Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start and completion date: 27 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals / tissue source

Species:
other: Eyes from adult cattle.
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:
other: 20% w/v solution in 0.9% w/v sodium chloride solution.
Controls:
no
Amount / concentration applied:
For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP compliance statement.

Duration of treatment / exposure:
The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Study Design
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of corneas:
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 ml of the test item preparation or control items were applied to the appropriate corneas.
The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the corne was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed.


Preparation of negative and positive control items.
The negative control item, 0.9 % w/v sodium chloride solution, was used as supplied.
The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Value:
0.6
Remarks on result:
no indication of irritation

Any other information on results incl. tables

In Vitro Irritancy Score

TheIn Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test Item

0.6

Negative Control

1.1

Positive Control

96.9

 

Criteria for an Acceptable Test

The positive control In VitroIrritancy Score was within the range of 65.9 – 140.8. The positive control acceptance criterion was therefore satisfied.

 

The negative control gave opacity of ≤4.7 and permeability≤0.080. The negative control acceptance criteria were therefore satisfied.

Table1     Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

16

2

1

0

 

0.064

 

 

18

2

2

0

 

0.067

 

 

19

2

2

0

 

0.080

 

 

 

 

 

0.0*

 

0.07 -

 

1.1

Positive Control

1

1

70

67

67.0

1.759

1.689

 

3

1

74

72

72.0

2.199

2.129

 

20

2

71

64

64.0

2.100

2.030

 

 

 

 

 

67.7·

 

1.949·

96.9

Test Item

22

1

2

1

1.0

0.101

0.031

 

23

3

2

-1

0.0

0.066

0.000

 

24

4

3

-1

0.0

0.099

0.029

 

 

 

 

 

0.3·

 

0.020·

0.6

 

OD= Optical density            * = Mean of the post-treatment -pre‑treatment values            ¨= Mean permeability                     ·= Mean corrected value

 

 


 

Table2     Corneal Epithelium Condition Post Treatment

 

 

Treatment

Cornea Number

Observation

Post Treatment

Negative Control

16

clear

18

clear

19

clear

Positive Control

1

cloudy

3

cloudy

20

cloudy

Test Item

22

clear

23

clear


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not classified, no category, not requiring classification to UN GHS or EU CLP.
Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and bioitem function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

 

 

Method

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS).

 

 

Interpretation

The test item is classified according to the prediction model below:

 

IVIS

CLASSIFICATION

≤ 3

No category. Not requiring classification to UN GHS or EU CLP

> 3; ≤55

No prediction of eye irritation can be made

> 55

Category 1. UN GHS or EU CLP Causes serious eye damage

 

Results

The In Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test Item

0.6

Negative Control

1.1

Positive Control

96.9

Conclusion

No category. Not requiring classification to UN GHS or EU CLP.