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Description of key information

in vitro Guideline studies for skin and eye irritation available.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.01.2016 - 21.01.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: keratinocytes
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 prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories,
Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 19. Jan. 2016
Batch no.: 21590
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test material: Tissue 1: 54.6 mg; Tissue 2: 54.0 mg
Negative control: 50 μL demineralised water
Positive control: 50 μL methyl acetate
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
2
Details on study design:
Pre-Tests:
Assessment of Direct Reduction of MTT by the Test Item:
The test item dimethyl-2,2'-azobisisobutyrate was tested for the ability of direct formazan reduction. To test for this ability, 49.7 mg of the solid test item were added to 1 mL of MTT reagent 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 h. 1 mL of MTT reagent plus 50 μL of H2O demin. was used as negative control.
The MTT reagent 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:
53.6 mg of the test item was added to 2 mL isopropanol, incubated in 6-well plates and placed on an orbital shaker for 3 h 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 OD for isopropanol, the OD of the test item solution was 0.003 (≤ 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 concentrate was diluted with the MTT solvent and the solution was stored at 2 - 8 °C in the dark. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, resp. negative control, resp. 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 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 80 – 100 % relative humidity for 17.25 h (16 – 24 h). 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 min. After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in 1-min-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 h 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 1-min-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 min 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 h 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 incubated for 180 min 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 3 h at room temperature.
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-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectral photometer at 570 nm.
Irritation parameter:
other: % tissue viability
Run / experiment:
1
Value:
12.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other: % tissue viability
Run / experiment:
2
Value:
10.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive 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. Therefore, the experiment is considered valid.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the conditions of the test system, dimethyl-2,2'-azobisisobutyrate is considered as eye irritant in the EpiOcularTM Eye Irritation Test.
Executive summary:

After treatment with the test item, the relative absorbance values were reduced to 11.8 %. This value is well below 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 1.8 (> 0.8 and < 2.5). 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 (< 20 %).

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

adverse effects on respiration are observed in an inhalatory acute toxicity study.

No effects are stated for the low-dose group (0.560 mg/L, aerosol).

For the mid-dose and high-dose groups it is stated:

Clinical signs of toxicity in animals exposed to 1.935 mg/L comprised abdominal and

intermittent respiration, nasal red discharge and red encrusted nose, salivation indicating a

local irritation effect.

Clinical signs of toxicity in animals exposed to 3.994 mg/L comprised abdominal respiration,

gasping respiration, intermittent and labored respiration indicating a local irritation effect.

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification for skin irritation /corrosion.

The available information is conclusive and sufficient for classification for eye irritation cat. 2.

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