(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

Test material information

Constituent 1

Reference substance name:

(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

EC Number:

857-673-6

Cas Number:

1879067-61-4

Molecular formula:

C48H52F4N4O4.2Br

IUPAC Name:

(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Test material form:

solid: particulate/powder

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

On the basis of solubility results, the test item was solubilized in DMSO at 200 mM. One stock formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 for the first run or using a dilution factor of 1.41 for the second and third runs, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
Test system KeratinoSens cells, Supplier Givaudan, Batch D1, absence of mycoplasm confirmed: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Cell seeding for testing - Cells were grown using general culture procedures up to 80-90% confluence, the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 10E+04 cells/mL, cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 10E+04 cells) per well taking care to avoid sedimentation of the cells during seeding, after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates
prior to test item addition.
Treatment - After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium, from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation, all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells, the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate: After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
Luminescence flash signal to evaluate induction signal - white plates
• After incubation, the supernatants from the white assay plates were discarded,
• the cells were washed once with D-PBS,
• a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
• the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
• a volume of 50 μL of the luciferase substrate was added to each well,
• 1 second after this addition, the luciferase signal was integrated for 2 seconds.
Absorbance signal to evaluate the cytotoxicity - transparent plate
• For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
• a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
• the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
• at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
• the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
• after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.
Result analysis - For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted. For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells. For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value. For wells in which a statistically significant gene-induction (using a student test, also called
T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
• Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
• EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
• IC50 and IC30: concentrations effecting a reduction of cellular viability by 50% and 30%,
• indication whether significant 1.5-fold gene induction occurred below the IC30.
The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose-response curves or large variation may lead to wrong extrapolations. Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

A clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

1.62

Cell viability:

89% at 2000 μM, ranging from 107 to 143% at all other test concentrations ranging from 45.67 to 1416 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: This parameter has no unit, therefore no unit was provided

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.38

Cell viability:

Ranging from 99 to 141% at all test concentrations in the range from 45.67 to 2000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: This parameter has no unit, therefore no unit was provided

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.62

Cell viability:

72% at 2000 μM, ranging from 113 to 149% at all other test concentrations in the range from 0.98 to 1000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: This parameter has no unit, therefore no unit was provided

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

All acceptance criteria were met in the three validated runs with the exception of the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" which was not fulfilled in the second validated run (i.e. Imax of 8.15). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in this run, this was considered not to have any impact on the validity of the results and the study was therefore considered as validated.

Any other information on results incl. tables

No geometric mean IC30 or IC50 was calculated since the cell viability was >70% in all runs performed. Since two of the three runs performed were considered as positive, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Applicant's summary and conclusion

Interpretation of results:

other: Positive result is considered in a weight-of-evidence approach

Conclusions:

The substance was positively tested in the KeratinoSens assay and considered to activate the Nrf2 transcription factor.

Executive summary:

The potential of the substance to activate the Nrf2 transcription factor was studied under GlP in the KeratinoSens assay to OECD TG 442D. The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent validated runs were performed as part of this study. All acceptance criteria were considered met in the three validated runs, the study was therefore considered as validated. This study was performed at tested concentrations ranging from 0.98 to 2000 μM in the first run, and from 45.67 to 2000 μM in the second and third runs, in culture medium containing 1% DMSO. At these tested concentrations, statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control in two of the three runs performed (at the concentration of 250 μM in the first run and at the two successive concentrations of 128 and 180.5 μM in the third run). The evaluation criteria for a positive response are met in two of the three runs performed, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.