Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-04-06 to 2017-04-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 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:
2013
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch No.: 16023LS4B5
Purity: 98.3%

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: from the slaughterhouse
- Transport: 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): 301.3 to 427.9 mg
Duration of treatment / exposure:
240 ± 10 minutes
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- 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) Fetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) 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.
- Cornea Selection: 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. Three corneas were selected at random for each treatment group.

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
Two experiments were performed. 3 for each treatment group

NEGATIVE CONTROL USED
- Negative Control: physiological saline

POSITIVE CONTROL USED
- Positive Control: 20% (w/v) Imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
- Application dose: 750 μL of the negative control and positive control, 301.3 to 427.9 mg for test item
- Exposure time: 240 ± 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red

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 microtiter plate 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:
>= 38 - <= 62
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
In both experiments, the corneas were clear after the 240 minutes of treatment with the test item. The corneal membranes were detached. No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

The corneas treated with the test item showed opacity values ranging from -1.6 to -1.0 and permeability values ranging from 2.895 to 4.815. Hence, the in vitro irritancy scores ranged from 42 to 71 after 240 minutes of treatment with the test item.

Since the individual in vitro irritancy scores were spread over 2 categories (42, 48 and 71), a repeat experiment was performed.

In experiment 2, the corneas treated with the test item showed opacity values ranging from 0.1 to 2.7 and permeability values ranging from 2.351 to 4.127. Hence, the in vitro irritancy scores ranged from 38 to 62 after 240 minutes of treatment with the test item.

The individual in vitro irritancy scores were spread over 2 categories (38, 41 and 62).

In both experiments it was observed that the cornea membranes were detached, resulting in a clear cornea and no effect on opacity. Therefore it is concluded that this substance should be labelled category 1.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, test item should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the most recent OECD guideline 437.

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes.

The test item was used as delivered and added pure on top of the corneas.

In the first experiment, the mean in vitro irritancy score was 54 after 240 minutes of treatment with the test item. Since the individual in vitro irritancy scores were spread over 2 categories (42, 48 and 71), a repeat experiment was performed.

In the second experiment, the mean in vitro irritancy score was 47 after 240 minutes of treatment with the test item. The in vitro irritancy score values were, like in the first experiment spread, over 2 categories (38, 41 and 62). However, since in both experiments it was observed that the cornea membranes were detached resulting in a clear cornea (and no effect on opacity), it is concluded that this substance should be labelled category 1.

In conclusion, test item should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.