Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: room temperature, darkness (Optimal temperature at 20°C)

Test animals / tissue source

Species:
other: Reconstructed human Cornea-like Epithelia
Details on test animals or tissues and environmental conditions:
Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration: Undiluted
Duration of treatment / exposure:
6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 25 minutes at room temperature - Post-exposure incubation period: 18 hours at standard culture conditions
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
MAIN TEST

- Pre-incubation of the tissues:

On the day of receipt, the tissues were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 19 hours and 55 minutes, at standard culture conditions.

After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.

- Treatment and post-treatment incubation of the tissues:

The test item was coated on a nylon mesh then applied during 6 hours at standard culture conditions, at the dose 19.4 mg or 19.5 mg to the entire surface of 2 living RhCE tissue replicates.

In the same experimental conditions, a positive control (Methyl acetate - Sigma, batch No. BCBV2084) and a negative control (distilled water - ADL Prochilab - Batch No. 180517) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 6 hours at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 5780818). The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish).
This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for an 18 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions.

The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 2 hours at room temperature in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.

The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results
Irritation parameter:
other: % mean viability of the tissues
Vehicle controls validity:
not applicable
Negative controls validity:
other: Results on-going
Positive controls validity:
other: Results on-going
Remarks on result:
other: Results on-going.

Applicant's summary and conclusion

Interpretation of results:
other: On-going
Conclusions:
The mean percent tissue viability of the RhCE replicates treated with test item not available (on-going). Therefore, no classification is proposed according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An OECD 439 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcularTM tissue model).

 

Test item was coated on a nylon mesh then applied , at the approximate dose of 19 mg to 2 DPBS pre-treated RhCE (EpiOcularTM tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

  

The mean percent tissue viability of the RhCE replicates treated with test item not available (on-going). Therefore, no classification is proposed according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.