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Diss Factsheets
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EC number: 941-661-3 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
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:
- 2 020
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- 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
Test material
- 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
- Reference substance name:
- Unidentified impurity
- IUPAC Name:
- Unidentified impurity
Constituent 1
impurity 1
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
Resultsopen allclose all
- 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
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.
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