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Diss Factsheets

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:
Augustus 2012 - May 2013
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
24 August 2009
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
457-080-9
EC Name:
-
Cas Number:
32940-15-1
Molecular formula:
C11H12O2
IUPAC Name:
5-methoxy-1,2,3,4-tetrahydronaphthalen-2-one
Test material form:
solid
Details on test material:
- Appearance: Ochre-yellow solid
- Storage condition of test material: In refrigerator (2-8°C) in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 12-EKIN-031 and 13-EKIN-011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temerature
- Temperature of other incubations: 37.0 ± 1.0°C (actual range 36.7 - 37.6°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per test substance

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: incubation with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium.
- N. of replicates: 3
- Method of calculation used:
OD: Optical densitiy, ku: untreated killed tissues, kt: treated killed tissues, nc: negative control
Non specific MTT reduction (NSMTT) = [(ODkt – ODku)/ODnc] * 100
True MTT metabolic conversion (TODtt) = [ODtv – (ODkt-ODku)]
% viability = [TODtt/ODnc] * 100

ACCEPTABILITY OF THE ASSAY
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

DECISION CRITERIA
A test substance is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test substance is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 μL (first experiment) or 43.3 to 68.9 mg (repeat experiment) of the undiluted test substance

NEGATIVE CONTROL
- Amount applied: 25 μL

POSITIVE CONTROL
- Amount applied: 25 μL
- Concentration: 5%
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
first experiment (molten test substance) and repeat experiment (solid test substance)
Remarks on result:
not determinable because of methodological limitations
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The non-specific reduction of MTT (NSMTT) by the test item was 67% and 57% of the negative control tissues for the molten test substance and solid est substance, respectively. Since the NSMTT by the substance was >30% the acceptability criteria of the assay are not met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the absolute means OD570 of negative control tissues (0.918 (molten) and 1.074 (solid) were within the laboratory historical control data range (0.576 - 1.352).
- Acceptance criteria met for positive control: Yes, the positive controls had a mean cell viability of 14% and 16% after 15 minutes exposure to molten and solid test substance, respectively.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically with the negative or positive control was less than 8%, indicating that the test system functioned properly. However, too many test substance remained on the dead and viable skin tissues after washing and cleaning the tissues and consequently non-specific staining by MTT caused a high NSMTT and a high SD between the three identically treated tissues (27% for molten test substance and 26% for solid test substance). Therefore the obtained viabilities are not reliable, and the test system is not suitable to detect the skin irritation potential of the substance.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the substance cannot be tested in the in vitro skin irritation test both when tested as liquid or as solid under the experimental conditions described in this report.
Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), the influence of the substance on the viability of human skin was tested according to OECD 439 guideline and in accordance with GLP principles. The test substance was applied directly to 0.38 cm2 cultured skin (25μL molten test substance in first experiment or 43.3 to 68.9 mg solid test substance in repeat experiment). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Adequate positive and negative controls were included. The test item did interact with MTT and in addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 67% (molten) and 57% (solid) of the negative control tissues with a high SD between the three identically treated tissues (26% and 27% for molten and solid test substance, respectively).Therefore the obtained viabilities are not reliable, and the test system is not suitable to detect the skin irritation potential of the substance.