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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted prior to the adoption of the OECD test Guideline No. 437 but followed the SOP used to validate the model. The absence of references to historical control data reduce the reliability of this assay, but this deviation is considered not to have affected the conclusions (unequivocal results)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
historical control data are not reported, some information on eye collection and preparation are missing
Qualifier:
according to
Guideline:
other: INVITTOX (UK) protocol no.98 "The Bovine Corneal Opacity and Permeability Assay"
Version / remarks:
February 1994
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Storage conditions: In the refrigerator at 2 – 8 °C, protected from light in N2 atmosphere

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, D-64395 Brensbach
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes and they were contained and transported in Hank’s BSS supplemented with streptomycin / penicillin at room temperature.
- Time interval prior to initiating testing: The bovine eyes were fetched from the slaughterhouse and stored in the refrigerator at about 2 - 8 °C until preparation of the corneas on the same day,
- indication of any existing defects or lesions in ocular tissue samples: none reported. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Hank’s BSS supplemented with streptomycin / penicillin

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):1 mL of test item was applied on each cornea.
Duration of treatment / exposure:
10 minutes ± 30 seconds at 32 ± 2 °C in a horizontal position
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for 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 Oring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete minimum essential medium (cMEM). The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 2 °C in a water-bath.
At the end of the incubation period, the medium was removed from both compartments and replaced with fresh cMEM, and the basal opacity was determined (t0). For measurement, the posterior compartment was plugged and the anterior compartment was unplugged.
The bovine eyes were fetched from the slaughterhouse and stored in the refrigerator at about 2 - 8 °C until preparation of the corneas on the same day,
The cornea of the freshly delivered eye was removed as described earlier and inserted in pre-cooled preservation medium. The corneas were stored individually in a minimum volume of 5 mL. The preservation medium was composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.

QUALITY CHECK OF THE ISOLATED CORNEAS : macroscopic examination

NUMBER OF REPLICATES : 3 corneas/group

NEGATIVE CONTROL USED : 0.9% (v/v) saline

POSITIVE CONTROL USED : Ethoxyethanol

APPLICATION DOSE AND EXPOSURE TIME : Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item or negative or positive control at a volume of 1.0 mL on the surface of the corneas and was incubated at 32 ± 2 °C in the water-bath, while the corneas were in a horizontal position.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE:
- Number of washing steps after exposure period: After the test item was rinsed off from the application side by changing cMEM several times, in minimum three times, fresh cMEM was added and opacity was measured (t10).

- POST-EXPOSURE INCUBATION: The corneas were then incubated at 32 ± 2 °C for further two hours in a vertical position, followed by a third opacity reading (t120).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneas, and displays a numerical opacity value. This value was recorded in a table. The opacitometer was calibrated 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 cornea.
After recording the basal opacity of all corneas, the mean value of all corneas were taken and any cornea deviating from this by more than 3 units, also –3 units is possible, was discarded. Sets of three corneas were used for treatment with the test item and the negative and positive controls.
- Corneal permeability: Following to the opacity readings, 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 0.5% (v/v) fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32 ± 2 °C. Medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean/3 corneas
Value:
0.13
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS = 0.92
Positive controls validity:
valid
Remarks:
IVIS = 45.06
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: the test item did not cause any opacity or permeability of the corneas compared with the results of the negative control

DEMONSTRATION OF TECHNICAL PROFICIENCY: not included in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: cannot be assessed
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not reported

Any other information on results incl. tables

Table 7.3.2/1: Individual and Mean Corneal Opacity and Permeability Measurements

Test Group

Opacity value =

Difference (t120-t0) of Opacity

Permeability at

490 nm (OD490)*

In vitro Score

Mean in vitroScore

Proposed in vitro Irritation Scale

 

 

Mean

 

Mean

 

 

 

Negative

Control

0

0

0.049

0.061

0.74

0.92

Non eye irritant

Negative

Control

0

0.046

0.69

Negative

Control

0

0.088

1.32

Positive control

32

0.566

40.49

Moderate eye irritant

Positive control

30

1.180

47.70

Positive control

28

1.265

46.98

Test item

0

0.000

-1.05

Non eye irritant

Test item

0

0.104

1.56

Test item

0

0.059

-0.12

 

Assay validity

The positive control elicited an In Vitro Irritancy Score of 45,06. Even if below the cut-off value of 53, it is close to the treshold and the since the negative control and test item results were not equivocal, the study was considered to be acceptable.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
With an IVIS < 3, the test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 and to the GHS.
Executive summary:

An in vitro eye irritation study was performed prior to the adoption of the OECD Guideline 437 (but follows the SOP used to validate the model), and in compliance with GLP to assess the corneal damage potential of test substance by means of the BCOP assay using fresh bovine corneae. 

After a first opacity measurement of the fresh bovine corneas (t0), the neat test item , the positive, and the negative controls were applied to corneas and incubated for 10 minutes at 32 ± 2 °C in cMEM (complete Minimum Essential Medium), supplemented with 10% FCS (Fetal Calf Serum). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t10). Further, the corneas were incubated for another 120 minutes at 32 ± 2 °C in medium, and opacity was measured a third time (t120).

The opacity measurement permeability of the corneas was determined by application of 1 mL of a fluorescein solution for about 90 minutes at 32 ± 2 °C. The liquid in the posterior chamber was measured spectrophotometrically.

The negative control (0.9% NaCl solution) showed neither an increase of opacity nor permeability of the corneas. The in vitro score was calculated as 0.92.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneas and therefore, is classified as moderate eye irritant. The in vitro score was calculated as 45.06. The positive controlelicited an In Vitro Irritancy Score of 45.06. Even if below the cut-off value of 53, it is close to the treshold and the since the negative control and test item results were not equivocal, the study was considered to be acceptable.

The test item did not cause any opacity or permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 0.13 and therefore, the test item was classified as non eye irritant.

 

With an IVIS < 3, the test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 and to the GHS.

The study is acceptable and satisfies the requirement for an in vitro eye irritation assay.