3-tert-butyl-1-(3,5-dicarboxyphenyl)-1H-pyrazol-5-aminium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

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:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E: In vitro skin sensitization assays addressing the Key Event on activation of dendritic cells on the adverse outcome pathway for Skin Sensitization.

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

reactivity check for each ATCC batch of cells & working cell bank, not each time cells thawed.Validation of cells reactivity check by 2 +ve controls (NiSO4 & DNCB) instead of one (DNCB).1st DRF assay at >1000 µg/mL if solubility allows.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 158: human cell line Activation Test (h-CLAT).

Version / remarks:

July 2015

Deviations:

yes

Remarks:

reactivity check performed for each ATCC batch of cells & each working cell bank, not each time frozen cells were thawed. Validation of cells reactivity ensured in each run by both positive controls (NiSO4 & DNCB) instead of one (DNCB)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

3-tert-butyl-1-(3,5-dicarboxyphenyl)-1H-pyrazol-5-aminium chloride

EC Number:

941-661-3

Molecular formula:

C15H18ClN3O4

IUPAC Name:

3-tert-butyl-1-(3,5-dicarboxyphenyl)-1H-pyrazol-5-aminium chloride

impurity 1

Reference substance name:

Unidentified impurity

IUPAC Name:

Unidentified impurity

In vitro test system

Details on the study design:

The study was divided in two successive phases, Dose-Range Finding assays (DRF) and a main test with a concentration series tested in successive runs

DRF:
The DRF consisted of two separated assays, for which the treatments were performed at the following final concentrations: 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 µg/mL.
Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then 50-fold diluted into cRPMI to obtain working solutions. The solutions were ready for treatment after adding 500 µL of solutions to the volume of THP-1 cell suspension in the plate (i.e. 500 µL) to achieve a further 2-fold dilution. A sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The treated plates were then incubated for 24 hours (± 30 minutes) in a humidified incubator set at 37°C and 5% CO2.
At the end of the treatment phase, an inspection under a light microscope was performed for each well. Then, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 µL of FACS buffer. Finally, cells were resuspended in 200 µL FACS buffer and the plate positioned into the plate-reader of the flow cytometer. A volume of 10 µL of Propidium Iodide (PI) solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

Main test:
The main test consisted of 6 successive runs (i.e. 3 out of the 6 were validated), with treatments performed at the following final concentrations: 279.08, 334.90, 401.88, 482.25, 578.70, 694.44, 833.33 and 1000 µg/mL.
Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest tested concentration of 1000 µg/mL, corresponded to the highest achievable non-cytotoxic concentration based on solubility of the test item.
All stock formulations were then 50-fold diluted into cRPMI to obtain working solutions. In parallel, the working solutions of both positive controls (DNCB and NiSO4) and vehicle control were prepared. All working solutions were used for treatment after adding 500 µL of working solutions to the volume of THP-1 cell suspension in the plate (i.e. 500 µL) to achieve a further 2-fold dilution. A sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The treated plates were then incubated for 24 hours (± 30 minutes) in a humidified incubator set at 37°C and 5% CO2.
At the end of the treatment phase, an inspection under a light microscope was performed for each well.
Cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer, blocked with 600 µL of blocking solution and incubated at 4°C for 15 minutes (± 1 minute).
After blocking, cells were split into 3 aliquots of 180 µL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each corresponding aliquot before incubation for 30 minutes (± 2 minutes) at 4°C.

Finally, cells were washed with 150 µL FACS buffer twice and re-suspended in 200 µL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 µL of PI solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Run C at 1000 µg/ml

Parameter:

other: RFI for CD86

Value:

78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Run C at 1000 µg/ml

Parameter:

other: RFI for CD54

Value:

126

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: Run E 694.4 µg/ml

Parameter:

other: RFI for CD86

Value:

195

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Run / experiment:

other: Run E at 694.4 µg/ml

Parameter:

other: RFI for CD54

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were fulfilled in the three validated runs (i.e. Runs C, E and F), the study was therefore considered as valid.

Each run was performed using the following concentrations (final concentrations in wells): 279.08, 334.90, 401.88, 482.25, 578.70, 694.44, 833.33 and 1000 µg/mL.
At these tested concentrations, the following results were obtained:
No precipitate/emulsion and no cell morphology modification were noted in treated wells, at any tested concentrations, in either of the three validated runs,
In Run C (Runs A et B invalidated): neither RFI(CD86), nor RFI(CD54) exceeded their respective positivity thresholds at any tested concentration. The run was therefore considered negative.
However, in Run E (Run D invalidated): RFI(CD86) exceeded the positivity threshold of 150 in 2 out of the 8 tested concentrations (i.e. at concentrations of 578.70 and 694.44 µg/mL). The run was therefore considered positive for the CD86 marker,
Since non-concordant results were obtained throughout the 2 previous validated runs, an additional run (i.e. Run F) was performed. In this Run F and as for Run C, neither RFI(CD86), nor RFI(CD54) exceeded their respective positivity thresholds at any tested concentration. This last run was therefore also considered negative.

Since two independent validated runs, out of the three, were negative for both markers, the overall h-CLAT prediction is considered negative

Applicant's summary and conclusion

Interpretation of results:

other: the test item was found to be negative in the h-CLAT test

Conclusions:

Since two independent validated runs, out of the three, were negative for both markers, the overall h-CLAT prediction is considered negative.