Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 June 2019 to 13 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

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
Details on test material:
Name: CAS 577978-76-8
Chemical Name: Polyglycidyl ether of 1,6-naphthalenediol-pxylyleneglycol (or p-xylyleneglycol dimethylether) condensation polymer
CAS number: CAS 577978-76-8
Batch/Lot number: 0950985
Description: Brown solid
Purity: 100%
Expiry date: 08 February 2020 (as per updated CoA)
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity).
Safety precautions: Enhanced safety precautions above the routine safety precautions (lab coat, gloves, safety glasses, face mask) will be applied considering the supplied safety data sheet to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
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 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then 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 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 acclimatization 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, acclimatization 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 acclimatization and treatment periods.
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 Charles River Laboratories Hungary Kft. 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 Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

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

THE BASELINE ASSESSMENTS
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% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in any eye. Following the equilibration period, the fluorescein retention was measured. Baseline 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:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg of the powdered test item was applied onto the entire surface of the cornea
Duration of treatment / exposure:
10 seconds from the end of the application
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
3 eyes per test group.
Details on study design:
Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the powdered test item and another three eyes with powdered Imidazole.

Test item removal
The time of application was noted, 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 solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 3x20 mL saline was performed at each time point when the test item and positive control material remaining on the cornea was observed.
Note: Physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 90352Y05-2, Expiry date: December 2021) was used for rinsing.

OBSERVATION
Observation and assessment of corneal effects
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.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
swelling at up to 75 min
Value:
4.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
swelling at up to 240 min
Value:
4.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
0.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 75 minutes after the post-treatment rinse.
The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study.

Any other information on results incl. tables

TEST ITEM

Observation

 

Value

 

ICE Class

 

Mean maximum corneal swelling at up to 75 min

4.9%

I

Mean maximum corneal swelling at up to 240 min

4.9%

I

Mean maximum corneal opacity

0.50

I

Mean fluorescein retention

0.17

I

Other Observations

 

Test item was stuck on all cornea surface after the post-treatment rinse. The cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

Overall ICE Class

3xI

 

Based on thisin vitroeye irritation study in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant. At the Sponsor request, the second run (tor confirm the negative outcome according to the OECD No 438 guideline) was not performed.

POSITIVE CONTROL

Observation

 

Value

 

ICE Class

 

Mean maximum corneal swelling at up to 75 min

8.6%

II

Mean maximum corneal swelling at up to 240 min

28.3%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

 

Imidazole 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

1xIII 2xIV

 

The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.

NEGATIVE CONTROL

Observation

 

Value

 

ICE Class

 

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the performed in vitro eye irritation assay (one run) in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (25 June 2018).

After the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds exposure time, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in this experiment. Thus, the study was considered to be valid.

No significant swelling (mean swelling ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on any eyes. No significant fluorescein retention change (severity 0.5 on one eye and no fluorescein retention change on two eyes) was noted. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

At the Sponsor request the second run (to confirm the negative outcome according to the OECD No. 438 guideline) was not performed.

Based on the performed in vitro eye irritation assay (one run) in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant.