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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: granular
Specific details on test material used for the study:
- Source and lot/batch No.of test material: CH1004
- Expiration date of the lot/batch: 21.03.18

Test animals / tissue source

other: kreatinocytes
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 pre-pared 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, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EACH
Day of delivery: 25. Jul. 2017
Batch no.: 23797

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): #01: 50.4 mg; #02: 49.9 mg
- Concentration (if solution):-

- Amount(s) applied (volume or weight with unit):-
- Concentration (if solution):-
- Lot/batch no. (if required): -
- Purity:-
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
Details on study design:
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, 49.4 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 reagent 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
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 53.3 mg of the solid test item were added to 1 mL of sterile H2O de-min. in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % rela-tive humidity for 1 hour. Moreover, 51.0 mg of test item were added to 2 mL isopropanol and incubated in a 6-well plate for 2 hours at room temperature.
After incubation, no colour development was visible. 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 concen-trate 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 % rela-tive 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 item (see table 7.2-a) were applied in duplicate in 1- min- intervals. At the beginning of each experiment (application of negative controls), a stopwatch was started. After dosing the last tissue, all plates were trans-ferred 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 in-cubated 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 190 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 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 tem-perature, 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-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectro-photometer at 570 nm.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: % tissue viability
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Irritation parameter:
other: % tissue viability
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Values for negative control and for positive control were within the range of historical data of the test facility.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Under the conditions of the test system, 1,1'-azobis(1-cyclohexanecarbonitrile) is considered non-eye irritant.
Executive summary:

One valid experiment was performed.

The test item1,1'-Azobis(cyclohexane-1-carbonitrile)was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to each tissue.

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 1.5. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 32.4 % (< 50 %).

The variation within tissue replicates was acceptable (< 20 %).


After treatment with the test item, the mean value of relative tissue viability was 115.5 %.

This value is well above the threshold for eye irritation potential (≤ 60 %).


 Under the conditions of the test,1,1'-Azobis(cyclohexane-1-carbonitrile)is considered non- eye irritant in the EpiOcularTMEye Irritation Test.