Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-06-22 to 2006-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to INVITTOX Protocol 98 (Bovine Corneal Opacity and Permeability Assay, February 1994) and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, April 1997 without deviations. Conducted in a manner similar to OECD guideline 437.
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
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 437
Deviations:
no
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T000268
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: solid
- Analytical purity: 100%
- Impurities (identity and concentrations): not applicable
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: BE A351
- Expiration date of the lot/batch: 2007-05-31
- Stability under test conditions: no data
- Storage condition of test material: at room temperature (20 +/5 deg C), light protected
- Other: solubility: <0.01 g/L in water; 0.13 g/L in 2-propanol; 0.28 g/L in ethanol; 0.46 g/L in methanol; 8.5 g/L in acetone; 24 g/L in N, N-dimethylformamide; 68 g/L in dichloromethane
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable

Test animals / tissue source

Species:
other: cow
Strain:
not specified
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 immediately after delivery in the laboratory and within four hours after slaughtering. Corneas were dissected from the eyes and were stored overnight in a refrigerator at about 4 deg C in a preservation composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.
- Age at study initiation: no data
- Weight at study initiation: not applicable
- Housing: not applicable
- Diet (e.g. ad libitum): not applicable
- Water (e.g. ad libitum): not applicable
- Acclimation period: not applicable


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): During the whole experiment, cornea holders and medium were maintained in a water-bath at 32 +/- 2 deg C.
- Humidity (%): not applicable
- Air changes (per hr): not applicable
- Photoperiod (hrs dark / hrs light): not applicable


IN-LIFE DATES: From: no data To: no data

Test system

Vehicle:
other: saline (0.9% sodium chloride)
Controls:
other: negative control (solvent) and positive control (imidazole)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL - aliquot
- Concentration (if solution): 20% suspension in saline


VEHICLE
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): 0.9% (w/v)
- Lot/batch no. (if required): 773609
- Purity: no data
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
- Three corneas were treated with the test substance, the positive control substance, or the solvent.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test substance was rinsed off from the application side by changed cMEM (Eagle’s minimal essential medium) several times until precipitates of the test substance could be observed no longer.
- Time after start of exposure: 240 minutes


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 vivo

Resultsopen allclose all
Irritation parameter:
other: in vitro score
Basis:
mean
Time point:
other: 240 minutes
Score:
-2.2
Remarks on result:
other: +/- 0.5
Irritation parameter:
other: Corrected opacity
Basis:
mean
Time point:
other: 240 minutes
Score:
-1.3
Remarks on result:
other: +- 0.6
Irritation parameter:
other: Corrected permeability
Basis:
mean
Time point:
other: 240 minutes
Score:
-0.06
Remarks on result:
other: +/- 0.022
Irritant / corrosive response data:
- Treatment of the corneas with the test item resulted in a mean in-vitro score of -2.2 +/- 0.5 after 240 minutes incubation, ranging from -2.7 to -1.7. The net value of the opacity score ranged from -1.7 to -0.7, the mean value was -1.3 +/- 0.6. The mean corrected permeability value of the corneas was -0.056 +/- 0.022, ranging from -0.070 to -0.030.

The in-vitro score of saline, used as negative control, was 2.1 +/- 1.4 (0.6 to 3.2) with the mean opacity value of 0.7 +/- 0.6 (0 to 1) and the mean permeability value of 0.096 +/- 0.056 (0.038 to 0.149).

The in-vitro score of the positive control (imidazole, 20%, dissolved in saline) was 83.4 +/- 13.2, confirming the validity of the study. The corrected mean value of the opacity was 52.3 +/- 11.8, ranging from 39.3 to 62.3. The corrected mean value of the permeability was 2.069 +/- 0.373, ranging from 1.651 to 2.369.

Any other information on results incl. tables

- According to the results obtained in this experiment, the criteria of a valid test were met.

- Before starting the permeability test, the dye solution sodium fluorescein was checked for its quality. The dye solution was valid for use if a dilution of the stock solution containing 10 ug/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.697.

Samples

Opacity

Permeability

In Vitro Score

Cornea no.

Treatment

t0min

t240min

t240min - t0min

Corrected

490 nm

Corrected

 

1

Negative Control: Saline

2

3

1

 --

0.149

-- 

3.2

2

1

2

1

 --

0.102

-- 

2.5

3

1

1

0

 --

0.038

-- 

0.6

Mean +/- SD

0.7 +/- 0.6

 --

0.096 +/- 0.056

-- 

2.1 +/- 1.4

 

4

Positive Control:Imidazole, 20%

2

42

40

39.3

2.283

2.187

72.1

5

1

64

63

62.3

2.465

2.369

97.9

6

2

58

56

55.3

1.747

1.651

80.1

Mean +/- SD

-- 

52.3 +/- 11.8

-- 

2.069 +/- 0.373

83.4 +/- 13.2

 

7

Test Substance: T000268, 20%

2

1

-1

-1.7

0.029

-0.068

-2.7

8

2

2

0

-0.7

0.027

-0.070

-1.7

9

2

1

-1

-1.7

0.066

-0.030

-2.1

Mean +/- SD

 --

- 1.3 +/- 0.6

-- 

-0.056 +/- 0.022

-2.2 +/- 0.5

 

Applicant's summary and conclusion

Conclusions:
Under the given test conditions, the test substance was considered to be non-eye irritant.
Executive summary:

not applicable