[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 29 2020 to November 26 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Molecular formula:

Cannot be adequately assigned

IUPAC Name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Molecular formula: C50H52CuN10Na2O20S8 (main component)
Molecular weight: 1479.02 (main component)
Purity of test item: 93.1%
Impurity
Water: 6.5%
EDTA-4Na: 0.4%
Supplier: FUJIFILM Corporation
Lot number: M-002P
Storage conditions: Store in the dark at room temperature.

Specific details on test material used for the study:

Not specified

Test animals / tissue source

Species:

chicken

Strain:

other:

Remarks:

Strain of chicken: ROSS 308

Details on test animals or tissues and environmental conditions:

Test System
Chicken heads collection and transport
Strain of chicken: ROSS 308
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
Only the eyes of healthy animals considered suitable for entry into the human food chain were used. Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed 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 (19.3 ºC to 20.1 ºC in first experiment and 19.4 ºC to 20.3 ºC in additional experiment) 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).
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 isotonic saline. Then the fluorescein-treated cornea was examined with a 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 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.
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, avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the super fusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were 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. 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 was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.
Identification
The eyes were identified by chamber number, marked on the door of the chamber.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03g of test item and positive control
30 μL negative control.

Duration of treatment / exposure:

Exposure period of 10 seconds from the end of the application the cornea surface

Duration of post- treatment incubation (in vitro):

30, 75 and 120 minutes for test item treated eyes.
30, 75, 120, 180 and 240 minutes for positive control treated eyes.

Number of animals or in vitro replicates:

3 replicates for test item per test performed.
3 replicates for positive control per test performed.
1 replicate for negative control per test performed.

Details on study design:

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 ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) was observed in the eyes during the first and the additional experiments. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

Test procedure

Treatment
After the zero reference measurements in each experiment, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test item, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.
The three positive control eyes were treated in a similar way with 0.03 g of Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 μL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

Test item removal
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 test item was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and additional experiments. Gentle rinsing with 20 mL saline was performed in all test item treated eyes after the 30 and 75 minutes of observation. The test item treated cornea surfaces were totally clear at 120 minutes after the post-treatment rinse in the first and additional experiments.
The positive control item was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and additional experiments. Gentle rinsing with 20 mL saline was performed in all positive control treated eyes after the 30, 75, 120 and 180 minutes of observation. The positive control treated cornea surfaces were not totally clear at 240 minutes after the post-treatment rinse in the first and additional experiments.

Observation and assessment of corneal effects
The control 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 ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse.

Retention of corneas
At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

Histopathology
Histopathology of the corneas was not performed as not borderline results were obtained, for which histopathology could provide final clarification. Corneas are discarded 2 months after the final report.

Demonstration of Proficiency
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

Additional experiment

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Additional experiment

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Additional experiment

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

First experiment

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

First experiment

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

percent corneal swelling

Run / experiment:

First experiment

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this ICET, Dye-2019 did not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either cases.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Dye-2019 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 item exposure) were determined at each of the above time points.
Based on the results of the first experiment the test item showed negative outcome (GHS NC). According to the Study Plan an additional experiment was necessary to confirm or discard the negative outcome.
The Imidazole (positive control) and test item Dye-2019 were grounded before use in the study. The test item and positive control were 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 item or positive control. Three test item treated eyes, three positive control treated eyes and one negative control treated eye (which was treated with 30 μL of NaCl 9g/L saline solution) were used in the first and additional experiments.
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, Dye-2019 did not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either cases. The overall ICE classes were once I (based on corneal swelling of 4 % within 240 minutes) and twice II (based on corneal opacity score of 0.7 and based on the fluorescein retention of 0.8) in the first experiment and the overall ICE classes were twice I (based on corneal swelling of 1 % within 240 minutes and based on the fluorescein retention of 0.0) and once II (based on corneal opacity score of 0.8) in the additional experiment.
The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in in the first and in the additional experiment. Furthermore, the three endpoints of the positive and the negative controls were in the historical control range. So, the positive and negative controls showed the expected results in the first and in the additional experiment. The experiments were considered to be valid.
In this ICET, the overall ICE classes for the test item were 1xI, 2xII in the first and 2xI, 1xII in the additional experiment.
According to the guideline OECD 438, Dye-2019 is categorized as “No Category”. Electronic copy 1 of 1