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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
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted: 25. June 2018
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
adopted 14. Feb. 2017
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This in vitro study was performed to assess the potential of the test item tert-butylbenzene to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” OECD 442D(Bauch et al. 2012).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: tert-butylbenzene
- IUPAC name: tert-Butylbenzene
- Molecular formula: C10H14
- Molecular weight: 134.22 g/mol
- Substance type: Organic
- Physical state: Liquid

In vitro test system

Details on study design:
-Solvent: dimethyl sulfoxide (DMSO).
-Negative control: DL-Lactic acid.
-Positive control: EGDMA (Ethylene glycol dimethylacrylate)
-Cell line: LuSens cell line (BASF SE), are stored in liquid nitrogen in the cell bank of LAUS GmbH.
-Cell cultivate condition: 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
-Cell measurement: Cytotoxicity Range Finder Assay cells
-Chemicals and Media: Ca2+/Mg2+-Solution for PBS, EDTA Solution (250 g/L), FCS (Fetal Calf Serum) Superior, Lysepuffer Glo Lysis Buffer 1X.
-Test vessels: 96-well plates.


PERFORMANCE OF THE STUDY:
Cytotoxicity Range Finder Test:
Method: measuring the cell viability with MTT.
Exposure time: 48h
Nominal concentrations of the test item: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM.

Dose Selection for Experiment I and II:
the following 12 nominal concentrations were chosen for experiment I and II:
33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM

Results and discussion

Positive control results:
Experiment I: Induced a clear effect with an induction value of 4.6 fold in comparison to the solvent control.
Experiment II: Induced a clear effect with an induction value of 4.0 fold in comparison to the solvent control.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

In vitro / in chemico

Results
Key result
Value:
< 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: luciferase induction is < 1.5 fold
Remarks:
luciferase induction is < 1.5 fold

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, tert-Butylbenzene, was negative in the LuSens assay and is therefore considered not to have the potential to ac-tivate the Nrf2 transcription factor (no sensitizing potential).
Executive summary:

Thisin vitrostudy evaluates the potential of the test itemtert-Butylbenzeneto activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay based on the OECD 442D Guidelinewith the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (250 µM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

No substantial and reproducible dose-dependent increase in luciferase induction equal or above 1.5 fold was observed in both experiments up to the maximal tested concentration of the test item.