Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 04 September 2018 and 04 September 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
EU method B.47 (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
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Batch: Y180510A
Purity: See Certificate of Analysis
Sponsor Description: Dark brown powder
Envigo Description: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:
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 sodium chloride 0.9% w/v
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test item: 0.75 mL of 20% w/v solution in sodium chloride 0.9% w/v
Negative control: 0.75 mL of Sodium chloride 0.9% w/v
Positive control: 0.75 mL of Imidazole, 20% w/v solution in sodium chloride 0.9% w/v
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Triplicate
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 65 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 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 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.

Application of Sodium Fluorescein
Following the 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 (5 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.
No histopathology was required for this study.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
24.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD492)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

5

5

0

 

0.002

 

 

2

5

5

0

 

0.002

 

 

3

3

4

1

 

0.001

 

 

 

 

 

0.3*

 

0.002¨

 

0.4

Positive
Control

4

7

90

83

82.7

1.830

1.828

 

5

4

85

81

80.7

1.700

1.698

 

6

3

54

51

50.7

0.908

0.906

 

 

 

 

 

71.3·

 

1.478·

93.5

Test Item

7

5

27

22

21.7

0.039

0.037

 

9

5

41

36

35.7

0.005

0.003

 

10

5

20

15

14.7

0.002

0.000

 

 

 

 

 

24.0·

 

0.014·

24.2

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

Corneal Epithelium Condition Post Treatment

Treatment

Cornea Number

Observation
Post Treatment

Negative Control

1

Clear

2

Clear

6

Clear

Positive Control

4

Cloudy

5

Cloudy

6

Cloudy

Test Item

7

Clear with some black staining

9

Clear with some black staining

10

Clear with some black staining

 

Corneal Epithelium Condition

The corneas treated with the test item were clear with some black staining post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

24.2

Negative Control

0.4

Positive Control

93.5

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction of eye irritation can be made.
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 test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v 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 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 Vitro Irritancy Score

Test Item

24.2

Negative Control

0.4

Positive Control

93.5

Conclusion

No prediction of eye irritation can be made.