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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Keratinocyte activation has been identified as one of the key events of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO (2012)10). The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes (LuSens, Bauch et al. 2012 and Ramirez et al. 2014, and 2016). It employs the reporter gene for luciferase under the control of an antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the up-regulation of the luciferase activity after 48 hours incubation with test substances. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway.

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes

Test material

Constituent 1
Chemical structure
Reference substance name:
Isobutyl vinyl ether
EC Number:
EC Name:
Isobutyl vinyl ether
Cas Number:
Molecular formula:
isobutyl vinyl ether
Specific details on test material used for the study:
Name of test substance: Isobutylvinylether
Test-substance No.:03/0186-5
Purity: ≥ 99.1 area-%
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the exposure period was guaranteed by the sponsor until 11 Sep 2019, and the sponsor holds this responsibility.
Physical state / color: liquid / colorless, clear
Storage conditions: ambient, avoid temperatures > 25 °C
Molecular weight: 100.16 g/mol

In vitro test system

Details on the study design:
Preparation of the cells:
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing.
Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 100000 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Twoindependent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

Test-substance application for MTT and luciferase assay:
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). After test substance 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 +/-1 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.

Visual inspections:
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 +/-1 hours in order to detect test-substance precipitates.

Luciferase assay:
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for ca. 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay MTT:
Cell culture medium was aspirated from all wells. Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well of the 96-well microtiter plate and incubated for at least further 2 hours in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

Cell line (LuSens):
Human transgenic keratinocyte cell line derived from HaCaT cells,
prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany.

Controls (LuSens):
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
Positive control (PC): ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells.

Test substance preparation for the LuSens:
The test substance was weighed and topped up with the chosen vehicle (DMSO) to achieve the required 100x concentration of the highest concentration (stock solution). Further concentrations were prepared as 100x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate) and were further diluted (1:25) in culture medium 3 to obtain 4x concentrations (dilution plate). The test-substance preparations were prepared by stirring.

Reason for the vehicle: The test substance was soluble in DMSO.

Results and discussion

In vitro / in chemico

Remarks on result:
other: does not have a keratinocyte activating potential

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed. From this it has to be concluded that test substance Isobutylvinylether does not have a keratinocyte activating potential.
Executive summary:

The keratinocyte activating potential of test substance Isobutylvinylether was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. No cytotoxicity was observed.

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The following results were observed:

At concentrations used in the main experiment the test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

In summary, after 48 hours of exposure to test substance Isobutylvinylether luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance Isobutylvinylether does not have a keratinocyte activating potential.