Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 12. Mar. 2018 to 26. Apr. 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
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:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Acid Orange 061 (a)
IUPAC Name:
Acid Orange 061 (a)
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
Commercially available EpiOcularTM kit was 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 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 cm^2.
- Origin: EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Myln-ské Nivy 73, 82105 Bratislava, Slovakia.
- Main Test:
Designation of the kit: OCL-200-EIT
Day of delivery: 13. Mar. 2018
Batch no.: 27027

Additional Test on Coloured Test Items:
Designation of the kit: OCL-200-EIT
Day of delivery: 24. Apr. 2018
Batch no.: 27036

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
-Tissue 1: 50.9 mg (Main test), 52.4 mg (Additional Test)
-Tissue 2: 51.1 mg (Main test), 53.7 mg (Additional Test)
Duration of treatment / exposure:
6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity
Duration of post- treatment incubation (in vitro):
18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity
Number of animals or in vitro replicates:
2 tissue replicates
Details on study design:
PRE-TESTS
-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, 53.0 mg of the solid 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 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 Items
52.4 mg of the test item was added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature.
As the extract solution was turbid, it was centrifuged (30 seconds at 16 000* g).
Then, two 200 µL aliquots of the supernatant 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 item solution was 0.474 (>0.08). The test item was possibly interacting with the photometrical measurement and an additional test on colourant controls was performed.
The additional test was performed in order to evaluate the amount of colour bound to the tissues. The test item was applied to two additional tissues (= colourant controls) and the test was performed in the same way as described for the main test, but no MTT assay was performed: instead of 300 µL MTT solution, 300 µL assay medium was used. The bound colour is extracted and the absorbance of the isopropanol extract was measured in the same fashion as in the MTT assay for coloured test items (without piercing the tissues).
As the colourant control result is ≤ 50% of the viable negative control, a data correction procedure was performed.

THE 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 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 Ca2+/Mg2+ -free-DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes. After that, 50 µL of the controls and a defined amount of the test item were applied in duplicate in one- minute- 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 at least three times with Ca2+/Mg2+ -free 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 180 minutes 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 was 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.
-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.

EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.
I) Calculation
-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
(Note: Corrected OD value of negative control corresponds to 100 % viability)
To calculate the relative absorbance, the following equation was used:
% Viability = [OD corrected test item/OD corrected mean negative control] x 100
- Additional Test for Coloured Test Items
% Viability (colourant-control): [OD corrected test item (colourant control)/OD corrected mean negative control] x 100 %

Results and discussion

In vitro

Results
Irritation parameter:
other: % viability mean (corrected)
Value:
106.9
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
VALIDITY CRITERIA
The experiment is considered as valid if:
- optical density OD of the negative control is > 0.8 and < 2.5
- mean relative viability of positive control is < 50 % of negative control viability
- the difference in % viability (compared with the negative control) of any two replicates is < 20 %
All validity criteria were met:
- optical density OD value of the negative control was 1.6.
- the positive control induced a decrease in the relative absorbance as compared to the negative control to 38.5 %.
- variation within the replicates was acceptable as: 2.9 % (negative control), 4.9 % (positive control) and 5.5 % (test item).

COMPARISON WITH HISTORICAL DATA
The means of negative control and positive control of all performed experiments were stated and compared with the values which were found in this study.
Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

EYE IRRITATION ASSESSMENT
-if % viability > 60 %: non eye irritant (No category UN GHS classification);
-if % viability ≤ 60 % serious eye damage/ eye irritant (No prediction can be made: Category 1 or Category 2 UN GHS classification).
The value obtained for tissue viability of test item is well above the threshold for eye irritation potential (≤ 60 %). Thus, the test item is not considered eye irritant in the EpiOcularTM Eye Irritation Test.

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC 1272/08)
Conclusions:
The test item is considered as not eye irritant in the EpiOcularTM Eye Irritation Test.
Executive summary:

In order to evaluate the potential of the test item to evoke eye irritation in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study, the EpiOcularTMEye Irritation Test was performed, according to the OECD Guideline 492 (2017).

As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement. Therefore, an additional test for intensely coloured test items was performed.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. 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 1.6. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 38.5 % (< 50 %). Variation within tissue replicates of negative control, positive control and test item was acceptable (< 20 %).

After treatment with the test item, the mean value of relative tissue viability was 106.9 %. The result of the additional test showed, that the test item colour did not influence the result of the study.

The value of relative tissue viability obtained is well above the threshold for eye irritation potential (≤ 60 %). Test items that induce values above the threshold are not considered eye irritant.

Thus, the test item is not considered eye irritant in the EpiOcularTMEye Irritation Test, under the conditions of the test.