Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016

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:

2018

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:

2013

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:

Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

EC Number:

305-928-2

EC Name:

Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

Cas Number:

95370-65-3

IUPAC Name:

DL-Proline, 5-oxo-, compd. with N2-coco acyl-L-arginine Et ester

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White to pale yellow powder
- Storage Conditions: Controlled room temperature (15 to 25 ºC, below 70 RH %)

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Number of animals: Not specified. Seven eyes were used in the experiment.
- Characteristics of donor animals: Approximately 7 weeks old. Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and transported to the testing laboratory 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).
- Time interval prior to initiating testing: The heads were received at the testing laboratory and processed within approximately 2 hours of collection.
- Indication of any existing defects or lesions in ocular tissue samples: 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.
- Indication of any antibiotics used: None specified.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 replicates

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 theorbit, 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.

EQUILIBRATION AND BASELINE RECORDINGS
- 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). Too much pressure on the eye by the clamp was avoided. 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 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, 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 corneal thickness changes (0.0 %) were observed in the eyes. 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.
-After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea.

NUMBER OF REPLICATES: Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

NEGATIVE CONTROL USED: 30 μL of physiological saline.

POSITIVE CONTROL USED: 30 mg of powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 30 mg of test material was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material for 10 seconds.

REMOVAL OF TEST SUBSTANCE:
- The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible.
- Additional gentle rinsing with 20 mL saline was performed at each time point when the test material or control material remaining on the cornea was observed. The test material treated eyes were rinsed additional gentle rinsing with 4x20 mL saline after treatment in each experiment.

OBSERVATION PERIOD:
- 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 slit-lamp microscope was used for the measurements.
- Morphological effects: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test material to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

EVALUATION:
- 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
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:
ΔCO at time t = CO at time t – CO at t=0
Mean ΔCOmax = [FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3
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
ΔCO 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:
ΔFR at time t = FR at time t – FR at t=0
Mean ΔFR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR 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:

cornea opacity score

Run / experiment:

mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Results 3

Irritation parameter:

other: maximum corneal swelling at up to 75 min (%)

Run / experiment:

mean

Value:

3.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Results 4

Irritation parameter:

other: maximum corneal swelling at up to 240 min (%)

Run / experiment:

mean

Value:

13.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Other effects / acceptance of results:

- The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments. The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.
- Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

HISTOPATHOLOGY
- Semi-quantitative microscopic evaluation was performed on the cornea in the assay.
- The negative control Physiological saline (Salsol solution 0.9 %) cornea showed no abnormalities.
- Positive control, 30 mg powdered Imidazole associated with moderate epithelial erosion without partly detached layer from basal membrane in 3/3 cases. No compromised basement membrane, Bowman’s and Descemet’s membrane, or as well as no stromal and endothelial changes were recorded.
- The test material produced from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.

VALIDITY OF THE TEST
- The positive control Imidazole was classified as severely irritating and the negative control Physiological saline was classified as non-irritating.
- The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

DISCUSSION
- To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections).
- Microscopic evaluation showed from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.
Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 2 (sub-category 2A).

Any other information on results incl. tables

Table 1: Summary of results

Observation	Test material		Positive control		Negative control
	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.8 %	I	10.3 %	II	0.0 %	I
Mean maximum corneal swelling at up to 240 min	13.1 %	II	27.7 %	III	0.0 %	I
Mean maximum corneal opacity	2.00	III	4.00	IV	0.0	I
Mean fluorescein retention	3.00	IV	3.00	IV	0.0	I
Other Observations	Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.		Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.		None
Overall ICE Class	1xII 1xIII 1xIV		1xIII 2xIV		3xI

 

Table 2: Summary table for UN GHS classification

Criteria for no category:	True/false
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoints classed as II	False
Test item were not stuck to the cornea at 240 minutes after post-treatment rinse	True
Criteria for Category 1:	True/false
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:	True/false
Based on the endpoints not classifiable for No Category and for Category 1	True
Particles of test item were stuck to the cornea and could not be washed off during the study.	False

Applicant's summary and conclusion

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of this study the test material was concluded to be classified as UN GHS eye irritant Category 2 (sub-category 2A).

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions with an in vitro eye irritation study performed in isolated chicken’s eyes.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test material was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg of 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, three positive control treated eyes and one negative control treated eye were examined. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. Moderate corneal opacity change (severity 2.0) was noted on all three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation assay in the isolated chicken eyes test with, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.