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

Endpoint summary

Description of key information

An Integrated Approaches to Testing and Assessment (IATA) was used to determine the skin sensitising potential of the substance.

The test material gave a negative response in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

The test material was found to be unsuitable for use in a DPRA test.

The test material was found to be negative in the h-CLAT test.

Results from the KeratinoSens assay and h-CLAT test can be used in combination to support the discrimination between skin sensitisers and non-sensitisers in the context of Integrated Approaches to Testing and Assessment (IATA). As both of these studies gave a negative result for skin sensitisation the test material is determined to not meet the criteria for classification as a skin sensitiser (UN GHS).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

other information

Study period:

18 July to 19 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours, protected from light. At the end of the incubation, the concentrations of residual peptides were evaluated by liquid chromatography with Ultra-Violet (HPLC/UV) detection at 220 nm.
Peptide reactivity was reported as percentage depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle).

Remarks on result:

not determinable

Other effects / acceptance of results:

The test item was dissolved at 100 mM in 1:1 (v/v) mixture of acetonitrile:milli-Q water.

All acceptance criteria were met, the study was therefore considered to be valid.

Chromatograms analysis of the co-elution samples indicated that the test item co-eluted with the cysteine peptide (i.e. 7083.5% interference). As a result, no mean percentage depletion value could be calculated for the cysteine peptide.

The calculated mean depletion value for the lysine peptide was 1.25%.

Since the test item co-eluted with the cysteine peptide (with a peptide peak that cannot be integrated) and since the determination of the peptide reactivity of a test item cannot be solely based on the percentage depletion of the lysine peptide, the analysis is therefore considered to be inconclusive.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of this study, the test item is considered as incompatible with the DPRA test method and consequently, no conclusion can be drawn regarding its peptide reactivity.

Reference 2

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

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.

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

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.

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

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

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

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

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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2019 to 19 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

June 2018

Deviations:

yes

Remarks:

Test item formulations were not filtered prior to treatment

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The test item was tested in two independent runs using cells from a different passage number. The plates were processed as described below:

Solubility test
Prior to the first treatment, a solubility test was performed in order to select the vehicle. When a solution or a stable dispersion was obtained in these vehicles, the formulations were 100-fold diluted in culture medium. Then, a visual inspection of the samples was performed immediately as well as after an overnight period of incubation at 37°C to evaluate the presence or absence of precipitate/emulsion.

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 104 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 104 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 preferentially (not exceeding 30 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:
− 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.

Positive control results:

All acceptance criteria were fulfilled in both runs.

Run / experiment:

other: 1

Parameter:

other: max. induction factor

Value:

1.13

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no potential to activate Nrf2 transcription factor

Run / experiment:

other: 2

Parameter:

other: max. induction factor

Value:

1.28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no potential to activate Nrf2 transcription factor

Other effects / acceptance of results:

All acceptance criteria were fulfilled in both runs. They were therefore considered to be valid.
Both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.
At these tested concentrations no precipitate/emulsion was observed at the end of the 48-hour treatment period, in any test item-treated wells and in either run,no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%). Therefore, neither IC30, nor IC50 was calculated in either run, no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control, at any tested concentrations and in either run, moreover, and for both runs, the Imax values were < 1.5 (i.e. 1.13 and 1.28 in the first and second runs, respectively) and thus no EC1.5 was calculated.

Interpretation of results:

other: the test item was negative in the KeratinoSens assay

Conclusions:

Under the experimental conditions of this study, the test item was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test material gave a negative response in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

The test material was found to be unsuitable for use in a DPRA test.

The test material was found to be negative in the h-CLAT test method.

Results from the KeratinoSens assay and h-CLAT test can be used in combination to support the discrimination between skin sensitisers and non-sensitisers in the context of Integrated Approaches to Testing and Assessment (IATA). As both of these studies gave a negative result for skin sensitisation the test material is determined to not meet the criteria for classification as a skin sensitiser (UN GHS).