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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:
weight of evidence
Study period:
From 2018-04-24 to 2018-05-07
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes

Test material

Constituent 1
Chemical structure
Reference substance name:
Quinazolin-4(1H)-one
EC Number:
207-735-8
EC Name:
Quinazolin-4(1H)-one
Cas Number:
491-36-1
Molecular formula:
C8H6N2O
IUPAC Name:
4-Hydroxyquinazoline
Test material form:
solid
Specific details on test material used for the study:
Batch No.: 20170707
Purity: 99.13%

In vitro test system

Details on the study design:
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

-Luciferase activity:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

-Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 over the weekend (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: first experiment
Parameter:
other: Imax
Value:
1.89
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item concentration is 2000 µM. The corresponding cell viability was 43.0%.
Key result
Run / experiment:
other: first experiment
Parameter:
other: EC1.5
Value:
613.76
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: second experiment
Parameter:
other: Imax
Value:
1.56
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item concentration is 1000 µM. The corresponding cell viability was 38.0%.
Key result
Run / experiment:
other: second experiment
Parameter:
other: EC1.5
Value:
853.66
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the first experiment, a max luciferase activity (Imax) induction of 1.89 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 43.0%. The lowest tested concentration with a significant luciferase induction >1.5 (1.67) was found to be 1000 µM. The corresponding cell viability was <70% (48.8%). The calculated EC1.5was<1000 µM (613.76).
In the second experiment, a max luciferase activity (Imax) induction of 1.56 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 38.0%. This was the only tested concentration with a significant luciferase induction. The calculated EC1.5was<1000 µM (853.66).
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
Executive summary:

The test was performed with OECD 442D, the in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 146.15 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM.

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.