5-oxo-DL-proline

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 October 2016 to 24 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

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:

5-oxo-DL-proline

EC Number:

205-748-3

EC Name:

5-oxo-DL-proline

Cas Number:

149-87-1

Molecular formula:

C5H7NO3

IUPAC Name:

5-oxo-DL-proline

Test material form:

solid: particulate/powder

Details on test material:

-White to pale yellow

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500 and ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption.
- Characteristics of donor animals: Approximately 7 weeks old
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and transfered to the testing laboratory at ambient temperatures at the earliest convenience. 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 testing laboratory and processed within 2 hours of collection.

Test system

Vehicle:

physiological saline

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):

Observations were 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:

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
-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 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.
-Preparation: 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.
-Examination and acclimatisation: The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position. 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, 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.

EQUILIBRATION AND BASELINE RECORDINGS
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
Negative control: One
Test material: Three
Positive control: Three.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 30 mg of test material / 30 µL negative control / 30 mg positive control, exposure time of 10 seconds.

METHOD
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 the negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.

REMOVAL OF TEST SUBSTANCE
-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.
Additional gentle rinsing with 20 mL saline was performed after treatment and at each time point when the test material or positive control material remaining on the cornea was observed. The test material treated eyes were rinsed with additional gentle rinsing with 2 or 3 x 20 mL saline after treatment in each experiment.

OBSERVATION PERIOD: Observations were made at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse, minor variations within approximately ± 5 minutes were considered acceptable.

METHODS FOR MEASURED ENDPOINTS:
- 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.
-Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10 % neutral buffered formalin) used for potential histopathology and stored at room temperature.

EVALUATION/ SCORING SYSTEM:
- 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 ] x100

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:

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

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the posttreatment 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

Where:
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:

percent corneal swelling

Run / experiment:

Experiment 1 Mean (at up to 75 min)

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 Mean (at up to 240 min)

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1 Mean

Value:

0.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 1 Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Results 5

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 2 Mean (at up to 75 min)

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.

Results 6

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 2 Mean (at up to 240 min)

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 2 Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 2 Mean

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.

Other effects / acceptance of results:

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 data range in experiment. This experiment was considered to be valid.

Positive control:
Experiment I
Mean maximum corneal swelling at up to 75 min: 12.0 %
Mean maximum corneal swelling at up to 240 min: 28.3 %
Mean maximum corneal opacity: 4.0
Mean fluorescein retention: 3.0
Other Observations: Imidazole 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.

Experiment II
Mean maximum corneal swelling at up to 75 min: 10.2 %
Mean maximum corneal swelling at up to 240 min: 25.7 %
Mean maximum corneal opacity: 4.0
Mean fluorescein retention: 3.0
Other Observations: Imidazole 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.

Negative control:
Experiment I
Mean maximum corneal swelling at up to 240 min: -1.6 %
All other parameters assessed: 0.0
Experiment II: 0.0 for all parameters assessed.

Any other information on results incl. tables

Summary of Results

Experiment 1
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2.2 %	I
Mean maximum corneal swelling at up to 240 min	2.2 %	I
Mean maximum corneal opacity	0.67	II
Mean fluorescein retention	0.50	I
Other Observations	Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.
Overall ICE Class	2 x I 1 x II
Experiment 2
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.6 %	I
Mean maximum corneal swelling at up to 240 min	1.7 %	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.33	I
Other Observations	Minimal amount of test item was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces (2/2) were cleared at 120 minutes after the post-treatment rinse.
Overall ICE Class	3 x I

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the study the test material is not irritating to the eye.

Executive summary:

The potential of the test material to cause eye irritation was determined in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions using the isolated chicken eye.

After the zero reference measurements, the eye was held in horizontal position and 30 mg test item 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, three positive control treated eyes and one negative control treated eye were examined.

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

Experiment I: No significant corneal swelling (mean ≤ 5 %) was observed during the four-hour observation period on test material treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on three eyes. Minimal amount of test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤ 5 %) was observed during the four hour observation period on test material treated eyes. No cornea opacity change was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Minimal amount of test material was stuck on two cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 120 minutes after the post-treatment rinse.

Under the conditions of the study the test material is not irritating to the eye.