2,2'-(naphthalene-1,4-diyl)bis(benzoxazole)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 12th to October 17th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

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)

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fluorescent Brightener 367

IUPAC Name:

Fluorescent Brightener 367

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03 g

Duration of treatment / exposure:

10 s then frinse with ca. 20 ml isotonic saline

Duration of post- treatment incubation (in vitro):

up to 240 minutes after post-treatment rinse.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
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 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 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 superfusion 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.


EQUILIBRATION AND BASELINE RECORDINGS
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%) were observed in the eyes during the first and additional experiment, finding considered as 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 effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

NUMBER OF REPLICATES: 3 eyes per test item, 3 eyes per positive control, 1 negative control eye

NEGATIVE CONTROL USED : yes

POSITIVE CONTROL USED : yes, imidazole

APPLICATION DOSE AND EXPOSURE TIME
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 substance, 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 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.

OBSERVATION PERIOD
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.

REMOVAL OF TEST SUBSTANCE
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 imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 ml saline was performed in all imidazole and test item treated eyes after the 30, 75, 120 and 180 minutes of observation. The imidazole and test item treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and additional experiment.


Evaluation
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

Cornea swelling was calculated according to the following formulae

CS at time t = 100 × (CT at time t – CT at t=0) CT at t=0
Mean CS at time t = (FECS(at time t)+ SECS (at time t) + TECS (at time t)) / 3

Remark:
CS = Cornea swelling
CT = Cornea thickness
FECS = First eye cornea swelling
SECS = Second eye cornea swelling
TECS = Third eye cornea swelling
Mean CS = The mean percentage of corneal swelling
at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse
at t=0 = Reference value

For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) 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.

Cornea opacity was calculated according to the following formulae
Scores were taken at any given timepoint according to:
Score Observation
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

ΔCO at time t = CO at time t - CO at t=0
Mean CO(at time t) = (FEΔCO (at time t)+ SEΔCO(at time t) + TEΔCO(at time t)) / 3

Remark:
CO = Cornea opacity
ΔCO = Difference between cornea opacity and cornea opacity reference value
FEΔCO = Difference between first eye cornea opacity and first eye cornea opacity reference value
SEΔCO = Difference between second eye cornea opacity and second eye cornea opacity reference value
TEΔCO = Difference between third eye cornea opacity and third eye cornea opacity reference value
Mean CO = The mean corneal opacity value
at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse
at t=0 = Reference value

Fluorescein retention was calculated according to the following formulae
Scores were taken at any given timepoint according to:
Score Observation
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

ΔFR at time t = FR at time t – FR at t=0
Mean FR = (FEΔFR (at time t) + SEΔFR(at time t) + TEΔFR at time t)) / 3

Remark:
FR = Fluorescein retention
ΔFR = Difference between fluorescein retention and fluorescein retention reference value
FEΔFR = Difference between first eye fluorescein retention and first eye fluorescein retention reference value
SEΔFR = Difference between second eye fluorescein retention and second eye fluorescein retention reference value
TEΔFR = Difference between third eye fluorescein retention and third eye fluorescein retention reference value
Mean FR = The mean fluorescein retention value
at time t = Observation time at 30 minutes after the post-treatment rinse
at t=0 = Reference value

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

I

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

I

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

II

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

II

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

fluorescein retention score

Run / experiment:

II

Value:

0.3

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 mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below.

First experiment:
Test item: Fluorescent Brightener 367
Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 3% I
Mean maximum corneal swelling at up to 240 min 4% I
Mean maximum corneal opacity 0.7 II
Mean fluorescein retention 0.0 I
Other observations none
Overall ICE Class 2×I, 1×II

Positive control: imidazole
Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 33% IV
Mean maximum corneal swelling at up to 240 min 40% IV
Mean maximum corneal opacity 4.0 IV
Mean fluorescein retention 3.0 IV
Other observations Corneal opacity score 4 was observed in three eyes eye at 30 minutes after the post-treatment rinse.
Overall ICE Class 3×IV

The positive control solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Negative control: NaCl (9 g/l saline)
Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 0% I
Mean maximum corneal swelling at up to 240 min 0% I
Mean maximum corneal opacity 0.0 I
Mean fluorescein retention 0.0 I
Other observations None
Overall ICE Class 3×I

The negative control NaCl (9 g/l saline) had no significant effects on the chicken eye in this study.

Additional experiment:
Test Item: Fluorescent Brightener 367

Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 2% I
Mean maximum corneal swelling at up to 240 min 3% I
Mean maximum corneal opacity 1.2 II
Mean fluorescein retention 0.3 I
Other observations None
Overall ICE Class 2×I, 1×II


Positive control: imidazole
Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 40% IV
Mean maximum corneal swelling at up to 240 min 44% IV
Mean maximum corneal opacity 4.0 IV
Mean fluorescein retention 3.0 IV
Other observations Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class 3×IV

The positive control solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Negative control: NaCl (9 g/l saline)
Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 0% I
Mean maximum corneal swelling at up to 240 min 0% I
Mean maximum corneal opacity 0.5 I
Mean fluorescein retention 0.0 I
Other Observations None
Overall ICE Class 3×I

The negative control NaCl (9g/l saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

Any other information on results incl. tables

ICE classification criteria for corneal swelling

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 for 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 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

Overall in vitro classifications

UN GHS Classification	Combinations of the 3 endpoints
No Category	3×I,
	2×I, 1×II
	1×I, 2×II
No prediction can be made	Other combinations
Category 1(Causes serious eye damage)	3×IV
	2×IV, 1×III
	2×IV, 1×II
	2×IV, 1×I
	Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
	Corneal opacity = 4 at any time point (in at least 2 eyes)
	Severe loosening of the epithelium (in at least 1 eye)

Applicant's summary and conclusion

Interpretation of results:

other: not eye irritant according to the CLP Regulation (EC 1272/2008)

Conclusions:

In this in vitro eye irritation study, using the Isolated Chicken Eye model with test item, no ocular corrosion or severe irritation potential was observed. The overall ICE class was 2×I, 1×II in the first and the additional experiment.

According to the guideline OECD 438, test item is not eye irritant.

Executive summary:

The isolated chicken eye test (ICET) was run according to OECD guideline 438.

Test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes.

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 substance exposure) were determined at each time points.

According to the first experiment results the test item showed negative outcome. Based on the OECD 438 additional experiment was necessary to confirm or discard the negative outcome.

The imidazole (positive control) was ground before use in the study. The test item and positive control 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 substance 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 saline solution were used in the first and additional experiment.

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.

The test item and imidazole were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and additional experiment. The imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and additional experiment.

In this ICET, test itemdid not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either cases. The overall ICE class was 2xI, 1xII in the first and the additional experiment.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

No ocular corrosion or severe irritation potential was observed. The overall ICE class was 2×I, 1×II in the first and the additional experiment.

According to the guideline OECD 438, test item does not require classification within the CLP Regulation (EC 1272/2008).