Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 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
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
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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cerium(3+) acetate
EC Number:
208-654-0
EC Name:
Cerium(3+) acetate
Cas Number:
537-00-8
Molecular formula:
C2H4O2.1/3Ce
IUPAC Name:
cerium(3+) triacetate
Test material form:
solid: granular
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: CEACK1/16
- Expiration date of the lot/batch: 17 October 2018

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

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: 0.9% w/v sodium chloride solution
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.: 3011424
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 67 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.
- 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 item 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 66.9 to 101.4.
- 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 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:
13.8
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
CORNEAL EPITHELIUM CONDITION
The corneas treated with the test material were clear post treatment. The corneas treated with the 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 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. he 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

5

2

 

0.029

 

 

2

3

5

2

 

0.036

 

 

3

3

4

1

 

0.029

 

 

 

 

 

1.7*

 

0.031**

 

2.1

Positive
Control

4

4

70

66

64.3

1.323

1.292

 

5

3

74

71

69.3

1.600

1.569

 

6

4

86

82

80.3

1.909

1.878

 

 

 

 

 

71.3***

 

1.579***

95.0

Test Material

7

5

16

11

9.3

0.111

0.080

 

8

3

19

16

14.3

0.043

0.012

 

9

3

21

18

16.3

0.029

0.000

 

 

 

 

 

13.3***

 

0.030***

13.8

 

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 of eye irritation can be made.
Conclusions:
Under the conditions of the study 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 IVIS 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. As a result, the study was valid.

Under the conditions of the study, the test material had an IVIS of 13.8 therefore no prediction of eye irritation can be made.

Categories Display