Willow, Salix alba, ext.

Endpoint summary

Description of key information

An in vitro skin irritation test of WWBE was performed in the reconstructed human epidermis model EPISKIN(TM) (SM), according to OECD Guideline 439. The study indicates that the test item is non-irritant to skin.

An in vitro eye irritation study of WWBE was performed in an isolated chicken eye test, according to OECD Guideline 438. No significant swelling and no significant cornea opacity change was observed on all three eyes. Moderate fluorescein retention change (severity 1 on one eye and severity 2 on two eyes) was noted on all three eyes. No other corneal effect was observed. Due to the moderate fluorescein retention observed, the study was not unambiguously negative. Accordingly, based on this study, white willow bark extract can neither be classified as causing severe eye damage (Category 1) nor as a non-irritant.

A confirmatory EpiOcular test according to OECD TG 492 was performed. Following exposure with the test item, after adjustment for non-specific controls, the mean cell viability in the EpiOcular test was 14.0%. This is below the threshold of 60%, therefore the test item is considered irritant to Reconstructed human Cornea-like Epithelium, indicating a classification as causing severe eye damage (Category 1) or as irritating (Category 2).

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:

From 11 FEB 2019 to 21 MAY 2019

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)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

reconstructed epidermis

Source species:

other: human

Vehicle:

unchanged (no vehicle)

Remarks:

test substance was grounded to fine powder

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small / Human Epidermis (SM/13)
- Tissue batch number(s): 19-EKIN-007
- Production date: not given in certificate of analysis
- suggested expiration date: 18 february 2019, according to certificate of analysis
- Date of initiation of testing: 13 february 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.6-24.4°C)
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed thoroughly with 25 mL PBS
- Observable damage in the tissue due to washing: not reported
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL, 2 mL per EpiSkin unit
- Incubation time: 3 hours at 37 °C and 5% CO2, protected from light
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Linear OD range of spectrophotometer:not reported

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: both
- Procedure used to prepare the killed tissues (if applicable): incubation in distilled water for 48 h, freezing
- N. of replicates : 2 living epidermis, 2 killed epidermis
- Method of calculation used: OD from non-specific MTT reduction is calculated by subtracting OD from killed epidermis treated with the test item from OD from killed tissues treated with the negative control. The OD of test item treated living epidermis is then corrected by the non-specific MTT reduction.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 15 minutes exposure and 42 hours post-treatment incubation is less than or equal to 50%.

- Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg, as powder



NEGATIVE CONTROL
- Amount(s) applied: 50 µL PBS

POSITIVE CONTROL
- Amount(s) applied: 50 µL SDS
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates in single experiment

Value:

111.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: yes. determined experimentally and considered in calculation of viability
- Colour interference with MTT: yes, determined experimentally. Not considered in calculation of viability because below threshold of 5%.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

The results indicate that the test item is non-irritant to skin

Executive summary:

An in vitro skin irritation test of the test material was performed in the reconstructed human epidermis model EPISKIN(TM) (SM). EPISKIN(TM) (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to OECD Guideline 439 under GLP conditions.

Disks of EPISKINTM (SM) (three units) were treated with the powdered test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units/control). Two additional disks were used to provide an estimate of colour contribution (SC living) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues, were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with White willow bark extract, the mean cell viability was 111.2% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test, the results indicate that the test item is non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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.

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

Irritation parameter:

cornea opacity score

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

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

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

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

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 17 SEP 2019 to 6 DEC 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Version from 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

Reconstructed Human cornea-like epithelium (tissues), EpiOcular (TM) from MatTek, Bratislava, Slovak Republic. The tissues were stored at 2-8ºC until use. Tissues were used within 72 hours after their production.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg of the test item was applied evenly to the unit surfaces

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours after rinsing

Number of animals or in vitro replicates:

2 replicates for the test titem, 2 negative controls, 2 positive controls.

Details on study design:

- Details of the test procedure used : The toxicity of test items (and thus their ocular irritation potential) was evaluated by the relative viability of the treated tissues compared to the negative control tissues. Viability was determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test item-treated cultures.

- RhCE tissue construct used, including batch number : Reconstructed Human cornea-like epithelium (tissues), EpiOcular (TM) from MatTek, Bratislava, Slovak Republic. Batch 30626.

