Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-20 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Appearance: Brown red solid
Expiry date: May 18 2018
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 1003398942

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was reduced in fine powder and applied directly to RhCE tissues.

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; test item was reduced in fine powder and applied directly to RhCE tissues.
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: 17 hours and 55 minutes 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 12 tissues in their 24-well shipping container 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 21 hours and 05 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:
As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg). The test item was applied to 2 DPBS pre-treated RhCE (EpiOcularTM tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes 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. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. 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 a 17-hour and 55-minute 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 by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
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 17 hours and 05 minutes at 6±3°C 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 measurement of formazan extracts at 570 nm was measured in triplicate samples 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
Run / experiment:
Main test
Value:
101.18
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 101.18% versus 26.37% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

Any other information on results incl. tables

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

0.802

0.769

0.848

90.74

100.00

18.53

0.746

0.758

2

1.130

0.926

109.26

0.827

0.821

Positive control

1

0.237

0.244

0.224

28.79

26.37

4.84

0.238

0.256

2

0.201

0.203

23.95

0.202

0.206

Test item

1

0.883

0.877

0.858

103.48

101.18

4.60

0.880

0.866

2

0.850

0.838

98.88

0.840

0.824

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.
Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was reduced in fine powder and applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 17-hour and 55-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 101.18% versus 26.37% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.