Willow, Salix alba, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 23 JAN 2019 to 22 MAY 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

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)

Version / remarks:

Version from 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

α-hydroxy-o-tolyl β-D-glucopyranoside

EC Number:

205-331-6

EC Name:

α-hydroxy-o-tolyl β-D-glucopyranoside

Cas Number:

138-52-3

Molecular formula:

C13H18O7

IUPAC Name:

(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[2-(hydroxymethyl)phenoxy]oxane-3,4,5-triol

Test material form:

solid: particulate/powder

Details on test material:

Name: White willow bark extract
CAS number: 84082-82-6

Specification:
Purity: 100% (UVCB substance)
Appearance: Brown powder
Odour: Characteristic
Sieve analysis: 100% pass 80 mesh

Loss on drying: 5.0 % max
Residue on ignition: 5.0 % max
Bulk density: 40 – 55 g/100ml
Extract solvent: Alcohol & water
Heavy metal: 10 ppm max
Lead (Pb): 2 ppm max
Arsenic (As): 2 ppm max
Cadmium (Cd): 2 ppm max
Mercury (Hg): 2 ppm max
Residual solvents: Complies with Eur.Pharm.


Microbiological:
===============
Total Plate Count: 1000 cfu/g max
Yeast & Mould: 100 cfu/g max
E. coli: Negative
Salmonella: Negative

Test animals / tissue source

Species:

chicken

Strain:

other: Ross 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: commercial abattoir for chicken intended for human consumption
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old
- Storage, temperature and transport conditions of ocular tissue: wrapped in tissue moistened with saline. Transport in closed box. Processing within 2 h of collection.
- indication of any existing defects or lesions in ocular tissue samples: Application of 2% (w/v) fluorescein solution onto the cornea surface for a few seconds, subsequently rinsed off with 20 mL physiological saline. Inspection of eyes with a hand-held slit lamp or slit lamp microscope for damage.

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, uniformly over the entire surface of the cornea

Duration of treatment / exposure:

10 seconds, counted from the completion of the application.

Duration of post- treatment incubation (in vitro):

up to 240 min

Number of animals or in vitro replicates:

3 eyes per treatment (test substance, positive control), 1 eye for negative control

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. 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 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) and the clamp 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.

Condition of eyes was again checked in the superfusion apparatus using a slit lamp microscope. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining or corneal opacity score were rejected. The cornea thickness was measured and 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
Acclimatization 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.

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 changes in thickness (1.6%) were observed in one eye. 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
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole.
NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
imidazole, as powder

APPLICATION DOSE AND EXPOSURE TIME
30 mg, 10 seconds exposure, beginning with termination of application

OBSERVATION PERIOD
up to 240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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 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:
- Corneal opacity: assessed with slit-lamp microscope
- Damage to epithelium based on fluorescein retention: assessed with slit-lamp microscope
- Swelling: measured with optical pachymeter on a slit-lamp microscope

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

DECISION CRITERIA: according to guideline

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 75 min

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 240 min

Value:

1.1

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

fluorescein retention score

Value:

1.67

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces treated with the positive control material were not cleared at 240 minutes after the post-treatment rinse.



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:
negative control:
max. corneal swelling, 75 min: -3.2% - 3.4%
max. corneal swelling, 75 min: -4.8% - 3.4%
max. corneal opacity change: 0.00 - 0.50
fluoresceine retention: 0.00 - 0.50
positive control:
max. corneal swelling, 75 min: -6.6% - 25%
max. corneal swelling, 75 min: -15.9% - 36.7%
max. corneal opacity change: 3.50 - 4.00
fluoresceine retention: 2.00 - 3.00

Any other information on results incl. tables

Test item results:

Chamber number¯	Corneal thickness (instrument units)																	Corneal opacity score							Fluorescein retention
Relative observation time (min)	-45	0	Change	30	change at 30	75	change corrected	change at 75	Max change up to 75	120	change at 120	180	change corrected	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max Δ Opac	0	30	Δ Flu ret
11	63	63	0.0%	63	0.0%	63	0.0000	0.0%	0.0%	63	0.0%	63		0.0%	63	0.0%	0.0%	0	0	0.5	0.5	0.5	0.5	0.5	0	2	2.0
12	63	63	0.0%	63	0.0%	63	0.0000	0.0%	0.0%	63	0.0%	64		1.6%	64	1.6%	1.6%	0	0.5	0.5	0.5	0.5	0.5	0.5	0	2	2.0
13	63	63	0.0%	63	0.0%	64	0.0159	1.6%	1.6%	64	1.6%	64		1.6%	64	1.6%	1.6%	0	0	0.5	0.5	0.5	0.5	0.5	0	1	1.0

Applicant's summary and conclusion

Interpretation of results:

other: Study results used for classification in weight of evidence approacha ccording to IATA or Serious Eye Damage and Eye Irritation

Conclusions:

The test item is not classified as a severe irritant (GHS category 1) and not classified as non-irritant. It is concluded that further information is required for classification.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (25 June 2018) in compliance with GLP.

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). Three test item 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 each experiment. Thus, the study was considered to be valid.

No significant swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. A mean corneal swelling of ≤5% leads to classificiation in ICE class I for this endpoint.

No significant cornea opacity change was observed on all three eyes (maximum severity 0.5 for each eye). The mean maximum severeity of 0.5 leads to classificiation in ICE class I for this endpoint.

Moderate fluorescein retention change (severity 1.0 on one eye and severity 2.0 on two eyes) was noted on all three eyes. The mean fluorescein retention score is 1.66, which corresponds to a classification as ICE class III for this endpoint (0.6 - 1.5 corresponds to class II, 1.6 - 2.5 corresponds to class III). No other corneal effect was observed.

The combination of the 3 ICE classes is: 2xII, 1x3. For this combination of ICE classes, no prediction can be made according to the guideline.

In consequence, based on these in vitro eye irritation assays in isolated chicken eyes, the test item is not classified as a severe irritant (GHS category 1) and not classified as non-irritant. It is concluded that further information is required for classification.