Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 09-September 22, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Test item: Yellow LF 6911
Batch No.: UGe-RS Kilo 1
Appearance: Yellow powder
Expiration date: 12 December 2018
Storage: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test animals / tissue source

Species:
chicken
Details on test animals or tissues and environmental conditions:
Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal
(19.4ºC to 20.3ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, 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).
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.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied in its original form, no formulation was required. The test item was finely ground before application.
Duration of treatment / exposure:
Start and end of experimental phase: June 10, 2016
Number of animals or in vitro replicates:
3
Details on study design:
isolated chicken eye test (ICET)
Removal of test item: 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.
The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.
The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 minutes of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.
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 .

Tool used to assess score: hand-slit lamp/biomicroscope/fluorescein
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
ICE Class was 3xI
Other effects / acceptance of results:
There was no visible damage on test system.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below.

 

Test Item: Yellow LF 6911

 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

2%

I

Mean maximum corneal swelling at up to 240 min

2%

I

Mean maximum corneal opacity

0.2

I

Mean fluorescein retention

0.2

I

Other Observations

None

Overall ICE Class1

3xI

 

 

Positive Control: Imidazole

 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

33%

IV

Mean maximum corneal swelling at up to 240 min

37%

IV

Mean maximum corneal opacity

4.0

IV

Mean fluorescein retention

3.0

IV

Other Observations

Cornea opacity score 4 was observed in two eyes at
75 minutes after the post-treatment rinse.

Overall ICE Class1

3xIV

 

The positive control Imidazole was classed as corrosive/severely irritating,UNGHS Classification: Category 1.

Applicant's summary and conclusion

Conclusions:
In this ICET, Yellow LF 6911 did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xI.

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with Yellow LF 6911, no ocular corrosion or severe irritation potential was observed.
According to the guideline OECD 438, Yellow LF 6911 is categorized as “No Category”.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Yellow LF 6911 by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and 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. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item Yellow LF 6911 and Imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount 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.

In this ICET, Yellow LF 6911 did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xI. Positive and negative controls showed the expected results. The experiment was considered to be valid.

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with Yellow LF 6911, no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, Yellow LF 6911 is categorized as “No Category”.