2-hydroxy-3-phenoxypropyl methacrylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted on 24 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 02-04 June 2015 Date on Certificate: 22 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-phenoxypropyl methacrylate

EC Number:

240-994-5

EC Name:

2-hydroxy-3-phenoxypropyl methacrylate

Cas Number:

16926-87-7

Molecular formula:

C13H16O4

IUPAC Name:

2-hydroxy-3-phenoxypropyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 171512
- Expiration date of the lot/batch: 23 December 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤70 RH%), protected from humidity (tight closed container).

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)
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 transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
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 Citoxlab Hungary Ltd. and processed within 2 hours of collection.

Selection and Preparation of eyes for the test
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.

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 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 avoiding 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, 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 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 conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were maintained at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Ex vivo observations made at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
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.

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 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 avoiding 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, 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 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 conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were maintained at controlled temperature (32±1.5°C) 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% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in the experiment. 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.

NUMBER OF REPLICATES
Three replications of each

NEGATIVE CONTROL USED
30 µL of physiological saline (0.9% (w/v) NaCl)

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
30 µL of Benzalkonium chloride solution 5% (w/v)

APPLICATION DOSE AND EXPOSURE TIME
30 µL

OBSERVATION PERIOD
240 minutes


REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Damage to epithelium based on fluorescein retention:
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:

A Haag-Streit Bern 900 slit-lamp microscope, with depth-measuring device no. 1 and slit-width setting at 9½, was used for the measurements.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum

Value:

0.67

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

Run / experiment:

Mean up to 75 min

Value:

0

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:

Mean up to 240 min

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:

fluorescein retention score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None noted

DEMONSTRATION OF TECHNICAL PROFICIENCY: N/A

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: N/A

Any other information on results incl. tables

5.1 TEST ITEM

 

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	0.67	II
Mean fluorescein retention	0.50	I
Other Observations	None
Overall ICE Class	2xI 1xII

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	7.4%	II
Mean maximum corneal swelling at up to 240 min	24.9%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Severe loosening of epithelium was observed in two eyes at 75 minutes and in one eye at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

The positive control Benzalkonium chloride solution (5% (w/v))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.

Tables of Individual Data are included in the attachments.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro eye irritation assay in isolated chicken eyes, PHPM is non- irritant.

Executive summary:

An in vitro eye irritation study of the test item PHPM was performed in isolated chicken’s eyes. The irritation effects of PHPM were evaluated according to the OECD No. 438 guideline (09 October 2017).

 

After the zero reference measurements, the eyes were held in a horizontal position and PHPM was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL of PHPM. The three positive control eyes were treated in a similar way with 30 µL Benzalkonium chloride solution 5 % (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

 

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 the experiment. Thus, the experiment was considered to be valid.

No corneal swelling was observed during the four-hour observation period on PHPM treated eyes. Slight cornea opacity change (severity 0.5 on two eyes and severity 1 on one eye) was observed in three eyes. No significant fluorescein retention change (severity 0.5) was noted in all three eyes. No other corneal effect was observed.

Summary Table for UN Classification 

Criteria for "No category" (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:	True

Criteria for "Category 1" (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity => 3 at 30 min (in at least 2 eyes)	False
Corneal opacity = 4 at any time point (in at least 2 eyes)	False
Severe loosening of epithelium (in at least 1 eye)	False

Criteria for "No prediction can be made" (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1	False
Particles of test item were struck to the cornea and could not be washed off during the study	False

Based on this in vitro eye irritation assay in isolated chicken eyes,  PHPM is non-irritant, UN GHS Classification: No Category.