- Doses of test chemical and control substances used : test item: 50 mg per tissue, negative control: 50 µL distilled water per tissue, positive control: 50 µL methyl acetate per tissue.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) :
Before exposure, tissues were kept in a controlled atmosphere (37°C, 5% CO2, 95% r.H.) for 30 min. Exposure was 6h in the same atmosphere. After rinsing, tissues were immersed in fresh assay medium for 25 min, followed by 18 hours of post-exposure incubation in the same controlled atmosphere.

- Description of any modifications to the test procedure : -

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : Direct MTT reduction was controlled by mixing test item with MTT solution and determining the MTT reduction (NSMTT). Colouring potential of the test substance was controlled by treating tissues and measuring OD in the same manner as for the main test, but without adding MTT solution (NSCliving; non-specific colour in living tissues). As the test item was identified as producing both direct MTT reduction and colour interference a third set of controls was performd with killed tissue (NSCKilled; non-specific colour in killed tissues)

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2 for each.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : OD was measured at 570 nm. The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 13 August 2018, calibration is valid until August 2020) at the required wavelength on the day of use.

- Description of the method used to quantify MTT formazan : The tissues were (except the two living and two killed colour control units which was incubated with pre-warmed Assay Medium) transferred to wells pre-filled with MTT solution and incubated for 3 hours at 37°C (± 2°C), 5% CO2 in a >95% humidified atmosphere. Formazan was extracted for 2 hours with isopropanol on an orbital shaker (~120 rpm).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : Evaluation criteria according to TG Guideline 402: Mean viability <= 60% corresponds to either Category 1 or Category 2. Mean viability > 60% corresponds to non-classification.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : negative control: mean OD: 1.944, standard deviation: 0.061, min OD: 1.889, max OD: 2.031, number of cases: 4; positive control: mean OD: 0.743, standard deviation: 0.204, min OD: 0.522, max OD: 1.016, number of cases: 4;

- Complete supporting information for the specific RhCE tissue construct used : The EpiOcular™ tissue constructs consisted of at least 3 viable layers of cells and a non-keratinized surface, showing a corneal-like structure analogous to that found in vivo. The EpiOcular™ human cell construct (MatTek Corporation) is a non-keratinized epithelium prepared from normal human keratinocytes, grown in such a way as to produce a membrane similar to a cornea. The use of EpiOcular™ cultures offers features appropriate for a model of ocular irritation. First, the model is composed of stratified human keratinocytes in a three-dimensional structure. Next, test materials can be applied topically to the model, so that all substances including water insoluble materials can be tested.

- Positive and negative control means and acceptance ranges based on historical data : see above
- Acceptable variability between tissue replicates for positive and negative controls : difference between two tissue replicates < 20%.
- Acceptable variability between tissue replicates for the test chemical: difference between two tissue replicates < 20%.

Irritation parameter:

other: Viability [%]

Run / experiment:

first and only run

Value:

14

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

29.8 % viability

Remarks on result:

positive indication of irritation

Additional controls for colored test items:

Optical Density (OD) and the calculated Non SpecificColour % (NSC_living%) of the Additional Control Tissues

 

Additional control	Optical Density (OD)			NSC%
		Measured	Blank corrected	(living)
Treated with	1	0.053	0.007	0.3
White willow bark extract	2	0.051	0.005
	mean	--	0.006

Notes:

1.         Mean blank value was 0.047.

2.         Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places)

 

 

 

 

Additional controls for interactions of the test item MTT:

Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT) of the Additional Control Tissues

 

Additional control	Optical Density (OD)			NSMTT	NSMTT%
		Measured	Blank corrected
Treated with	1	0.154	0.108	0.055	3.5
White willow bark extract	2	0.161	0.114	0.061
	mean	--	0.111	0.058
Treated with	1	0.104	0.058	--
Distilled water	2	0.095	0.049
	mean	--	0.053

Notes:

1.     Mean blank value was 0.047.

2.     Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

 

Additional controls using dead tissue (epidermis treated by freeze-thawing)

Optical Density (OD) and the calculatedNon SpecificColour % (NSC_killed%) of the Additional Control Tissues

 

Additional control	Optical Density (OD)			NSC%
(killed epidermis)		Measured	Blank corrected	(killed)
Treated with	1	0.053	0.006	0.3
White willow bark extract	2	0.051	0.005
	mean	--	0.005

Notes:

1.     Mean blank value was 0.047.

2.     Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

 

Viability measurements

Optical Density (OD) and the calculated relative viability % of the samples

 

