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Toxicological information

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:
30 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Validated in vitro study conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Description: extremely pale yellow liquid
Storage conditions: approximately 4 °C in the dark

Test animals / tissue source

Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
The test material was applied as evenly as possible to the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C).
Observation period (in vivo):
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment
Number of animals or in vitro replicates:
None, in vitro test. Three eyes were treated with test material, two additional eyes remained untreated for control purposes.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C). Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye. The untreated eyes were similarly washed and used for control purposes.
- Time after start of exposure: 10 seconds

SCORING SYSTEM:
Method for Evaluation of Ocular Irritation by Slit-Lamp Biomicroscopic Examination - McDonald - Shadduck Score System:

CORNEA
The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. Severity of corneal cloudiness is graded as follows:
0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stroma.
1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit-lamp. The underlying
structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse illumination, underlying structures are clearly visible.
3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination the underlying structures are just visible.
4 = Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With diffuse illumination, the underlying structures cannot be seen.

The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4.
0 = Normal cornea with no area of cloudiness
1 = 1 to 25 % area of stromal cloudiness
2 = 26 to 50 % area of stromal cloudiness
3 = 51 to 75 % area of stromal cloudiness
4 = 76 to 100 % area of stromal cloudiness

FLUORESCEIN
The use of fluorescein is a valuable aid in defining epithelial damage for fluorescein staining. The area can be judged as a 0 to 4 scale using the same terminology as for corneal cloudiness. The intensity of fluorescein staining can be divided into a 0 to 4 scale.
0 = Absence of fluorescein staining
1 = Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but are not completely obliterated.
4 = Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be seen.

REFERENCE:
Hackett R B and McDonald T O, Eye Irritation. In: Advances in Modern Toxicology: Dermatoxicology. 4th ed. (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749 815.


TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Mean 240 minutes
Value:
>= 8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean 240 minutes
Value:
>= 4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean 60 minutes
Value:
>= 28.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean 120 minutes
Value:
>= 59.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean 240 minutes
Value:
>= 166.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Other effects / acceptance of results:
Corneal Opacity:
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test eyes 60 and 120 minutes following treatment with moderate loss of transparency 180 and 240 minutes following treatment.
No corneal effects were noted in the control eyes during the study period.

Corneal Thickness:
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3.
Corneal swelling of the test eyes during the study period was considerably greater than that observed in the control eyes over the same period.

Corneal Condition:
The condition of the corneal epithelium following treatment is given in Table 4.
Pitting of the corneal epithelium was noted in all test eyes 60 and 120 minutes following treatment with sloughing of the corneal epithelium 180 and 240 minutes following treatment.
The condition of the corneal epithelium of the control eyes appeared normal during the study period.

Fluorescein Uptake:
Individual scores for fluorescein uptake are given in Table 5.
Slight fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Any other information on results incl. tables

Interpretation of Results:

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*

REET Cut‑Off Value

Maximum Corneal Opacity (Corneal Cloudiness x Area)

> or = 4

Maximum Fluorescein Uptake (Intensity x Area)

> or = 4

Mean Corneal Swelling (mins): 60, 120, 240

> or = 25 %

Corneal Epithelium Observations

Any with pitting, mottling or sloughing

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

((mean corneal thickness post-treatment) – (mean corneal thickness post equilibration) /(mean corneal thickness post equilibration)) x 100

- A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 Minute post treatment observation periods.

- A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.


*= Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant

Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal Opacity

Fluorescein Uptake

Corneal Swelling (%)

Condition of Corneal Epithelium

Test Eyesa

Control Eyesb

Cldy x Area

Int x Area

60mins

120 mins

240 mins

60 mins

120 mins

240 mins

8+

4+

28.5+

59.3+

166.2+

3.1

3.6

3.9

pitting/sloughing+

Applicant's summary and conclusion

Interpretation of results:
other: EU Criteria:
Conclusions:
Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. 

0.1 mL of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ± 1.5 °C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9 % Sodium Chloride).

Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.