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Toxicological information

Skin irritation / corrosion

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
other: white to off-white powder
Details on test material:
- Particle size distribution: 100 % particle size < 12 µm (method: Laser Diffraction; Certificate of Analysis)
Particle size parameter determined with a Malvern Mastersizer 2000 (Non-GLP determination)
D10% = 1.18 µm
D50% = 7.72 µm
D90% = 16.00 µm
D: Diameter; 10, 50, 90: percentage cumulative
- Mass median aerodynamic diameter (MMAD): 1.595 µm
- Geometric standard deviation (GSD): calculated as 2.09
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH, Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 5 october 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material : at room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture

OTHER SPECIFICS: IsoQure UR 300

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM (EPI-200, Lot no. 25876) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.
Justification for test system used:
Skin irritation by cytotoxic action of substances with direct human skin contact refers to the production of reversible damage to the skin following the application of a test item for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)]. This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach.
Vehicle:
other: Dulbecco’s phosphate buffered saline
Remarks:
For better contact of the test item to the skin, the skin surface was moistened .
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200)
- Tissue batch number(s): Lot no. 25876, Keratinocyte strain 00267

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: at the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Observable damage in the tissue due to washing: -
- Modifications to validated SOP: -

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 25 mg IsoQure UR 300 were added to 1 mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 87.4-110.8 % (negative control); 1.3-10.2% (positive control)
- Barrier function: The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration.
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- No. of replicates : each of the three tissues in 2 replicates
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): D PBS. 30 µL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):5% aqueous sodium dodecyl sulphate (SDS)



Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 min
Number of replicates:
3

Test system

Details on study design:
TEST SITE
- Area of exposure: surface
- % coverage: 0.63 cm2

REMOVAL OF TEST SUBSTANCE
- Washing : washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS
- Time after start of exposure: 42 h

OBSERVATION TIME POINTS
(indicate if minutes, hours or days)

SCORING SYSTEM:
- Method of calculation: According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2 )
mean tissue viability > 50% non-irritant (NI).

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test group
Value:
99.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 60 min exposure, followed 42h
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control (D-PBS)
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 60 min exposure, followed 42h
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control (5% SDS )
Value:
7.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: min exposure, followed 42h
Other effects / acceptance of results:
The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
The standard deviation determined for all triplicates was below the limit of acceptance of 18%. Hence, all acceptance criteria required were fulfilled.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, IsoQure UR 300 tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).
Executive summary:

A key in vitro study was performed to determine cytotoxic properties of IsoQure UR 300 to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin. The EpiDermTM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. IsoQure UR 300 was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS).D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium. The mean viability of cells exposed to IsoQure UR 300 was 99.7% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. IsoQure UR 300 was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%. The standard deviation of all triplicates determined was below the limit of acceptance of 18%.Hence, all acceptance criteria were fulfilled.