Substance	Optical Density (OD)			TODTT	Viability
		Measured	Blank corrected		(% RV)
Negative Control:	1	1.573	1.527	-	93.5
Distilled water	2	1.787	1.740	-	106.5
	mean	--	1.633	-	100.0
Positive Control:	1	0.593	0.546	-	33.4
Methyl acetate	2	0.473	0.426	-	26.1
	mean	--	0.486	-	29.8
Test Item:	1	0.273	0.227	0.169	10.3
White willow bark extract	2	0.393	0.346	0.288	17.6
	mean	--	0.286	0.228	14.0

Notes:

1.        Mean blank value was 0.047.

2.         Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3.         TOD_TT: the measured values were corrected for non-specific controls (NSMTT value was 0.058,NSC_livingvalue was 0.006 and NSC_killedvalue was 0.005, therefore the final correction was 0.058 (rounded to three decimal place)). TOD_TT= NSMTTOD-NSClivingOD+ NSC killed OD.

 

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:

Following exposure with the test item, the mean cell viability was 14.0% compared to the negative control (after adjustment for non-specific controls). This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiOcular™ model test, the results indicate that the test item is probably irritant to eyes (GHS Classification: Category 2) or causes serious eye damage (Category 1).

Executive summary:

The purpose of this study was to evaluate the eye hazard potential of the test item, based on its ability to induce cytotoxicity in the EpiOcularTM cornea epithelial model, as measured by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] assay.

Disks of EpiOcular™ (two units) were treated with the powdered test item and incubated at 37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2 in a >95% humidified atmosphere. After the incubation, MTT solution was added to the units and incubated for a further 3 hours to determine cell viability. The precipitated formazan crystals were then extracted using  isopropanol and quantified spectrophotometrically at 570 nm.

Distilled water and Methyl acetate treated epithelium tissues were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed Epithelium units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled), were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability is less or equal (≤) to 60% of the negative control, the test item is considered to be irritant to Reconstructed human Cornea-like Epithelium.

Following exposure with the test item, the mean cell viability was 14.0% compared to the negative control (after adjustment for non-specific controls). This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiOcular™ model test, the results indicate that the test item is irritant to eyes (GHS Classification: Category 2) or causes serious eye damage (Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin:

An in vitro skin irritation test of WWBE was performed in the reconstructed human epidermis model EPISKIN(TM) (SM). EPISKIN(TM) (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The study was performed according to OECD Guideline 439 under GLP conditions.

Following exposure with White willow bark extract, the mean cell viability was 111.2% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test, the results indicate that the test item is non-irritant to skin.

Eye:

An in vitro eye irritation study of WWBE was performed in isolated chicken’s eyes. The study was performed according to the OECD No. 438 guideline (25 June 2018) under GLP conditions.

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. No significant cornea opacity change (severity 0.5) was observed on all three eyes. Moderate fluorescein retention change (severity 1 on one eye and severity 2 on two eyes) was noted on all three eyes. No other corneal effect was observed.

Based on these results, the test item is not classified as causing serious eye damage (Category 1) and not classified as non-irritant.

In addition, an EpiOcular test using reconstructed human Cornea-like Epithelium was performed according to OECD TG 492 with the registered substance.

Following exposure with the test item, the mean cell viability (after adjustment for non-specific controls) was 14.0% compared to the negative control. This is below the threshold of 60%, therefore the test item was considered as being irritant to reconstructed human Cornea-like Epithelium. The experiment met the validity criteria, therefore the study was considered to be valid.

Based on the results of this study, WWBE is probably irritant to eyes (UN GHS Classification: Category 2) or causes serious eye damage (Category 1).

Justification for classification or non-classification

Skin:

Based on the results of the OECD 439 study, White willow bark extract is not classified for skin irritation according to Regulation (EC) No. 1272/2008.

Eye:

Assessment of the classification is based on the OECD guidance document on an Integrated Approach on Testing and Assessment (IATA) for serious eye damage and eye irritation (ENV/JM/MONO(2017)5).

The IATA uses 9 assessment modules to conclude on classification.

Module 1, 2 and 3 (Existing human, animal and in vitro data from OECD adopted test methods on eye hazard):

No existing human or animal data with relevance for classification is available for WWBE. In vitro testing was performed using an isolated chicken eye test (ICE) and a test with a reconstructed human Cornea-like Epithelium (EpiOcular). The results of these studies do not allow for direct classification of WWBE. However, they provide sufficient evidence for classification based on a WoE assessment (see Module 9).

