Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
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:
Jun. 2018
Deviations:
yes
Remarks:
Minor non-critical deviation related to the use of a 24-well plate for negative and positive controls.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: According to OECD TG492, the EpiOcularTM eye irritation test is able to correctly identify chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.

- Description of the cell system used: 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 cul-tured 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

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): neat
Duration of treatment / exposure:
Exposure time: 28 minutes.
Duration of post- treatment incubation (in vitro):
None.
Number of animals or in vitro replicates:
2 replicates
Details on study design:
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
- Designation of the kit: OCL-200-EIT
- Day of delivery: 07. May 2019
- Batch no.: 30605


Pre-tests:
1) 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 test item were 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 ≥ 95% relative humidity for 3 hours. 1 mL of MTT solution plus 50 µL of water 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.

2) Assessment of Coloured or Staining Test Items:
50 µL of the 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.


Main test:
Preparations:
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 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 ≥ 95% relative humidity for hours.

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 way 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, all plates were 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 respec-tive 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.

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 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At last, the two replicates of the test item were thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert.
The replicates of the negative and positive control 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 plates were firmly sealed to avoid evaporation of the solvent and stored in the refrigerator overnight. On the next day the 6-well-plate and the 24-well-plate were 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.

Results and discussion

In vitro

Results
Irritation parameter:
other: % tissue viability
Run / experiment:
mean of 2 replicates
Value:
93.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the EpiOcularTM test at LAUS GmbH was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

ACCEPTANCE OF RESULTS:
- Mean optical density of negative control: 1.9 -- acceptance criterion: > 0.8 and < 2.5 --> accepted
- % Mean relative viability of positive control: 29.0% -- acceptance criterion: < 50% of negative control --> accepted
- Variation within replicates: 0.3% (negative control), 0.5% (positive control), 3.6% (test item) -- acceptance criterion: < 20% --> accepted



Any other information on results incl. tables

Historical data

 Parameter

Optical Density

Negative Control

Relative Tissue Viability

Positive control

 

Demineralised water 

Methyl acetate

Exposure time 

30 minutes 

Mean 

1.873 

 32.1%
Standard deviation  0.278  7.5% 
 Range 1.167 - 2.437   12.4 - 57.2%
 This study 1.871  29.0% 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item "Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides" is found to be non-eye irritant in the EpiOcularTM Eye Irritation Test.
Executive summary:

Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

After treatment with the test item, the mean value of relative tissue viability was reduced to 93.8 %. This value is above the threshold for eye irritation potential (≤ 60%).

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.9 (> 0.8 and < 2.5).

The positive control induced a decrease in tissue viability as compared to the negative control to 29.0%. Variation within the replicates of the controls and the test item was acceptable (< 20%).

For these reasons, the result of the test is considered valid.