Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-04-25 to 2006-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study performed according to INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay", dated February 1994 and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997 without deviations and similar to OECD Guideline 437 which was adopted after study completion.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: INVITTOX protocol no. 98 "Bovine Corneal Opacity and Permeability Assay"
Deviations:
no
Remarks:
The testing facility indicated that the protocol was followed without deviation.
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK
Deviations:
no
Remarks:
The testing facility indicated that the protocol was followed without deviation.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
White to light yellow powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00405251ZT000841PUA421
- Expiration date of the lot/batch: 2006-06-30
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (range of 20 +/-5 deg C), light protected
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: Strong stirring with a magnetic stirrer resulted in a suspension. Until administration, the suspension was stirred with a magnetic stirrer.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

FORM AS APPLIED IN THE TEST (if different from that of starting material): 20% concentration in saline.

Test animals / tissue source

Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Freshly isolated bovine eyes were collected from the abattoir: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland. After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank’s balanced salt solution containing penicillin/streptomycin and then transported for further preparations. The eyes were used immediatley after delivery in the laboratory and within four hours after slaughtering.



Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20%

VEHICLE/NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL-aliquots
- Concentration (if solution): 0.9%

POSITIVE CONTROL
- Amount(s) applied (voumne or weight with unti): 0.75 mL
Duration of treatment / exposure:
240 min 32 +/- 2 deg C
Duration of post- treatment incubation (in vitro):
After the final opacity measurement was performed efter 240 minutes of treatment, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32 +/- 2 deg C.
Number of animals or in vitro replicates:
Three corneas per treatment group.

Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined for macroscopically defects. Those presenting defects such as vascularization, pigmentation, opacity, scratches were discarded. Each cornea was dissected from the eye using scalpel and round scissors. A rim of about 2-3 mm of tissue (sclera) was left for stability and handling of the cornea. All corneas used were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for defects listed before.
Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium overnight in a refrigerator at about 4 °C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, NA-bicarbonate and Taurine. Shortly before use, Dextran was added.
The isolated corneas were mounted in a corneal holder with the endothelial side against the sealing ring (O-ring) of the posterior half of the holder. The cornea was gently flattened over the O-ring, but stretching had to be avoided. The anterior half of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. Care was taken to assure no air bubbles were present within the compartments.

For equilibrium, the corneas in the holder were incubated for about one hour at 32 °C +- 2 °C in a water bath.

At the end of the incubation period, the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged.

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test item was introduced onto the surface of the cornea. Corneas were incubated for 240 minutes at 32 ± 2°C.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test item could be observed no longer, fresh cMEM was replaced in both compartments and opacity was measured (t240min).
- Time after start of exposure: 240 minutes

To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step. Corneal permeability is quantified by measuring the amount of fluorescein sodium dye diffusing into the medium in the posterior chamber. Fresh cMEM was added to the posterior chamber and 1 mL of the fluorescein sodium dye solution, 0.5% dissolved in Dulbeco's phosphate-buffered saline, was placed in the anterior compartment after removing the medium. The optical density of an aliquot of the mixed medium from the posterior chamber was measured by photometry at 490 nm. The dye solution is valid for use if a dilution of the stock solution containing 10 ug/mL shows an optical density (OD490) of 1.610 to 1.910.

METHODS FOR MEASURED ENDPOINTS:
-CORNEAL OPACITY:
The opacitometer determined changes in the light transmission passing through the corneas and displayed a numerical opacity value. The opacitometer was calibrated with a standardized opaque polyester sheet as described in a manual, and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.

After recording the basal opacity of all corneas, the mean value of all corneas was calculated. No cornea deviated from this by more than +-3 units and no cornea was discarded. Sets of three corneas were used for treatment with the test substance, the negative and positive controls, respectively.

Medium was completely removed from the anterior compartment and replaced by the test substance, positive or negative control. The anterior compartment was plugged. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test substance and was incubated by positioning in a water bath at 32 °C +- 2 °C.

Permeability Determination:
Following the opacity readings after treatment, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water bath at 32 deg. +- 2 deg. C. Medium from the posterior compartment was removed with a 5 mL- syringe, well mixed and transferred to a cuvette of 10 mm path length, and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

Criteria for Determination of a Valid Test:
The test was acceptable if the positive control caused an in-vitro score greater than 55. According to the results obtained in this experiment, the requirement was met.

SCORING SYSTEM:
Opacity:
The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (t240min-t0min).

The average change in opacity of the negative control corneas was calculated and this value subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

The mean corrected opacity value of each treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition.

Permeability:
The corrected OD490 value of each treated cornea or positive control corneas was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

The mean corrected permeability values of each treatment group were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.

In Vitro Score Calculation:
The following formula was used to determine the In Vitro Score:
In-Vitro Score = opacity value + (15 X OD490 value)

The In-Vitro Score was calculated for each individual treatment and positive control cornea. The mean In Vitro Score value of each treated group was calculated from the individual In-Vitro Score Values:

Negative control:
In-Vitro Score = opacity value + (15 X OD490 value)

Positive control and test substance cornea:
In-Vitro Score = corrected opacity value + (15 X corrected OD490 value)

Depending on the score obtained, the test substance was classified into one of the following categories:
In-Vitro Score: (Proposed In-Vitro Irritation Scale)
0-3 (non eye irritant)
3.1-25 (mild eye irritant)
25.1-55 (moderate eye irritant)
55.1 -80 (severe eye irritant)
> 80.1 (very severe eye irritant)


TOOL USED TO ASSESS SCORE:
- An opacitometer was used to determine the changes in light transmission passing through the corneas. The opacitometer was calibrated with a standardized opaque polyester sheet as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.
- To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
Test item after 240 minutes of exposure
Value:
115.1
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Range of IVIS score of test item 111.8 to 119.8
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
Test item after 240 minutes of exposure
Value:
82.3
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Range of corneal score of test item: 74.3 to 95.3
Irritation parameter:
other: permeability value mean
Run / experiment:
Test item after 240 minutes of exposure
Value:
2.183
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Range of permeability value of test item 1.633 to 2.497
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: 3.3 (2.5 to -4.7)
positive control: 105.2 (96.8 to 109.8)

mean opacity scores (range):
negative control: 2.7 (2 to 4)
positive control: 77.3 (67.3 to 86.3)

mean permeability scores (range):
negative control: 0.042 (0.036 to 0.045)
positive control: 1.865 (1.507 to 2.100)

Acceptance of results:
The test will be acceptable if the positive control has caused an in-vitro score greater than 55. According to the results obtained in this experiment, the requirement was met.

Any other information on results incl. tables

Before starting the permeability test, the dye solution sodium fluorescein was checked for its quality. The dye solution is valid for use, if a dilution of the stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.770.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the given test conditions, the test substance is considered to be a very severe eye irritant.