Number:
Molecular formula:
Not applicable for UVCB
Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester

Test animals / tissue source

other: EpiOcular™
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : Commercially available EpiOcular™ kit
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 23. Jul. 2019
Batch no.: 30618
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 EpiOcular™ 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.
The certificate of analysis is included in the study report. No contaminants were detected. Tissue viability and the barrier function tests were within the acceptable ranges and indicated appropriate formation of the mucosal barrier and a viable basal cell layer.

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 at 37 ± 1°C
Duration of post- treatment incubation (in vitro):
120 min at 37 ± 1°C
Number of animals or in vitro replicates:
in duplicates for each treatment and control group
Details on study design:
- Details of the test procedure used
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 item, 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 ≥ 95% relative humidity for 1 h. 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 ≥ 95% relative humidity for 16 hours and 58 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 ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and the test item were applied in duplicate in one-minute- intervals. This was done in such a fashion that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue of each plate, the plate was transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. At the end of the 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 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 ≥ 95%
relative humidity.
After the post-treatment incubation, the MTT assay was performed.
- Description of the method used to quantify MTT formazan :
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 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. At last, the test item inserts were thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The inserts of the controls were set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plates were firmly sealed to avoid evaporation of the solvent and then stored in the refrigerator overnight. On the next day the plate was shaken for 2 hours at room temperature, protected from light. The inserts of the test item were pierced with an injection needle, taking care that all colour is extracted and the inserts were then discarded.
The inserts of the controls were discarded without piercing the tissues, as they were extracted in a 6-well-plate. 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 (Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH) at 570 nm. The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test item
To calculate the relative tissue viability, the following equation was used:
% Viability = [(OD corrected of test item or positive control) / (OD corrected of mean negative control)]
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
Eye hazard potential is assessed using the following criteria (according to guideline):
% Viability > 60%: non eye irritant / UN GHS classification: no category
% Viability ≤ 60%: at least eye irritant / UN GHS classification: No prediction can be made (category 1 or 2)
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : The values for negative control and for positive control were within the range of historical data of the test facility.
- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct : yes, historical data available and included in the report
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals :The validity of the EpiOcular test at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested.
- Positive and negative control means and acceptance ranges based on historical data :
Optical density negative control: mean 1.870, SD 0.272, range: 1.167 - 2.437
Relative tissue viability positive control: mean 32.1%, SD 7.3%, range: 12.4 - 57.2%
- Acceptable variability between tissue replicates for positive and negative controls : < 20%
- Acceptable variability between tissue replicates for the test chemical: <20%

Results and discussion

In vitro

Irritation parameter:
other: % tissue viability
mean value of two tissues (tissue 1 / tissue 2)
Run / experiment:
28 min exposure
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Visible damage on test system: not detected

DEMONSTRATION OF TECHNICAL PROFICIENCY: All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcular test was demonstrated.

- Acceptance criteria met for negative control: yes; Mean OD of negative control found = 2.0
- Acceptance criteria met for positive control: yes ; % mean relative viability of positive control found = 41.2%
- Variation within replicates: negative control 1.7%, positive control 3.3%, test item 6.2%;
The values for negative control, positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Any other information on results incl. tables

Table 1: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate 1 2 3 4 5 6 7 8 Mean
Absorbance 0.033 0.034 0.034 0.033 0.034 0.034 0.034 0.035 0.034

Table 2: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Negative Control  Positive Control Fatty acids, C18-
unsaturated, 1,6
Hexanediol Diester
Tissue 1  1 2.053 0.882 2.001
2 1.973 0.868 2.016
Tissue 2  1 1.965 0.818 1.880
2 1.996 0.802 1.892

Table 3: Mean Absorbance Negative Control, Positive Control and Test Item (corrected with mean absorbance value of isopropanol)

Designation  Negative Control  Positive Control Fatty acids, C18-
unsaturated, 1,6
Hexanediol Diester
Mean – blank
(Tissue 1) 
1.979 0.841 1.975
Mean – blank
(Tissue 2) 
1.947 0.776 1.852

Table 4: % Viability Positive Control and Test Item

Designation  Positive Control  Fatty acids, C18-unsaturated,
1,6 Hexanediol Diester
% Viability (Tissue 1)  42.8 100.6
% Viability (Tissue 2)  39.5 94.3
% Viability Mean  41.2 97.5

Table 5: Historical Data

Parameter  Optical Density
Negative Control
Relative Tissue Viability
Positive Control
Demineralised H2O  Demineralised H2O  Methyl acetate
Exposure time  30 minutes
Mean  1.870 32.1%
Standard deviation  0.272 7.3%
Range  1.167 - 2.437  12.4 - 57.2%
Study 19062801G891  1.963 41.2

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Under the conditions of the conducted test, the test substance did not possess irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.