Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2017
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 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:
yes
Remarks:
(see "Principles of method if other than guideline")
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:
yes
Remarks:
(see "Principles of method of other than guideline")
Principles of method if other than guideline:
The positive control group had an overall IVIS of 121.2. This was marginally higher than the criteria range set for an acceptable test. However, as the score was only marginally exceeded, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Fine, dark grey powder
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of this study 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. It is assumed that the formulation was stable for this duration.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: 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: sodium chloride 0.9% w/v
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20% w/v

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration: 0.9% w/v
- Batch no.: 3012488
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes with sodium fluorescein
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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: Sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Imidazole 20% w/v in sodium chloride 0.9% w/v 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 materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: 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. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 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.
- 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)
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 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 CRITERIA
- The test was acceptable if the positive control (20% w/v Imidazole ) 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 50.8 to 100.4.
- The test was acceptable if the negative control (0.9% w/v sodium chloride solution) 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 solids the negative control limit for opacity should be ≤ 5.4 and for permeability ≤ 0.070.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
3.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not valid
Other effects / acceptance of results:
CORNEAL EPITHELIUM CONDITION
The corneas treated with the test material or negative control were clear post treatment. The corneas treated with the positive control were cloudy post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied, this was reported as a deviation.
- The negative control gave opacity of ≤ 5.4 and permeability ≤ 0.070. The negative control acceptance criteria were therefore satisfied.

A summary of the results is displayed in Table 1.

Any other information on results incl. tables

 Table 1: Summary of results

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

 

0.014

 

 

2

3

3

0

 

0.016

 

 

3

5

5

0

 

0.018

 

 

 

 

 

0.3*

 

0.016**

 

0.6

Positive
Control

4

5

107

102

101.7

2.215+

2.199

 

5

4

91

87

86.7

1.845+

1.829

 

6

3

92

89

88.7

1.760+

1.744

 

 

 

 

 

92.3***

 

1.924***

121.2

Test Material

7

3

7

4

3.7

0.076

0.060

 

8

4

7

3

2.7

0.041

0.025

 

9

4

7

3

2.7

0.029

0.013

 

 

 

 

 

3.0***

 

0.033***

3.5

OD = Optical density      

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

** = Mean permeability               

*** = Mean corrected value     

+ = 1 in 5 dilution performed

Applicant's summary and conclusion

Interpretation of results:
other: EU Criteria: no prediction of eye irritation can be made
Conclusions:
Under the conditions of the study, the test material had an IVIS of 3.5 therefore no prediction of eye irritation can be made.
Executive summary:

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

During the study the test material was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive controls 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).

The positive control In Vitro Irritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied, this was reported as a deviation. The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.

Under the conditions of the study, the test material had an IVIS of 3.5 therefore no prediction of eye irritation can be made.