Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
Principles of method if other than guideline:
Based on the in vitro eye irritation assays in isolated chicken eyes with erbium gadolinium yttrium zirconium oxide, the test item was not classified as a severe irritant and not classified as non-irritant. It was therefore concluded that further information was required for classification. To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections in total).
Semi-quantitative microscopic evaluation was performed on the corneas in the ICET. The classification of histopathology findings was performed based on two publications:
- Prinsen et al. (2011). Toxicology in Vitro 25, 1475-1479
- Cazelle et al. (2014). Toxicology in Vitro 28, 657-666
- Atlas of histopathological lesions of Isolated Chicken Eyes, Wijnands and Prinsen, June 2015
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PEGYZ-23
- Expiration date of the lot/batch: 31 March 2021
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% relative humidity), protected from light.

OTHER SPECIFICS
Description: pink powder (the colour was determined by visual inspection upon arrival at test facility)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personal health and safety. Possibly irritative.
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The solubility of the test item in physiological saline was tested prior to the experiment (30 mg test item in 1 mL physiological saline). The test item did not dissolve in physiological saline.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, the test item was applied as supplied, no formulation was required.

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old and 2.55 kg
- Storage, temperature and transport conditions of ocular tissue: Heads were collected after slaughter in a commercial abattoir by a slaughter house technician and transported to CiToxLAB Hungary Ltd. 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).
- Time interval prior to initiating testing: within approximately 2 hours after collection

EYES SELECTION AND PREPARATION
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, 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
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 too much pressure on the eye by the clamp was avoided. 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 that 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, and 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.

Test system

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

POSITIVE CONTROL
- Amount applied: 30 mg

NEGATIVE CONTROL
- Amount applied: 30 µL
- Concentration (if solution): 0.9% w/v NaCl
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
Observations were done until 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
3 test item treated eyes, 3 positive control treated eyes and 1 negative control eye
Details on study design:
BASELINE ASSESSMENTS
At the end of the acclimatisation 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. Slight changes in thickness (0.0% to 1.7%) were observed in the eyes, which is considered 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 effect after treatment. All eyes were considered to be suitable for the assay.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% (w/v) NaCl), batch number 52532Y05-2, manufactured by B.Braun Pharmaceuticals AG

POSITIVE CONTROL USED
Imidazole, CAS number 288-32-4, batch number SLBH7184V, manufactured by Sigma-Aldrich Co.

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 item was applied onto the centre of the cornea. The 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 positive control eyes were treated in a similar way with 30 mg of powdered imidazole. The negative control eye was treated with 30 µL of physiological saline.

REMOVAL OF TEST SUBSTANCE
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 item if possible.
Additionally, gentle rinsing with 20 mL saline was performed at each time point when the test item and positive control material remained on the cornea.

OBSERVATION
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.

RETENTION OF CHICKEN'S EYES
At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: Reanal, Batch number: KTM14051, Expiry date: October 2017). The corneas were used for histopathology and stored at room temperature.

METHODS FOR MEASURED ENDPOINTS:
Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

SCORING SYSTEM:
- Mean corneal swelling (%):
Corneal 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 CS at time t = [FECS(at time t) + SECS(at time t) + TECS(at time t)] / 3
With:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (> 12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Mean maximum opacity score:
ΔCO(at time t) = CO(at time t) – CO(at t=0)
Mean ΔCOmax = [FECOmax(30 min to 240 min) + SECOmax(30 min to 240 min) + TECOmax(30 min to 240 min)] / 3
With:
C(at time t) = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO(at t=0) = baseline cornea opacity
ΔCO(at time t) = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30 min to 240 min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

- Mean fluorescein retention score at 30 minutes post-treatment:
ΔFR(at time t) = FR(at time t) – FR(at t=0)
Mean ΔFR = [ FEFR (30 min) + SEFR(30 min) + TEFR(30 min) ] / 3
With:
FR(at time t) = fluorescein retention at 30 minutes after the post-treatment rinse
FR(at t=0) = baseline fluorescein retention
ΔFR(at time t) = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

