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Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Stearic anhydride

EC Number:

211-318-6

EC Name:

Stearic anhydride

Cas Number:

638-08-4

Molecular formula:

C36H70O3

IUPAC Name:

octadecanoic anhydride

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

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 ≥ 95% 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 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 ≥ 95% 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 incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidi-ty.
After the post-treatment incubation, the MTT assay was performed.

The following amounts were applied to the tissues:

Table 7.2 a Exact amounts of Test Item

Replicate Amount
Tissue 1 50.5 mg
Tissue 2 49.9 mg

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent tissue viability 

Run / experiment:

average of tissues 1+2

Value:

116.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 2

Value:

119.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 1

Value:

113.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). In this case no prediction can be made and further testing with other suitable test methods is re-quired.

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

Executive summary:

One valid experiment was performed.

The test item Stearinsäureanhydrid 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 two tissue replicates.

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 < 2.8, OD was 1.4. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 34.5% (< 50%).

The variation within tissue replicates of the controls and the test item was acceptable (< 20%).

 

After treatment with the test item, the mean value of relative tissue viability was 116.8%.

This value is above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.