ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2019 to 27 August 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

Test guideline 1

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

EC Number:

806-801-9

Cas Number:

1539267-56-5

Molecular formula:

C25H25O4P

IUPAC Name:

ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White to off white.
- Storage conditions: Controlled room temperature (15 - 25 °C, =70 % relative humidity), protected from light.

Specific details on test material used for the study:

The solubility of the test material in physiological saline was tested prior to the Experiment I (30 mg test material in 1 mL physiological saline). The test material did not dissolve in physiological saline.

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
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 the Test Facility 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 the Test Facility and processed within 2 hours of collection in each experiment.
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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg

Duration of treatment / exposure:

10 seconds from the end of the application

Number of animals or in vitro replicates:

One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED 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.
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.

EQUILIBRATION AND BASELINE RECORDINGS
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.
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. No changes in thickness (0.0 %) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES : One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

NEGATIVE CONTROL USED : 30 µL of physiological saline

POSITIVE CONTROL USED: 30 mg powdered imidazole

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. In each experiment, 30 mg of the test material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material, taking care not to damage or touch the cornea.
In each experiment a 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 test material and another three eyes with powdered imidazole in each experiment.

REMOVAL OF TEST MATERIAL
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 at least 20 mL physiological saline solution at ambient temperature in each experiment, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 3 x 20 mL physiological saline was performed at each time point when the test material or positive control material remaining on the cornea was observed in each experiment.

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. A Haag-Streit BP 900® slit-lamp microscope was used for the measurements.
Any morphological effects were recorded.

EVALUATION
A test is considered acceptable if the concurrent negative or vehicle/solvent controls and the concurrent positive controls are identified as GHS No Category and GHS Category 1, respectively.

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

Where:
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).

Cornea opacity was calculated according to the following formulae:

¿CO at time t = CO at time t – CO at t=0

Mean ¿COmax = [FECOmax(30min to 240min) + SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3

Where:
CO 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
¿CO 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(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

¿FR at time t = FR at time t – FR at t=0

Mean ¿FR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
¿FR 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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

(up to 75 min)

Run / experiment:

Experiment I and Experiment II (mean)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

(up to 240 min)

Run / experiment:

Experiment I (mean)

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Remarks:

(up to 240 min)

Run / experiment:

Experiment II (mean)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I (mean)

Value:

0.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II (mean)

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I (mean)

Value:

0.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II (mean)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

EYE IRRITATION RESULTS
The test material showed no significant corneal effect in the first experiment. As the test material was solid, the negative results were confirmed by a second experiment. The second experiment confirmed the negative results.
The positive control (Imidazole) was classified as inducing serious eye damage (Category 1) as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
The negative control Physiological saline was not classified for eye irritation or serious eye damage as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

MORPHOLOGICAL EFFECTS
In experiment I the test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.
In experiment II the test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 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 in each experiment.
No other morphological effect was observed in the study.

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 control data range in each experiment. The number of eyes used for each treatment group was adequate. These experiments were considered to be valid.

Any other information on results incl. tables

Test Material – Experiment I

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.	2.2 %	I
Mean maximum corneal opacity change	0.67	I
Mean flouorescein retention change	0.83	I
Other observations	Test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.
Overall ICE class	1 x I 2 x II

 

Test Material – Experiment II

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 change	0.5	II
Mean flouorescein retention change	0.0	II
Other observations	Test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse.
Overall ICE class	3 x I

 

Positive Control – Experiment I

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	9.6 %	II
Mean maximum corneal swelling at up to 240 min.	25.5 %	III
Mean maximum corneal opacity change	4.0	IV
Mean flouorescein retention change	3.0	IV
Other observations	Imidazole was stuck on all cornea surfaces (3/3) 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 III 2 x IV

 

Positive Control – Experiment II

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	10.9 %	II
Mean maximum corneal swelling at up to 240 min.	29.3 %	III
Mean maximum corneal opacity change	4.0	IV
Mean flouorescein retention change	3.0	IV
Other observations	Imidazole was stuck on all cornea surfaces (3/3) 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 III 2 x IV

 

Negative Control – Experiment I

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 change	0.0	I
Mean flouorescein retention change	0.0	I
Other observations	None
Overall ICE class	3 x I

 

Negative Control – Experiment I

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 change	0.0 %	I
Mean flouorescein retention change	0.0 %	I
Other observations	None
Overall ICE class	3 x I

 

Historical Control Data: Negative Control – Physiological Saline

Observation	Min. Value	Max. Value
Mean maximum corneal swelling at up to 75 min.	-3.2 %	3.4 %
Mean maximum corneal swelling at up to 240 min.	-4.8 %	3.4 %
Mean maximum corneal opacity change	0.0	0.5
Mean flouorescein retention change	0.0	0.5
Number of cases	497

 

Historical Control Data: Positive Control – Imidazole

Observation	Min. Value	Max. Value
Mean maximum corneal swelling at up to 75 min.	-6.6 %	25.0 %
Mean maximum corneal swelling at up to 240 min.	-15.9 %	36.7 %
Mean maximum corneal opacity change	3.50	4.00
Mean flouorescein retention change	2.00	3.00
Number of cases	221

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not eye irritating

Conclusions:

Under the conditions of the study, the test material was found to be non-irritant, not requiring classification for eye irritation or serious eye damage.

Executive summary:

The eye irritation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions.

The in vitro study was performed in isolated chicken eyes. In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, 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). In the study, three test material treated eyes were examined in each experiment, furthermore three positive control treated eyes and one negative control treated eye were examined in each experiment.

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 each experiment. The number of eyes used for each treatment group was adequate. Thus, the experiments were considered to be valid.

Experiment I: No significant corneal swelling (mean = 5 %) was observed during the four-hour observation period on all test material treated eyes. Slight corneal opacity change (severity 0.5 on two test material treated eyes and severity 1.0 on one test material treated eye) was noted on test material treated eyes. Slight fluorescein retention change (severity 0.5 on one test material treated eye and severity 1.0 on two test material treated eyes) was noted on test material treated eyes. Test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.

No other corneal effect was observed.

Experiment II: No corneal swelling was observed during the four-hour observation period on all test material treated eyes. No significant corneal opacity change (severity 0.5 on all test material treated eyes) was noted on test material treated eyes. No fluorescein retention change was observed on all test material treated eyes. Test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse. No other corneal effect was observed.

Based on these in vitro eye irritation results in the isolated chicken eyes tests, the test material was concluded to be non-irritant, not requiring classification for eye irritation or serious eye damage.