Module 4 and 5 (Other existing animal and in vitro data on eye hazard from non-OECD adopted methods):

No other existing data with relevance for classification are available.

Module 6 (Existing data indicating skin corrosion):

In vitro testing determined that WWBE is not classified for skin corrosion, therefore no implicit risk of serious eye damage can be derived.

Module 7 (Physico-chemical properties):

WWBE does not have physico-chemical properties that implicitly constitute a risk of serious eye damage.

Module 8 (Non-testing data on serious eye damage and eye irritation):

As the registered substance is a UVCB, no (Q)SAR models or structure based expert systems are applicable. No data from similar substances that would allow a read-across or grouping is available.

Module 9 (WoE assessment on all collected information):

At the time of this assessment, no validated in vitro method is available that allows the identification of substances with classification as Category 2. All validated tests only allow conclusion whether the test item requires classification as Category 1 or does not require classification for eye irritation or serious eye damage.

Prior to in vitro testing, no information was available to make a prediction on the eye damage potential of WWBE. An isolated chicken eye (ICE) test was performed first, as it is compatible with a top-down approach, as well as with a bottom-up approach: in case of a negative result, the ICE allows to conclude directly on no need for classification. In case of a sufficiently strong positive result, the ICE also allows to conclude directly that classification for serious eye damage is warranted.

The combined results of the 3 test criteria of the ICE were slightly above the threshold for non-classification.

As the result of the ICE is very close to non-classification, as the next step, an assay with reconstructed human cornea-like epithelium (EpiOcular) was performed with WWBE. The EpiOcular test allows direct conclusion on non-classification in case of a sufficiently low reduction of cell viability, but does not allow conclusions on classification as Category 1 or Category 2. In the EpiOcular test, WWBE led to a viability of 14%, which is clearly below the threshold for non-classification (60%).

Therefore, no direct conclusion on classification is possible based on the obtained information from in vitro studies.

Classification is however possible with an WoE assessment on all collected information:

Both in vitro studies agree that it is not adequate to conclude on non-classification for WWBE. The viability determined in the EpiOcular test is of little value to differentiate between Category 1 and Category 2. In the validation study (EC EURL ECVAM (2014)), very poor correlation between known Category 1 & Category 2 substances and the magnitude of viability reduction in the ICE was observed.

The test results of the ICE are of significantly higher value for a decision on classification. The ICE has 3 test endpoints (corneal swelling, opacity, fluorescein retention) which are all scored on an integer scale from I to IV (i.e. ICE class), with I representing the lowest potential for irritation. WWBE scored I for corneal swelling and opacity and III for fluorescein retention. Classification into Category 1 requires a score of IV in at least 2 of the 3 endpoints. These classification criteria are clearly not met as the results obtained for WWBE are far from it. However, because of the high false negative rate for Category 1 substances in this assay (OECD (2018)), the failure to meet the criteria for classification as Category 1 is not indicative of classification as Category 2.

In contrast, the scores of WWBE are very close to the requirements for non-classification (either of the score combinations 3xI, 2xI+1xII, 2xII+1xI). This is even more apparent when considering how the score III for fluorescein retention is derived: 3 treated chicken eyes are scored regarding their fluorescein staining intensity as 0, 0.5, 1.0, 2.0, 3.0. For WWBE the eyes were scored as 1.0, 2.0, 2.0 (i.e. mean score 1.67). A mean score up to 1.5 scleads to a score of II for this endpoint, which has been missed by the smallest possible margin. In other words, had any of the treated eyes scored the next lower category for fluorescein retention, the requirements to conclude on non-classification would have been met (scores 2xI+1xII).

Taken together, the WoE assessment provides sufficient certainty to conclude that WWBE has to be classified as irritating to eyes (Category 2).

References:

EC EURL ECVAM. (2014). The EURL ECVAM -Cosmetics Europe prospective validation study of Reconstructed human Cornea-like Epithelium (RhCE)-based test methods for identifying chemicals not requiring classification and labelling for serious eye damage/eye irritation: Validation Study Report. EUR 28125 EN; doi:10.2787/41680. Available at: [http://publications.jrc.ec.europa.eu/repository/handle/JRC100280].

OECD (2018). Guideline for Testing of Chemicals No. 438. Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification. Organisation for Economic Cooperation and Development, Paris. Version from 25 June 2018. Available at: http://www.oecd.org/env/testguidelines.