Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May 2020 - 26 August 2020

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

EC Number:

950-492-4

Cas Number:

2361378-62-1

Molecular formula:

C21-23H39-43NO3

IUPAC Name:

Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

impurity 1

Reference substance name:

Amides, C16-18, N-(hydroxyethyl)

EC Number:

309-819-0

EC Name:

Amides, C16-18, N-(hydroxyethyl)

Cas Number:

101226-97-5

IUPAC Name:

Amides, C16-18, N-(hydroxyethyl)

impurity 2

Reference substance name:

Zirconium compounds

IUPAC Name:

Zirconium compounds

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity test date: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was applied in its original form (although it was ground to a fine powder in a mortar and pestle).

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: H-9600 Sárvár, Rábasömjéniutca 129., Hungary)

- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. 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 Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.`

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): 30mg

VEHICLE: unchanged (no vehicle)

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

30, 75, 120, 180 and 240 minutes after the post-treatment rinse

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 for positve control group
1 for negative control group
3 for test group

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.

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.

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. If the selected eyes were appropriate for the test, acclimatization 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 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 significant corneal thickness changes (-1.6% was observed on two eyes in Experiment I and -1.6% was noted on one eye or 1.6% was noted on eye in Experiment II) and no corneal thickness changes were observed in the other eyes in the Experiments. 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 :
3 for positve control group
1 for negative control group
3 for test group

NEGATIVE CONTROL USED
physiological saline [B. Braun Pharmaceuticals SA (194948162)]

POSITIVE CONTROL USED
powdered Imidazole [Acros Organics (A0386897)]

APPLICATION DOSE AND EXPOSURE TIME
30mg for 10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, Additional gentle rinsing with 3*20 mL saline was performed at each time point when the positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
Haag-Streit BP 900® slit lamp microscope was used for the measurements. Corneal thickness and corneal opacity were measured at all time points. For opacity determination, the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used.

- Macroscopic morphological damage to the surface: In each experiment 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. No other morphological effects were observed in each experiment in any group.

SCORING SYSTEM:
- Mean corneal swelling (%) Corneal swelling is determined from corneal thickness measurements made with an optical pachymeter on a slit lamp microscope. It is expressed as a percentage and is calculated from corneal thickness measurements according to the guideline formulae.
- Mean maximum opacity score : See below.
- Mean fluorescein retention score at 30 minutes post-treatment : See below.

DECISION CRITERIA: As per OECD 438 criteria.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

up to 75min

Run / experiment:

Experiment I

Value:

1.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

Experiment I

Value:

-7.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class II

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 5

Irritation parameter:

percent corneal swelling

Remarks:

up to 75min

Run / experiment:

Experiment II

Value:

-1.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 6

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment II

Value:

-4.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0.33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The mean corneal swelling at 240 min was -7.6%. As no severe loosening of the epithelium and no other morphological effects were observed, the mean maximum corneal swelling is considered to be slight and therefore classified as ICE class: II. In each experiment 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. No other morphological effects were observed in each experiment in any group.

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro eye irritation test in isolated chicken eyes, the test item is not an eye irritant.

Executive summary:

In an in vitro eye irritation test in isolated chicken eyes (ICET) assay (20/071-038CS), isolated chicken eyes were exposed to the test item (92.4%) for 10 seconds. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and powdered Imidazole was used for the positive control. 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. 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. Any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

In each experiment 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.  No other morphological effects were observed in the study in any group.

In Experiment I, slight corneal swelling change (mean = -7.6%) was observed during the four-hour observation period on test item treated eyes (As no severe loosening of the epithelium and no other morphological effects were observed, the mean maximum corneal swelling is considered to be slight and therefore classified as ICE class: II.). Minimal cornea opacity change (mean = 0.33) was observed. No fluorescein retention change (mean = 0.0) was noted. No other morphological effects were observed.  The Overall ICE Class was 2xI 1xII. In experiment II, minimal corneal swelling change (mean = -4.3%) was observed during the four-hour observation period on test item treated eyes. Minimal cornea opacity change (mean = 0.50) was observed. Minimal fluorescein retention change (mean = 0.33) was noted. No other morphological effects were observed. The Overall ICE Class was 3xI.  Therefore, based on these results, the test item was a non-irritant.