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Skin sensitisation

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skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study, validated method

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: Dentritic cell activation assay (MUSST) performed as described in Python F. et al (2007), Toxicol Appl. Pharmacol. 220(2), 113-24 and Bauch C. et al. (2011), Toxicology in Vitro 25, 1162 – 1168.
Principles of method if other than guideline:
Measurement of CD 86 expression as marker for stimulation of human dentritic cells (Myeloid U937 Skin Sensitization Test, MUSST)
GLP compliance:
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
In accordance with Annex XI of EC legislation 1907/2006 in-vivo testing is not required if the endpoint is adequately covered by in-vitro testing.

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
ethyl 2-cyano-3,3-diphenylprop-2-enoate
Test material form:
Details on test material:
- Physical state: solid/white

In vitro test system

Details on the study design:
Provider of U937 cells: German Resource Center for Biological Material (DSMZ, Germany, catalog no.: ACC 5).

Culture medium: RPMI 1640 (Gibco): with L-Glutamine, 25mM HEPES + 10% FBS + 1% Penicillin/Streptomycin

Vehicle: DMSO (final conc. 0.25%)

Culture conditions: 37°C, ca. 5% CO2, ≥ 90% humidity

Number of passages prior to testing: 5-13

For substance incubation, cells were seeded in 96-well microtiter plates (100 μL of 0.5x10exp6 cells/mL cell suspensions).

Treatment was performed by adding 100 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 0.25x10exp6 cells/mL. After application the plates were sealed with semi-permeable plate
sealers in order to prevent evaporation of the test substance.
The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours.

Two independent experiments were performed. In each experiment, duplicates of each treatment were tested. If contradictory results were obtained in the first and second experiments, further experiments were conducted.

A visual inspection for test-substance precipitates was performed for each test-substance concentration prior to application. In addition, each well was inspected under a microscope after the exposure period of 48 hours in order to detect irregularities in cell morphology or test-substance precipitates.

After visual inspection the cells were transferred into V-shaped 96-well microtiter plates and centrifuged. Supernatants were discarded. The cells were washed once with PBS with 5% FBS at 4°C. Cells were resuspended in 100 μL PBS (with 5% FBS) and labeled for 30 min at 4°C in the dark with 5 μL IgG-FITC (isotype control) or 5 μL anti-CD86-FITC antibody.
Following incubation, cells were washed twice with PBS (with 5% FBS) and once with PBS and were then resuspended in 200 μL PBS. For cell viability analysis, cells were stained with propidium iodide (1.25 μg/mL final concentration in PBS) for 5 min at 4°C in darkness. Fluorescence intensity was analyzed using flow cytometry.
Positive control:
other: Ethylene diamine (70 μg/mL)

Results and discussion

Positive control results:
Ethylene diamine gave the expected induction of CD86. For details, see table 3

In vitro / in chemico

Remarks on result:
other: CD86 value = 1
Outcome of the prediction model:
negative [in vitro/in chemico]

Any other information on results incl. tables

The test substance was tested in a concentration range of 0.88 to 28.00 µg/mL. No decrease in cell viability below 70% was observed. In both experiments no induction of the expression of CD 86 was observed. The test substance was not active in the MUSST assay. Precipitation occurred after 48 h at 28.0 µg/ml.

Applicant's summary and conclusion

Interpretation of results:
other: no activation of dentritic cells in vitro