Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
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:
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:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
-White powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature over silica gel, in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test material was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution. The test material was formulated within 2 hours of being applied to the test system.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: A local abattoir as a by-product from freshly slaughtered animals
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle typically 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: 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: 0.9 % (w/v) sodium chloride solution
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20 %

VEHICLE
- Concentration (if solution): 0.9 % (w/v)
- Batch no.: 3011424
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND 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 (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
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 material and three corneas to the positive control material.

NUMBER OF REPLICATES: 3

VEHICLE CONTROL USED: 0.9 % (w/v) sodium chloride solution

POSITIVE CONTROL USED: Imidazole 20 % (w/v) in 0.9 % (w/v) sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes

TREATMENT METHOD:
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material 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 test material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1°C for 240 minutes.

REMOVAL OF TEST MATERIAL
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red.

POST-EXPOSURE INCUBATION: 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°C for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken after the test material had been removed and each cornea was visually observed.
- Corneal permeability: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96 well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- 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
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 material induced a response through only one of the two endpoints.

DECISION CRITERIA:
The test material was classified according to the following prediction model;
- IVIS ≤ 3: No category, not requiring classification to UN GHS or EU CLP;
- IVIS > 3 - ≤ 55: No prediction of eye irritation can be made;
- IVIS > 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

ACCEPTABILITY OF THE ASSAY
- 20 % w/v Imidazole 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 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 66.9 to 101.4.
- 0.9 % w/v sodium chloride solution 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 2014 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤ 4.1 and for permeability ≤ 0.105.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
9.3
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
A summary of individual and mean corneal opacity and permeability measurements can be seen in Table 1.

- CORNEAL EPITHELIUM CONDITION
The corneas treated with the test material were slightly cloudy post treatment. The corneas treated with the negative control material were clear post treatment. The corneas treated with the positive control material were cloudy post treatment.

- ACCEPTANCE OF RESULTS
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table1: Individual and Mean Corneal Opacity and Permeability Measurements

 

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

3

4

1

 -

0.029

 -

2

3

3

0

 -

0.021

 -

3

3

3

0

 -

0.020

 -

 Mean

 -

 -

0.3*

 -

0.023**

 -

0.7

Positive
Control

4

3

73

70

69.7

1.860

1.837

 -

5

3

74

71

70.7

1.570

1.547

 -

6

2

73

71

70.7

1.422

1.399

 -

Mean

 -

 -

70.3***

 -

1.594***

94.2

Test Material

7

3

13

10

9.7

0.058

0.035

 -

8

2

13

11

10.7

0.033

0.010

 -

9

4

11

7

6.7

0.033

0.010

 -

 Mean

 -

 -

9.0***

 -

0.018***

9.3

 

OD= Optical density        

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

** = Mean permeability               

*** = Mean corrected value       

                   

Applicant's summary and conclusion

Interpretation of results:
other: No prediction on eye irritation can be made
Conclusions:
Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.
Executive summary:

The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 437 and EU method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) test method.

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 material is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test material was applied to the corneas at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 minutes, the study was performed in triplicate. Negative and positive control materials were tested concurrently, in triplicate also. 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).

The in vitro irritancy score for the test material was 9.3 and as a result it falls into the band of IVIS > 3 - ≤55 in the classification system and so no prediction of eye irritation can be made.

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.

Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.