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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:
weight of evidence
Study period:
Oct 24-26, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Council Regulation (EC) No 761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed skin produced by SkinEthic Laboratories
Source strain:
other: adult human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: in vitro skin model RHE
- Tissue batch number(s): 12022A1001

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: a minimum of 25 mL PBS; excess PBS was removed by gently shaking the inserts and blotting the bottom with the blotting paper
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 hours
- Spectrophotometer: plate spectrophotmeter
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD > 0.7 (acceptance criteria), OD = 1.116 (result)
- Barrier function: 4 h < ET50 < 9 h (acceptance criteria), 4.5 h (result)
- Morphology: Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum. At least 4 viable cell layers present. Absence of significant histological abnormalities. (acceptance criteria), confirmed, 5/6 cell layers (result)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Method of calculation used: A direct interaction with the MTT is detected if the test material solution clearly turns blue or purple.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 42 minutes exposure is less than or equal to 50%
- The test substance is considered to be not irritating to skin if the viability after 42 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS solution
Duration of treatment / exposure:
42 min (+/- 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (+/- 1 hour)
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Run 1/tissue 1
Value:
1.479
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Run 1/tissue 2
Value:
1.426
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Optical density
Run / experiment:
Run 1/tissue 3
Value:
1.081
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1/mean value
Value:
87.71
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: none

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:
Under the experimental conditions reported, the test material is not irritanting to skin.
Executive summary:

The in vitro study was performed to assess the irritation potential of the test material by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.
Triplicates of the human skin model RHE (Reconstructed Human Epidermis model) were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5% sodium dodecylsulphat solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue.
Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.
After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) > 1.2 and < 2.5 for the treatment interval thus showing the quality of the tissues.
The tissue viability after treatment with the test item was higher than 50 % (mean viability: 87.7 %). Therefore, the test item is not considered to possess an irritant potential.