[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February the 17th. 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

26 July 2013

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

The test item was applied in its original form, no formulation was required, but it was ground finely in a mortar and pestle before use in the study.

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Species of chicken: COBB 500
- Source: TARAVIS KFT. 9600 Sárvár. Hungary
- Head collection: collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current).
- Transport to laboratory: the heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.9 ºC to 20.5 ºC) during the transport.
- Source of eye: all eyes used in the assay were from the same groups of eyes collected on one specific day.
- Health check: the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item was applied in a volume of 0.03 g by powdering the entire surface of the cornea of isolated chicken eyes

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control eyes and one negative control eye were used in this study

Details on study design:

EYE SELECTION
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 ml isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

EYE PREPARATION
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0 % to 3 %) were observed in the eyes, a finding considered as normal when maintaining enucleated eyes.
Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NEGATIVE CONTROL USED
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 μl from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

POSITIVE CONTROL USED
The three positive control eyes were treated in as test item group with 0.03 g Imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The substance was applied in a volume of 0.03 g by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eyes.

REMOVAL OF TEST SUBSTANCE
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 ml saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
Test item was stuck on the corneas surface at 30 minutes after the post-treatment rinse. The gentle rinsing with additional 20 ml saline was performed after 30 minutes of observation. The cornea surfaces were totally cleared at 75 min after the post-treatment rinse.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

SCORING SYSTEM
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

The effects were divided into four categories for each endpoint:
I = none
II = slight
III = moderate
IV = severe

Cornea opacity . score
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

Fluorescein retention . score
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

ICE Class

Run / experiment:

mean

Value:

1 - 2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In in vitro eye irritation study using the isolated chicken eyes, no ocular corrosion or severe irritation potential was observed. The test item was not a severe irritant.

Positive and negative control values were within the corresponding historical control data ranges.

Any other information on results incl. tables

Test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2 %	I
Mean maximum corneal swelling at up to 240 min	3 %	I
Mean maximum corneal opacity	0.7	II
Mean fluorescein retention	1.2	II
Overall ICE Clas		1xI, 2xII

Other Observations: the test item was stuck on the cornea surfaces at 30 minutes after the post-treatment rinse. The cornea surfaces were totally cleared at 75 min after the post-treatment rinse.

Negative control

bservation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Overall ICE Clas		3xI

Other Observations: none.

Positive Control: Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	17 %	III
Mean maximum corneal swelling at up to 240 min	28 %	III
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	2.8	IV
Overall ICE Clas		1xIII, 2xIV

Other Observations: cornea opacity score 4 was observed in three eyes at 30 minutes after the posttreatment rinse.

Applicant's summary and conclusion

Interpretation of results:

other: substance does not cause ocular corrosion or severe irritation

Conclusions:

Test item is not classified as an ocular corrosive or severe eye irritant.

Executive summary:

The purpose of the Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes.The experiment was conducted following the procedures outlined into the OECD guideline 438. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. The damage by the test substance was assessed by determination of corneal swelling, opacity, fluorescein retention and morphological effects. These parameters were evaluated pre-treatment and starting at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. The test item and Imidazole (positive control) were applied in a volume of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μl saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 ml saline solution at ambient temperature and this procedure was repeated for each eye.

The substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Conclusion

Test item is not classified as an ocular corrosive or severe eye irritant.