Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
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:
yes
Remarks:
see "Any other information on materials and methods incl. tables" for details
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Name: Extract of Chardon marie without support
Batch/Lot number: VDO06205A
Appearance: Light yellow brown cloudy oil
Purity: Considered as 100%
Retest date: 13 July 2019
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.


Name: Extract of Chardon marie without support
Batch/Lot number: ABT01059A
Appearance: Brown/orange turbid oil
Purity: Considered as 100%
Retest date: 19 July 2018
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Reference: MPB-693061 / MPF-00024688
Batch number: VDO06126E
Provider: Pierre Fabre
Reference: GD 1508
Certificate of analysis: Yes
MSDS: Yes
Type: Raw material
Form: Liquid
Nature: Complex mixture
Colour: Yellowish
Storage conditions: Stored at room temperature
Requested test conditions: To be tested at 100 % (as is)
Sent quantity: 10 g
Expiry / Retest date: 27 November 2016
Specific details on test material used for the study:
*Batch ABT01059A was used in Experiment I, batch VDO06205A was used in Experiment II.
**No correction for purity of the test item was applied.
*** The storage conditions of test item were changed by the Sponsor request. The test item was stored in refrigerator (2-8oC) from the test item arrival to 19 September 2018.

Test item solubility
The solubility of the test item in physiological saline was tested prior to the experiment (~30 μL test material in 1 mL physiological saline). The test item did not dissolve in physiological saline.

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
CHICKEN HEADS COLLECTION AND TRANSPORT
Strain of chicken: ROSS 308
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 heads transported to Citoxlab Hungary Ltd. 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 Citoxlab Hungary Ltd. and processed within 2 hours of collection in each experiment.

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 of 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 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.

Eyes examination and acclimatization time
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.

Identification
The eyes were identified by chamber number, marked on the door of the chamber.

THE BASELINE ASSESSMENTS
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 each 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test material: 30 μL of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
Postive control: 30 μL of Benzalkonium chloride solution (25% (w/v) in Experiment I and 5% (w/v) in Experiment II).
Negative control: 30 μL of physiological saline (0.9% (w/v) NaCl)
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
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.
Number of animals or in vitro replicates:
3 eyes per treatment - test material, positive and negative controll (9 in total)
Details on study design:
Treatment
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 μL of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 μL of Benzalkonium chloride solution (25% (w/v) in Experiment I and 5% (w/v) in Experiment II). The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl) solution in each experiment.
One eye was treated with physiological saline, three eyes with the test item and another three with Benzalkonium chloride solution in each experiment.
Note: The negative and positive controls were also part of a concurrent study (Citoxlab study code: 18/177-038CS) performed in the same experimental period using the same batch of chemicals.

Test item removal
The time of application was noted, 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 solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Note: Physiological saline (Manufacturer: B.Braun Pharmaceuticals SA, Lot number: 80353Y05-1, Expiry date: 31 December 2020 in each experiment) was used for rinsing.

OBSERVATION
Observation and assessment of corneal effects
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. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Retention of chicken’s eyes
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, Manufacturer: Reanal, Batch number: KTM21771, Expiry date: November 2019) was used for potential histopathology and stored at room temperature.

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) x100

Remark:
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

Remark:
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

Remark:
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

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
2
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
2
Value:
0.33
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
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.
Details of data interpretation for Isolated Chicken Eye (ICE) Class are given in Appendix 2 and 3. 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.

Any other information on results incl. tables

Test item 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

 

3.8 %

I

Mean maximum corneal opacity

 

1.00

II

Mean fluorescein retention

 

0.33

I

Other Observations

 

None

Overall ICE Class

 

2xI 1xII

 

Test item experiment 2

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

 

1.6 %

 

I

 

 

Mean maximum corneal swelling at up to 240 min

 

3.8 %

I

Mean maximum corneal opacity

 

1.00

II

Mean fluorescein retention

 

0.33

I

Other Observations

 

None

Overall ICE Class

 

2xI 1xII

 

Based on thesein vitroeye irritation in the isolated chicken eyes tests with Extract of Chardon marie without support, the test item is non-irritant, UN GHS Classification: No Category.

