Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-04-12 (experimental starting date and experimental completion date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Non-GLP study conducted according to INVITTOX protocol no. 98 "Bovine Corneal Opacity and Permeability Assay" and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK without deviations. The method used is equivalent with OECD guideline 437 with acceptable deviations.

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
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK without deviations.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD guideline 437 Bovine corneal Opacity and Permeability test method for identifying Ocular Corrosives and Severe Irritants
Deviations:
yes
Remarks:
different minimal essential medium used and sodium fluorescein dye used instead of phenol red
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T001310
- Substance type: no data
- Physical state: solid
- Analytical purity: 100%
- Lot/batch No.: 00465047 RT001310G4A851
- Expiration date of the lot/batch: 2006-06-30
- Stability under test conditions: no data
- Storage condition of test material: room temperature (20 +/- 5 °C), light protected
- Other: no data

Test animals / tissue source

Species:
other: bovine corneas
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST SYSTEM: freshly isolated bovine cornea
Source: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland

Collection of bovine eyes: 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 preparation. The eyes were used immediately after delivery in the laboratory and within four hours after slaughtering.

Preparation of corneas:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity, and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2-3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for the defects listed above.
Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. The posterior compartment had to be filled to return the cornea to its natural convex position. Care must be taken to assure no air bubbles were present within the compartments.
For equilibration, 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.



Test system

Vehicle:
physiological saline
Controls:
other: positive (Imidazole, 20%) and negative (physiol saline 0.9%) control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% in saline


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

Duration of treatment / exposure:
4 hours
Observation period (in vivo):
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). To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step.
Number of animals or in vitro replicates:
corneas per group: 3
test groups: 1
negative control groups: 1
positive control groups: 1
Details on study design:
Preparation of the test item solution:
The test item was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in a suspension. Until administration, the suspension was stirred with a magnetic stirrer.

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

OPACITY MEASUREMENT:
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 item, the negative and positive controls, respectively.
Medium was completely removed from the anterior compartment and replaced by the test item, 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 item and will be incubated in a horizontal positioning 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 1ml of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32°C +/-2°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.

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)
in vitro score 0 - 3: non eye irritant
in vitro score 3.1 - 25: mild eye irritant
in vitro score 25.1 - 55: moderate eye irritant
in vitro score 55.1 - 80: severe eye irritant
in vitro score > 80.1: very severe eye irritant

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
126.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD=6.9; range 122.1 to 134.3
Irritation parameter:
other: permeability score
Value:
0.02
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD=0.011; range 0.009 to 0.030
Other effects / acceptance of results:
Treatment of the corneas with the test item resulted in a mean in-vitro score of 126.3 + /- 6.9 after 240 minutes incubation, ranging from 122.1 to 134.3. The net value of the opacity score ranged from 122 to 134; mean value 126 + /- 6.9. The mean corrected permeability value of the corneas was 0.020 + /- 0.011, ranging from 0.009 to 0.030.

The in-vitro score of saline, used as negative control, was 0.4 +/- 1.1 (-0.8 to 1.4) with the mean opacity value of 0 +/- 1.0 (-1 to 0) and the mean permeability value of 0.025 +/- 0.012 (0.013 to 0.037).

The in-vitro score of the positive control (imidazole, 20%, dissolved in saline) was 77.9 +/- 10.0, confirming the validity of the study. The corrected mean value of the opacity was 58.7 +/- 4.6, ranging from 56 to 64. The corrected mean value of the permeability was 1.282 +/- 0.440, ranging from 0.776 to 1.575.

Any other information on results incl. tables

Before starting the permeability test, the sodium fluorescein dye solution was checked for quality. The dye solution is valid for use if the stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.703.

According to the results obtained in this experiment, the test was considered acceptable.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item was found to be a very severe eye irritant according to the criteria mentioned in the study report and should be classified for eye damage category 1 (irreversible effects on the eye) according to the criteria of the CLP Regulation (EC) No 1272/2008 .