Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/07/2018 - 12/07/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
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:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the appropriate corneas.
Duration of treatment / exposure:
10 minutes.
Duration of post- treatment incubation (in vitro):
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
Number of animals or in vitro replicates:
3 replicates.
Details on 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 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 (EMEM) without phenol red 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 EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer .Three corneas were randomly 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 EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item 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 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 65.1.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.041.

For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 65.1.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.041.
The positive control In Vitro Irritancy Score was within the range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.041. The negative control acceptance criteria were therefore satisfied.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
0.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and PermeabilityMeasurements

 

 

 

Treatment

 

Cornea Number

Opacity

Permeability (OD492)

In VitroIrritancyScore

 

Pre-Treatment

 

Post-Treatment

 

Post Incubation

Post-Incubation

-Pre-Treatment

Corrected Value

 

Corrected Value

 

NegativeControl

1

5

4

5

0

 

0.007

 

 

2

5

4

4

0

 

0.004

 

 

3

3

3

3

0

 

0.004

 

 

 

 

 

 

0.0*

 

0.005¨

 

0.1

 

PositiveControl

4

5

34

38

33

33.0

1.227

1.222

 

5

4

31

35

31

31.0

1.450

1.445

 

6

4

31

34

30

30.0

2.120

2.115

 

 

 

 

 

 

31.3·

 

1.594·

55.2

 

 

Test Item

7

4

4

3

-1

0.0

0.046

0.041

 

9

5

4

4

-1

0.0

0.011

0.006

 

10

4

4

4

0

0.0

0.007

0.002

 

 

 

 

 

 

0.0·

 

0.016·

0.2

Corneal Epithelium Condition Post Treatment and PostIncubation

 

 

Treatment

 

Cornea Number

Observation

Post Treatment

Post Incubation

 

 

Negative Control

1

Clear

Clear

2

Clear

Clear

3

Clear

Clear

 

 

Positive Control

4

Cloudy

Cloudy

5

Cloudy

Cloudy

6

Cloudy

Cloudy

 

 

Test Item

7

Clear

Clear

9

Clear

Clear

10

Clear

Clear

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
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 biochemical function of the bovine cornea in 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 EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP 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/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 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 an In Vitro Irritancy Score (IVIS).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

UN GHS

≤ 3

No Category

>3; ≤ 55

No prediction can be made

> 55

Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In VitroIrritancy Score

Test Item

0.2

Negative Control

0.1

Positive Control

55.2

Conclusion

 

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