Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Storage: room temperature 15-25 °C, continuously protected from all light

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to the testing laboratory at ambient temperature at the earliest convenience.
- After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the testing laboratory and processed within approximately 2 hours of collection.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µg

Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
3 replicates for the test material and positive control treated eyes and a single negative control treated eye.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- 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.
- Preparation of eyes: 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
- The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 2 or 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
- The appropriate number of eyes was selected and after being placed in the superfusion apparatus, 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, any eye with cornea thickness deviating more than 10% from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. Temperature of the circulating water was verified to ensure that all chambers were in the range of 32 ± 1.5°C during the acclimatisation and treatment periods.
- The base line assessments: At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t = 0) for each individual eye. The cornea thickness of the eyes should not increase by more than 5 - 7% between the -45 and the zero time. Slight changes in thickness (+2 to -3%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test material related effects after treatment; the location of any minor findings were marked on the record sheet as a drawing. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES: 3 replicates for the test material and positive control treated eyes and a single negative control treated eye.

NEGATIVE CONTROL USED
- Sodium chloride (Salsol solution 0.9%)

POSITIVE CONTROL USED
- Imidazole

APPLICATION DOSE AND EXPOSURE TIME
- 30 µg of test material was uniformly applied by powdering the entire surface of the cornea, taking care not to damage or touch the cornea with the application equipment. The positive control eyes were treated in a similar way with 30 μg of imidazole. The negative control eye was treated with 30 μL of isotonic saline.

REMOVAL OF TEST MATERIAL
- The time of application was observed, then after an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all the residual test material if possible.

OBSERVATION PERIOD
- The control eye and test eyes were evaluated pre-treatment and at approximately 30, 60, 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 measured on two occasions, at base line (t = 0) and 30 minutes after the post-treatment rinse.

TREATMENT OF DATA
- Cornea swelling was calculated according to the following formulae:
CS at time t = (CT at time t –CT at t=0 / CT at t=0) x 100
Mean CSmax at up to 75 min = [FECSmax(30min to 75min)+ SECSmax(30min to 75min) + TECSmax(30min to 75min)] / 3
Mean CSmax at up to 240 min = [FECSmax(30min to 240min)+ SECSmax(30min to 240min) + TECSmax(30min to 240min)] / 3
CS = cornea swelling
CT = cornea thickness
FECS = first eye cornea swelling
SECS = second eye cornea swelling
TECS = third eye cornea swelling
max(30min to 75min) = maximum swelling of the individual eye at 30 to 75 minutes max
(30min to 240min) = maximum swelling of the individual eye at 30 to 240 minutes
- Small negative numbers for swelling following application are counted as zero (larger negative numbers due to erosion invalidate the swelling evaluation, but indicate a severe effect)

- Cornea opacity was calculated according to the following formulae:
CO at time t = CO at time t – CO at t=0
Mean COmax = [FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity max
(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes

- Fluorescein retention was calculated according to the following formulae:
FR at time t = FR at time t – FR at t=0
Mean FR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3
FR = fluorescein retention
FEFR = first eye fluorescein retention
SEFR = second eye fluorescein retention
TEFR = third eye fluorescein retention

STORAGE OF CORNEAS
- At the end of the procedures, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin) for potential histopathology and stored.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Other effects / acceptance of results:
- Results of this in vitro eye irritation study, in isolated chicken eyes, suggest that the test material was not irritating.
- The positive control, imidazole, was classed as severely irritating, GHS Classification: Category 1.
- The negative control, sodium chloride 0.9%, had no significant effects on the chicken eyes in this study.

Any other information on results incl. tables

Table 1: Study Results

Observation

Test Material

Positive Control

Negative Control

Value

ICE Class

Value

ICE Class

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1 %

I

11 %

II

1 %

I

Mean maximum corneal swelling at up to 240 min

1 %

I

12 %

II

1 %

I

Mean maximum corneal opacity

0.00

I

4.00

IV

0.00

I

Mean fluorescein retention

0.17

I

2.83

IV

0.00

I

Other Observations

Minimal test material was stuck on the cornea after the post-treatment rinse. The cornea surface was clear 240 min after the post-treatment rinse

The positive control material was stuck on the cornea surface after the post-treatment rinse. The cornea surface was not cleared 240 min after the post-treatment rinse.

None

Overall ICE Class

3 x I

1 x II, 2 x IV

3 x I

 

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU Criteria.
Conclusions:
Under the conditions of this study, the test material was not irritating to eyes.
Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 438, under GLP conditions.

An in vitro eye irritation study of the test material was performed in chicken’s eyes.

After the zero reference measurements, 30 μg of the test material was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 μg imidazole. The negative control eye was treated with 30 μL of isotonic saline. The eyes were examined for a period of 4 hours following treatment.

The results suggested that the test material was not irritating. The positive control, imidazole, was classed as severely irritating, GHS Classification: Category 1. The negative control, sodium chloride 0.9%, had no significant effects on the chicken eye in this study.

Under the conditions of this study, the test material was not irritating to eyes.