Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 February 2015 to 07 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Violet powder
- Storage condition: Controlled room temperature (15 to 25 °C, below 70 RH %), protected from light and humidity
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety

Test animals / tissue source

Species:
other: Chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to laboratory at earliest convenience.
- After collection, 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). Heads were processed within approximately 2 hours of collection.

SELECTION AND PREPARATION OF EYES FOR THE TEST

EYES 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 physiological saline.
The fluorescein- treated cornea was examined with a 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 THE 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.

EYES EXAMINATION AND ACCLIMATISATION TIME
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, avoid 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. 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. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological 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 all 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 it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatisation and treatment periods.

IDENTIFICATION
The eyes were identified by chamber number, marked on the door of the chamber.

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 change by more than 5 % between the -45 min and the zero time. No changes in thickness (0.0 %) 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 item related effect after treatment. All eyes were considered to be suitable for the assay.

Test system

Vehicle:
physiological saline
Controls:
other: Positive and Negative control eyes
Amount / concentration applied:
TEST MATERIAL
- The test material was applied in powdered form in an amount of 30 mg
Duration of treatment / exposure:
- Exposure period: 10 seconds
Observation period (in vivo):
The control eyes 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 approximately ±5 minutes were considered acceptable.
Number of animals or in vitro replicates:
Three test material-treated eyes, three positive control-treated eyes and one negative control-treated eye were examined.
Details on study design:
REMOVAL OF TEST SUBSTANCE
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove as much of the residual the test material as possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test material or control material remaining on the cornea was observed.

SCORING SYSTEM
ICE Classification System

TOOL USED TO ASSESS SCORE
Haag-Streit Bern 900 slit-lamp microscope

Results and discussion

Results of in vivo studyopen allclose all
Irritation parameter:
other: Corneal swelling (value as a percentage)
Basis:
mean
Time point:
other: at up to 75 mins
Score:
0.5
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Corneal swelling (value as a percentage)
Basis:
mean
Time point:
other: at up to 240 mins
Score:
0.5
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Corneal opacity (value as a percentage)
Basis:
mean
Score:
1
Remarks on result:
other: ICE Class II
Irritation parameter:
other: Fluorescein retention (value as a percentage)
Basis:
mean
Score:
1
Remarks on result:
other: ICE Class II
Irritant / corrosive response data:
The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification. Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Any other information on results incl. tables

Table 1: Eye Irritation Results of the Test Material

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.5%

I

Mean maximum corneal swelling at up to 240 min

0.5%

I

Mean maximum corneal opacity

1.00%

II

Mean fluorescein retention

1.00%

II

Other Observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse

Overall ICE Class

1 x I 2 x II

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant.
Executive summary:

An in vitro eye irritation study of the test material was performed in isolated chicken’s eyes. The irritation effects of the test material were evaluated in accordance to the standardised guidelines OECD 438, EU Method B.48 and under GLP conditions.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of 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 with 30 mg Imidazole. The negative control eye was treated with 30 μL of physiological saline (Salsol solution, 0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined.

No significant corneal swelling was observed during the four hour observation period.

Corneal opacity change (severity 1) and fluorescein retention change (severity 0.5 or 1 or 1.5) was noted on all test material treated eyes; particles of test material were stuck to the cornea and could not be washed off during the study. The effects were clearly greater that a negative effect, but not sufficient to classify as severe. The particles stuck to the cornea could potentially result in mechanical damage in vivo.

Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant.