Willow, Salix alba, ext.

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

α-hydroxy-o-tolyl β-D-glucopyranoside

EC Number:

205-331-6

EC Name:

α-hydroxy-o-tolyl β-D-glucopyranoside

Cas Number:

138-52-3

Molecular formula:

C13H18O7

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

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

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

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


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

In vitro test system

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

Results and discussion

In vitro

Results

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

Applicant's summary and conclusion

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.