Silybum marianum, ext.

Endpoint summary

Description of key information

Skin Corrosion/Irritation

The viability mean of the tissues treated with the test item MPB-693061 / MPF-00024688 at 100 % was measured to be higher than 50% by in vitro evaluation of acute skin irritation using the reconstructed human epidermis SkinEthicTM RHE model, therefore the test item MPB-693061 / MPF-00024688 is classified as potentially non-irritant.

Eye Damage/Irritation

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.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September 2016 - 09 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Foreskin

Source strain:

not specified

Details on animal used as source of test system:

Information not available

Justification for test system used:

Recommended in the OECD test guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

The evaluation of the in vitro cutaneous tolerance was performed on reconstructed human epidermis SkinEthicTM RHE model (0.5 cm2) provided by SkinEthic Laboratories.
Every unit of the reconstructed epidermis consists of a stratified and differentiated epidermis obtained from human keratinocytes. Cells are grown on inert polycarbonate filter on chemically defined medium, for 17 days.
Reconstructed epidermis was maintained in agar medium supplied by SkinEthic Laboratories during the transport. On receipt of the epidermis kits on day 17, the colour of the agar medium was checked. The epidermises were transferred onto 2 ml of growth medium, and incubated at least 2 hours (37°C, 5% CO2)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material: 16 μl
Negative control: 16 μl of PBS
Positive control: 16 μl of SDS at 5 % in water

Duration of treatment / exposure:

42 min ± 1 min at room temperature

Duration of post-treatment incubation (if applicable):

Recovery period at 37±3 °C, 5±1% CO2: 42 hours ± 1 hour

Number of replicates:

3 replicates of the test item, negative controll and positive control.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

standard deviation 6.2%

Other effects / acceptance of results:

Solubility test
The requested test conditions specify that the test item has to be tested at 100%, thus, no solubility assay was performed.

Condition of test item for exposure
The requested test conditions specify that the test item has to be tested at 100%, thus, no dilution was made. The test item was tested neat and, at this step, weighing data of the test item were documented.
The application of test item was achieved with a positive displacement pipette and the solutions were spread uniformly over the surface of the epidermises by the application of a nylon mesh.

MTT-interaction of the test item
No direct interaction of the test substance with the solution of MTT was observed.

Colouring potential of the test item
The test item did not color the tissues nor perturb the OD measurements during the MTT assay.

Interpretation of results:

GHS criteria not met

Conclusions:

The viability mean of the tissues treated with the test item MPB-693061 / MPF-00024688 at 100 % was measured to be higher than 50% by in vitro evaluation of acute skin irritation using the reconstructed human epidermis SkinEthicTM RHE model, therefore the test item MPB-693061 / MPF-00024688 is classified as potentially non-irritant.

Executive summary:

The skin irritation potential was tested in vitro using the reconstructed human epidermis model SkinEthicTM RHE which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin.

 

For that purpose, cell viability of the epidermis was measured by enzymatic conversion of the vital MTT dye into a blue formazan salt that is quantitatively measured after extraction from tissues after exposure of the epidermises to the test item for a standardized

time period.

 

Relative viability was evaluated by comparison to a negative control and expressed as a percentage (%). Irritant test substances were identified by their ability to decrease cell viability below defined threshold levels.

 

This method was granted regulatory approval for skin irritation testing: OECD (2015),Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

The viability mean of the tissues treated with the test item MPB-693061 / MPF-00024688 at 100 % was measured to be higher than 50% byin vitroevaluation of acute skin irritation using the reconstructed human epidermis SkinEthicTM RHE model, therefore the test item MPB-693061 / MPF-00024688 is classified as potentially non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details

GLP compliance:

yes (incl. QA statement)

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.

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.

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

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.

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.

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

The skin irritation potential was tested in vitro using the reconstructed human epidermis model SkinEthicTM RHE which in its overall design closely mimics the biochemical and physiological properties of the upper parts of the human skin.

 

For that purpose, cell viability of the epidermis was measured by enzymatic conversion of the vital MTT dye into a blue formazan salt that is quantitatively measured after extraction from tissues after exposure of the epidermises to the test item for a standardized time period.

 

Relative viability was evaluated by comparison to a negative control and expressed as a percentage (%). Irritant test substances were identified by their ability to decrease cell viability below defined threshold levels.

 

This method was granted regulatory approval for skin irritation testing: OECD (2015),Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

The viability mean of the tissues treated with the test item MPB-693061 / MPF-00024688 at 100 % was measured to be higher than 50% by in vitro evaluation of acute skin irritation using the reconstructed human epidermis SkinEthicTM RHE model, therefore the test item MPB-693061 / MPF-00024688 is classified as potentially non-irritant.

Eye Damage/Irritation

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

 

Justification for classification or non-classification

Based on the available study data, the substance does not require classification and labelling in accordance with Regulation 1272/2008.