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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This in vitro study is performed to assess the potential to activate the Nrf-2 transcription factor of the test item by using the genetically modified keratinocyte cell-line “LuSens” (LuSens, Bauch et al. 2012).
The LuSens ARE-Nrf2 luciferase test method (abbreviated as “LuSens test”) is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The assay differs in some points from the OECD guideline. Since July 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.
The LuSens test employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with test substances. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010). In order to conclude on the skin sensitization potential of the test substance, a LuSens test, comprising at least two independent and valid experiments will be performed.
The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- State of aggregation: contains polymer which is not soluble after dessication
- Particle size distribution: not appropriate (liquid)
- Mass median aerodynamic diameter (MMAD): not appropriate
- Geometric standard deviation (GSD): not appropriate
- Shape of particles: not appropriate
- Surface area of particles: not appropriate
- Crystal structure: not appropriate
- Coating: not appropriate
- Surface properties: surface tension see appropriate section of IUCLID
- Density: see appropriate section of IUCLID
- Moisture content: see above
- Residual solvent: see above
- Activation: not appropriate
- Stabilisation: not required as long as treated properly and instructions of manufacturer are followed
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance, and no significant differences in the purity profile exist
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- Reactivity of the test substance with the solvent: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

In vitro test system

Details on the study design:
Negative Control
DL-Lactic acid (CAS no. 50-21-5) will be used as negative control in a final concentration of 5000 µM (nominal, real test item concentrations may vary ± 10 %). For that a 100 x stock solution (500 mM) in DMEM will be prepared. Afterwards this stock solution will be further diluted (1:25) in medium no. 3. Another 1:4 dilution will be achieved by adding 50 µL of the 1:25 dilution to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor is then 1:100. The stock solution as well as the dilutions will be freshly prepared on the day of treatment.
Positive Control
Since EGDMA (Ethylene glycol dimethylacrylate, CAS no. 97-90-5)) is not soluble in DMEM at the required concentration, p-Phenylenediamine (CAS no. 106-50-3) will be used as positive control in a final concentration of 80 µM (nominal, real test item concen-trations may vary ± 10 %). For that a 100 x stock solution (8 mM) in DMEM will be pre-pared. Afterwards this stock solution will be further diluted (1:25) in medium no. 3. Another 1:4 dilution will be achieved by adding 50 µL of the 1:25 dilution to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor is then 1:100. The stock solution as well as the dilutions will be freshly prepared on the day of treatment.
Solvent Control
As solvent control, DMEM will be used in a final concentration of 1 % in medium no.3.

Demonstration of proficiency
Prior to routine use, the validity of the LuSens test at LAUS GmbH was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.
For all control substances historical data are available , which demonstrates the reliability and the validity of those substances.

Cytotoxicity Range Finder Test (CRFT)
In order to determine the cytotoxic potential of a test item, a cytotoxicity range finder test (CRFT) will be performed. In this CRFT no luciferase induction will be measured. For the main experiments (including luciferase expression measurement), the concentrations to be used, will be adapted to the results of the CRFT.
At the time of seeding for the test the cells should be 80-90 % confluent. Cells are washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA and trypsinized (by adding Trypsin/EDTA to the flask and waiting until cells have detached). To stop this reaction, medium no. 2 will be added. After centrifugation (5 min at 380 * g), the supernatant will be discarded and the cells will be resuspended in medium no. 2. Cells are quantified and the cell suspension is adjusted to 83 000 (± 10 %) cells per mL. 120 µL of cell suspension (≙ 10 000 cells) are seeded as follows:
The cells are seeded in a flat bottom 96 well plate and incubated for 24 ± 2 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2. No cells should be seeded into the well for blank.
After the incubation time the medium will be removed from the cells and 150 µL medium no. 3 will be added to each well. Afterwards 50 µL of each single test item concentration and the controls will be added to the cells in triplicates (only test item concentrations). In total 12 concentrations will be tested in the CRFT. 12 wells will be used for solvent control, 6 wells will be used for growth control (cells + medium no.3), 3 wells will be used for negative control, 2 wells for positive control and 1 well for blank. The plate will be sealed with breathable tapes to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plate will be incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the viability assay the MTT working solution will be prepared by mixing 9 parts of me-dium no. 3 with 1 part of MTT solution. The cell culture medium will be removed from all wells of the 96 well plate and 200 µL MTT working solution will be added to each well. The plates will be incubated for 2 h ± 15 min at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution will be removed and 100 µL of lysis buffer will be added to each well. The plate will be agitated for 4- 10 min before it will be measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values are transferred in a validated spreadsheet for the calculation of the viability

Results and discussion

Positive control results:
Detailed results will be supplied in near future

In vitro / in chemico

Results
Parameter:
other:
Remarks on result:
other: not available
Remarks:
Detailed results will be supplied in near future.
Other effects / acceptance of results:
Detailed results will be supplied in near future

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Detailed results not available at the moment
Conclusions:
skin sensitizing
Executive summary:

A study on the sensitizing potential of the test item with the “LuSens ARE-Nrf2 Luciferase Test Method” according to Draft OECD Guideline 442D “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation” showed skin sensitizing potential. Detailed results will be supplied in near future.