Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyl ethylene carbonate
EC Number:
700-261-7
Cas Number:
4427-96-7
Molecular formula:
C5H6O3
IUPAC Name:
Vinyl ethylene carbonate
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes
Number of animals or in vitro replicates:
Three corneas for each treatment group.
Details on study design:
PREPARATION OF CORNEAS :
The isolated corneas were stored in a petri dish with cMEM (Earle's Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

SELECTION OF CORNEAS:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

QUALITY CHECK OF THE ISOLATED CORNEAS :
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

NUMBER OF REPLICATES :
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED : Yes, physiological saline.

POSITIVE CONTROL USED : Yes, ethanol.

APPLICATION DOSE AND EXPOSURE TIME : 750 μL; 10 min

POST-INCUBATION PERIOD: yes, incubated for 120 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed with MEM with phenol red (Earle's Minimum Essential Medium) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
-0.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: A mean in vitro irritation score
Other effects / acceptance of results:
OTHER EFFECTS:
The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: Yes. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -0.7 to 0.2. The corneas treated with the negative control item were clear after the 10 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 33 to 60. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from -1.5 to -0.5 and permeability values ranging from -0.004 to 0.001. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.5 to -0.5 after 10 minutes of treatment with the test item.

Applicant's summary and conclusion

Interpretation of results:
other: unclassified
Conclusions:
The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage).

The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied as it is (750 μL) directly on top of the corneas.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.9 after 10 minutes of treatment.

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.