 

Positive control experiment 1

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

 

13.4%

III

 

Mean maximum corneal swelling at up to 240 min

 

33.9%

IV

Mean maximum corneal opacity

 

4.00

IV

Mean fluorescein retention

 

3.00

IV

Other Observations

 

None

Overall ICE Class

 

3xIV

The positive control Benzalkonium chloride solution (25% (w/v)) was classified as severely irritating in Experiment I, UN GHS Classification: Category 1.

Positive control experiment 2

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

 

10.2%

II

 

Mean maximum corneal swelling at up to 240 min

 

30.0%

III

Mean maximum corneal opacity

 

3.83

IV

Mean fluorescein retention

 

2.83

IV

Other Observations

 

Severe loosening of epithelium was observed in one eye at 75 minutes, loosening of epithelium was observed in one eye at 120 minutes and severe at 180 minutes on the same eye and beginning loosening of epithelium was observed in one eye at 180 minutes after the post-treatment rinse.

 

Overall ICE Class

 

1x III2xIV

The positive control Benzalkonium chloride solution (5 % (w/v)) was classified as severely irritating in Experiment II, UN GHS Classification: Category 1.

 

Negative control experiment 1

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

 

0.0

I

Mean fluorescein retention

 

0.0

I

Other Observations

 

None

Overall ICE Class

 

3xI

 

Negative control experiment 2

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

 

0.0

I

Mean fluorescein retention

 

0.0

I

Other Observations

 

None

Overall ICE Class

 

3xI

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category in both cases.

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 each experiment. This study was considered to be valid.

Negative Control: Physiological Saline

Observation

Minimum value

Maximum value

Maximum corneal swelling at up to 75 min

 

-3.2%

3.4%

Maximum corneal swelling at up to 240 min

 

-4.8%

3.4%

Maximum corneal opacity change

 

0.00

0.50

Fluorescein retention

 

0.00

0.50

Number of studies

 

416

 

Positive Control: 5% (w/v) Benzalkonium chloride solution

Observation

Minimum value

Maximum value

Maximum corneal swelling at up to 75 min

 

-8.5%

27.0%

Maximum corneal swelling at up to 240 min

 

-10.7%

38.3%

Maximum corneal opacity change

 

2.50

4.00

Fluorescein retention

 

1.50

3.00

Number of studies

 

234

 

MORPHOLOGICAL EFFECTS

In the positive control group, severe loosening of epithelium was observed in one eye at 75 minutes, loosening of epithelium was observed in one eye at 120 minutes and severe at 180 minutes on the same eye and beginning loosening of epithelium was observed in one eye at 180 minutes after the post-treatment rinse in Experiment II.

No other morphological effect was observed in the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes with Extract of Chardon marie without support, the test item was non-irritant, UN GHS Classification: No Category.
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 each experiment, after the zero reference measurements the eye was held in horizontal position and 30 μL 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 μL Benzalkonium chloride solution (25% (w/v) in the first experiment and 5% (w/v) in the second experiment). The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution) in each experiment. Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined in each experiment.

 

Due to a technical error, the final concentration of the positive control material (benzalkonium chloride solution) was 25% (w/v) in Experiment I. As this way the proper sensitivity of the system (positive response at the required concentration (5% (w/v) of the positive control material) could not be proved, the experiment did not fulfil all the criteria of the relevant OECD guideline. Therefore, an additional experiment (Experiment II) was performed to ensure validity of the overall study.

 

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

 

Experiment I:No significant swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1) was observed on all three eyes. No significant fluorescein retention change (severity 0.5 on two eyes and no fluorescein retention change on one eye) was noted on the treated eyes.

 

Experiment II:No significant swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1) was observed on all three eyes. No significant fluorescein retention change (severity 0.5 on two eyes and no fluorescein retention change on one eye) was noted on the treated eyes.

Based on these in vitro eye irritation assays in isolated chicken eyes with Extract of Chardon marie without support, the test item was non-irritant, UN GHS Classification: No Category.

Summary for UN Classification Experiment I and II

Criteria for “No category” (all true)

 

3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoints classed as I and 2 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 stuck to the cornea and could not be washed off during the study:

False