Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 December, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(2010)
Deviations:
no
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
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. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approx. 750 µL per cornea

NEGATIVE CONTROL
- Amount applied: 750 µL of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µL per cornea
Concentration: 10% (w/v) Benzalkonium Chloride
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
Three corneas / substance or control.
Details on study design:
TEST SITE
- Isolated bovine cornea, incubation at 32°C.

REMOVAL OF TEST SUBSTANCE
- Washing: yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed (possible pH effects of the test substance on the corneas were recorded) by incubation for 120 minutesat 32°C. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated (incubation 90 minutes at 32°C).

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Opacity and permeability values were also evaluated independently.

TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is defined as non irritant, IVIS > 3 ≤ 55 mild to moderate and IVIS ≥ 55 is defined as a corrosive or severe irritant

ACCEPTABILITY OF THE ASSAY:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean_time point: 10 min
Value:
-1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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 -3 to 0 and permeability value was -0.002 for all three corneas. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -3.0 to 0.0 after 10 minutes of treatment with the test item.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Conclusions:
The test item is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles, as the test item induced an IVIS ≤ 3.
Executive summary:

The test item was screened for the eye irritation and corrosion potential using the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles.

Adequate negative and positive controls were included and 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, opacity and permeability, resulting in a mean in vitro irritancy score of -1.0 after 10 minutes of treatment. Based on the results, it is concluded that the test item is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Regulation (EC) 1272/2008.