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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
from 26 Septembre 2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:040802#
- Expiration date of the lot/batch: 06.08.2016
- Purity test date:08/01/2015
- Purity: 99.17%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable in water, instable after repeated contact to air


TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final preparation of a solid:moistened with 100 +/- 5 µl of 0.9% NaCl solution, to ensure good contact surface.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Episkin SM
Source strain:
other: 09-KERA-009, 08-KERA-008, 08-KERA-001, 10-KERA-005
Details on animal used as source of test system:
2nd passage
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (SkinEthic)
- Tissue batch number(s): 16-EKIN-004
- Production date: 26 January 2016
- Shipping date:26 January 2016
- Delivery date:26 January 2016
- Date of initiation of testing: 05/01/2016
- Experimental Starting date: 26 January 2016
- Experimental Completion date: 28 January 2016


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable):37 +/- 1°C, 5%CO2/ 95% air (incubator)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: with 25 ml of Phosphate Buffered Saline and 15 times
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/ml in phosphate buffered saline (PBS) (stock solution) : 0.3 mg/ml (1:9 DMEM-based medium (MTT medium)
- Incubation time: 3 hours +/- 15 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 Nm
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: absolute OD570 for blank was 0.042 (mean of 2 aliquots) for these experiment.
historical data absolute OD570 for blank was 0.044 (SD 0.002 / n=21) / Relative viability PC = 5.0 % (SD 1.9, n=21) and a maximal difference viability of 8% (SD = 7.4, n=197). Historical control data were generated in 2014-2015.

- Barrier function: IC50 = 2mg/ml (SDS concentration, MTT test, n=14 – specification ≥ 1.5 mg/ml)
- Morphology: histology scoring = 21.8 +/- 0.3, CV = 1.3% (HSE stained vertical paraffin sections, n=6 – specification ≥ 19.5)
- Contamination: Free of bacteria, fungus and mycoplasma
- Reproducibility:

NUMBER OF REPLICATE TISSUES: duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No MTT direct interference notified during the pretest
- 2 killed tissues/exposure periods
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 2
- 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%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 +/-2 mg +100 +/-5 µl NaCl 0.9%

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 0.9%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution):
Duration of treatment / exposure:
3 min, 60 min and 4 hours
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2 replicates for each exposure time for the test item and the negative control

Test system

Vehicle:
unchanged (no vehicle)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min Experiment
Value:
106
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min Experiment
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4 hours experiment
Value:
98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES for each experiments. Mean absolute OD (570 nm) of the two negative control tissues of every treatment period is between 0.6 and 1.5
- Acceptance criteria met for positive control: YES mean relative tissue viability of the 2 positive control tissues of the 4 hours treatment period is less or equal to 20%
- Acceptance criteria met for variability between replicate measurements: In the range of 20-100% viability and for ODs superior to 0.3, the difference between each two replicates must be less or equal to 30%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the in vitro skin corrosion test on a human skin model (EPISKIN-SM), following the EC guideline Method B.40 BIS, the test substance is classified as no-corrosive.
Executive summary:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EPISKIN-SM™, comprising a reconstructed epidermis with a functional stratum corneum. In the present study the substance was applied topically to the EPISKIN-SM™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCl. The test item showed no non-specific MTT-reducing or water-colouring potential. The test item is no MTT-reducer and has no colouring potential, therefore no additional controls were necessary. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 35% (98%) after 4 h treatment. Relative mean tissue viability was 100% after 60 min treatment and 106% after 3 min treatment. The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was ≤ 30% (1.0% - 14.9%).