DECISION CRITERIA:
ICE classification criteria for corneal thickness:
Mean Corneal Swelling (%) = ICE Class
0 to 5 = I
>5 to 12 = II
>12 to 18 ( >75 min after treatment ) = II
>12 to 18 ( <=75 min after treatment ) = III
>18 to 26 = III
>26 to 32 ( >75 min after treatment ) = III
>26 to 32 ( <=75 min after treatment ) = IV
>32 = IV

ICE classification criteria for corneal opacity:
Mean Maximum Opacity Score = ICE Class
0.0 – 0.5 = I
0.6 – 1.5 = II
1.6 – 2.5 = III
2.6 – 4.0 = IV

ICE classification for criteria for mean fluorescein retention:
Mean fluorescein Retention Score at 30 minutes post-treatment = ICE Class
0.0 – 0.5 = I
0.6 – 1.5 = II
1.6 – 2.5 = III
2.6 – 3.0 = IV

Assessment of the general IN VITRO eye irritancy and regulatory UN GHS classification:
- No Category = 3×I or 2×I, 1×II
- Category 1 = 3×IV or 2×IV, 1×III or 2×IV, 1×II or 2×IV, 1×I or Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) or Corneal opacity = 4 at any time point (in at least 2 eyes) or Severe loosening of epithelium (in at least 1 eye)
- No prediction can be made = Other combinations
In the case where the result indicates Non-irritant or Corrosive/Severely Irritating, then the test item can be classified.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
mean maximum
Run / experiment:
up to 75 min after treatment / test item
Value:
2.2
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
Remarks:
mean maximum
Run / experiment:
up to 240 min after treatment / test item
Value:
5.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class: II
Irritation parameter:
cornea opacity score
Remarks:
mean maximum
Run / experiment:
test item
Value:
0.5
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
Remarks:
mean
Run / experiment:
test item
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class: I
Irritation parameter:
histopathological observations
Run / experiment:
test item
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: classified as Category 2B under GHS
Other effects / acceptance of results:
TEST ITEM
Test item was stuck on all cornea surfaces after the post-treatment and could not be rinsed off. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse. The particles stuck to the cornea could mask corneal effects underneath the particles and could potentially result in mechanical damage in vivo.
The overall ICE class for test item is: 2xI, 1xII.
Hence, the test item is not classified as a severe irritant and not classified as non-irritant. It was concluded that further information was required for classification.

Semi-quantitative microscopic evaluation was performed on the cornea in the ICET. The test item produced very slight erosion of the epithelium in 4/6 cases, and slight intensity in 2/6 sections. Slight necrosis of the top epithelial layer was seen in 1/6 cases. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, erbium gadolinium yttrium zirconium oxide was classified as Category 2B (under GHS).

POSITIVE CONTROL
Mean maximum corneal swelling at up to 75 min: 10.9% = ICE class II
Mean maximum corneal swelling at up to 240 min: 28.3% = ICE class III
Mean maximum corneal opacity score: 4.00 = ICE class IV
Mean fluorescein retention score: 3.00 = ICE class 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.
The overall ICE Class for positive control is 1xIII; 2xIV.
The positive control (imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

Histopathology: Positive control was associated with severe epithelial erosion in 3/3 cases. No compromised Bowman’s or basement membrane as well as no endothelial changes were recorded.

NEGATIVE CONTROL
Mean maximum corneal swelling at up to 75 min: 0.0% = ICE class I
Mean maximum corneal swelling at up to 240 min: 0.0% = ICE class I
Mean maximum corneal opacity: 0.00 = ICE class I
Mean fluorescein retention: 0.00 = ICE class I
Other Observations: None
The overall ICE Class for negative control is 3xI.
The negative control, physiological saline, was classified as non-irritating, UN GHS Classification: Non-classified.

Histopathology: the negative control cornea showed no abnormalities.

VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range for this type of experiment. The experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
Category 2B (mildly irritating to eyes) based on GHS criteria
Remarks:
and based on supplemental histopathology data
Conclusions:
Based on the in vitro eye irritation assays in isolated chicken eyes, the test item was not classified as a severe irritant and not classified as non-irritant. To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed very slight erosion of the corneal epithelium in 4/6 cases, and slight intensity in 2/6 sections. Slight necrosis of the top epithelial layer was seen in 1/6 cases. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, erbium gadolinium yttrium zirconium oxide was classified as Category 2B under GHS.

Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 2B.