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 on 15 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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)
Version / remarks:
26 July 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:
EC No. 440/2008
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:
liquid: viscous
Details on test material:
Identification:
1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Appearance/Physical state:
clear, colorless, viscous liquid
Batch:
WA 1508
Purity:
100% UVCB
Expiry date:
01 January 2021
Storage conditions:
room temperature, in the dark
Specific details on test material used for the study:
Identification: 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Batch: WA 1508
Purity: not supplied
Physical state/Appearance: clear colorless viscous liquid
Expiry Date: 01 January 2021
Storage Conditions: room temperature in the dark

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
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:
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 cornea.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 Minutes
Number of animals or in vitro replicates:
3 corneas per treatment
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 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 (Annex 1). 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 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 three 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 posttreatment 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.

Data Evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

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.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed post treatment.

Data Interpretation
The test item was classified according to the following prediction model:

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

Criteria for an Acceptable Test
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 2015 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 27.2 to 53.4.

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 2015 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤2.8 and for permeability ≤0.115.



Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. 76.3
Negative controls validity:
valid
Remarks:
0.5
Positive controls validity:
valid
Remarks:
65.5
Remarks on result:
positive indication of irritation

In vivo

Irritant / corrosive response data:
Corneal epithelium condition
The corneas treated with the test item were cloudy 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

In vitro irritancy scores:
The in vitro irritancy score for the test item was 76.3
The in vitro irritancy score for the negative control was 0.5
The in vitro irritancy score for the positive control was 65.5
Other effects:
No other effects were observed

Any other information on results incl. tables

Criterion for an acceptable test

The positive control In VitroIrritancy Score was above the range of 27.2 to 53.4. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.

The negative control gave opacity of ≤2.8 and permeability ≤0.115. The negative control acceptance criteria were therefore satisfied.

Table 1 Individual and Mean Corneal Opacity and Permeability Measurements when needed in this detail (delete when editing):

Treatment

Cornea Number

 

 

Opacity

 

 

Permeability (OD)

In Vitro

Irritancy

Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre-Treatment

Corrected

Value

 

Corrected

Value

Negative

Control

2

3

3

3

0

 

0.013

 

 

3

3

4

4

1

 

0.016

 

 

5

3

3

3

0

 

0.013

 

 

 

 

 

 

0.3*

 

0.014

 

0.5

Positive

Control

1

5

37

38

33

32.7

2.070

2.056

 

4

4

37

43

39

38.7

1.670

1.656

 

6

3

41

41

38

37.7

2.140

2.126

 

 

 

 

 

 

36.3

 

1.946

65.5

Test Item

7

3

30

72

69

68.7

0.515

0.501

 

8

3

35

68

65

64.7

0.613

0.599

 

9

3

36

71

68

67.7

0.774

0.760

 

 

 

 

 

 

67.0

 

0.620

76.3


OD= Optical density            

* = Mean of the post-incubation -pre‑treatment values          

**= Mean permeability                     

***= Mean corrected value

Table 2 Corneal Epithelium Condition Post Treatment and Post Incubation:

Treatment

Cornea Number

Obser

vation

 

Post Treatment

 

Post Incubation

Negative Control

2

Clear

 

Clear

3

Clear

 

Clear

5

Clear

 

Clear

Positive Control

1

Cloudy

 

Cloudy

4

Cloudy

 

Cloudy

6

Cloudy

 

Cloudy

Test Item

7

Cloudy

 

Cloudy

8

Cloudy

 

Cloudy

9

Cloudy

 

Cloudy

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Category 1. UN GHS or EU CLP Causes serious eye damage.
Executive summary:

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 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 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 anIn VitroIrritancy Score (IVIS).  

 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 Vitrro Irritancy Score
 Test Item  76.3
 Negative Control  0.5
 Positive Control 65.5

 

Conclusion

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