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EC number: 287-719-5 | CAS number: 85567-07-3
- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-09-05 to 2017-09-26
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)"
- Version / remarks:
- July 29, 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- 2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt
- EC Number:
- 287-719-5
- EC Name:
- 2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt
- Cas Number:
- 85567-07-3
- Molecular formula:
- C36H42Cl2KN6NaO28S8
- IUPAC Name:
- 2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt
- Test material form:
- solid
- Details on test material:
- Batch: 1283067001
Constituent 1
- Specific details on test material used for the study:
- purity: 97.9%
In vitro test system
- Details on the study design:
- - Test system: human cell line
- Source species: human
- Cell type: human monocytic leukemic cell line
- Cell source: other: THP-1 cells (ATCC® TIB-202TM)
- Justification for test system used:
The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.
- Vehicle: 0.9% NaCl
- Details on test system: CD54 and CD86 Expression:
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.
- Evaluation of the Results:
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs.
In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 μg/mL for 0.9% NaCl solution; 1000 μg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.
- Acceptance criteria:
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.
- Deviations:
deviations did not influence the quality or integrity of the present study
- Control samples:
-- solvent control: 0.9% NaCl
-- solvent control for the positive control: DMSO
-- positive control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL
- Amount/concentration applied:
-- dose finding assay 1: 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81 μg/mL
-- dose finding assay 2: 5000, 2500, 1250, 625.0, 312.5, 156.25, 78.13, 39.06 μg/mL
-- main experiment 1, 2 and 3: 1705.1, 1420.9, 1184.1, 986.7, 822.3, 685.2, 571.0, 475.9 μg/mL
- Duration of treatment / exposure:
24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
- Number of replicates:
2 replicates per independant run
3 independent experiments
Results and discussion
- Positive control results:
- The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (335% experiment 1; 296% experiment 2, 278% experiment 3) and 200% for CD54 (447% experiment 1; 290% experiment 2, 368% experiment 3) were clearly exceeded.
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- other: Exp. 1 / CD86
- Parameter:
- other: Relative Fluorescence Intensity % (RFI %)
- Value:
- 82
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: Exp. 1 / CD54
- Parameter:
- other: Relative Fluorescence Intensity % (RFI %)
- Value:
- 171
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: Exp. 2 / CD86
- Parameter:
- other: Relative Fluorescence Intensity % (RFI %)
- Value:
- 72
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: Exp. 2 / CD54
- Parameter:
- other: Relative Fluorescence Intensity % (RFI %)
- Value:
- 291
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: Exp. 3 / CD86
- Parameter:
- other: Relative Fluorescence Intensity % (RFI %)
- Value:
- 72
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: Exp. 3 / CD54
- Parameter:
- other: Relative cell viability (%)
- Value:
- 335
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- Acceptance criteria were met
Any other information on results incl. tables
CD54 and CD86 Expression Experiment 1:
Sample | Concentration (μg/mL) |
Cell viability (%) | Relative Fluorescence Intensity % (RFI %) |
||
CD86 | CD54 | CD86 | CD54 | ||
Medium Control |
97.2 | 97.4 | 100 | 100 | |
Solvent Control |
97.6 | 97.5 | 108 | 85 | |
DNCB | 4 | 80.4 | 81.8 | 335 | 447 |
Test Item | 1705.09 | 13.7 | 11.4 | 31 | 32 |
1420.91 | 40.5 | 42.3 | 14 | 26 | |
1184.09 | 64.1 | 63.2 | 5 | 28 | |
986.74 | 83.9 | 81.5 | 4 | 52 | |
822.28 | 89.3 | 89.1 | 2 | 75 | |
685.24 | 94 | 94.7 | 13 | 131 | |
571.03 | 96 | 95.6 | 48 | 171 | |
475.86 | 95.2 | 95.7 | 82 | 137 |
CD54 and CD86 Expression Experiment 2:
Sample | Concentration (μg/mL) |
Cell viability (%) | Relative Fluorescence Intensity % (RFI %) | ||
CD86 | CD54 | CD86 | CD54 | ||
Medium Control | 93.8 | 94 | 100 | 100 | |
Solvent Control | 92.2 | 92.1 | 104 | 105 | |
DNCB | 4 | 79.2 | 76.2 | 296 | 290 |
Test Item | 1705.09 | 56.5 | 57.1 | -3 | 52 |
1420.91 | 80.2 | 79.9 | 4 | 102 | |
1184.09 | 88.9 | 86.9 | 9 | 261 | |
986.74 | 90.9 | 91 | 10 | 291 | |
822.28 | 93 | 91.8 | 38 | 228 | |
685.24 | 92.3 | 92.9 | 52 | 142 | |
571.03 | 91.5 | 92.4 | 72 | 108 | |
475.86 | 93.9 | 91.8 | 65 | 110 |
The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in the first experiment. In the second experiment the expression of the cell surface marker CD54 was upregulated to 291%. The upregulation above the threshold of 200% was observed at a concentration of 986.74 μg/mL. To verify the results a third experiment was performed.
CD54 and CD86 Expression Experiment 3:
Sample | Concentration (μg/mL) |
Cell viability (%) | Relative Fluorescence Intensity % (RFI %) | ||
CD86 | CD54 | CD86 | CD54 | ||
Medium Control | 97.5 | 95 | 100 | 100 | |
Solvent Control | 96 | 96.2 | 129 | 112 | |
DNCB | 4 | 85.5 | 84.2 | 278 | 368 |
Test Item | 1705.09 | 52.4 | 50.3 | 5 | 27 |
1420.91 | 72.5 | 69.9 | 1 | 27 | |
1184.09 | 72.4 | 74.4 | 7 | 63 | |
986.74 | 82.8 | 84.5 | 38 | 226 | |
822.28 | 91.3 | 91.3 | 28 | 245 | |
685.24 | 94.8 | 94.1 | 37 | 335 | |
571.03 | 95.1 | 94.1 | -49 | 102 | |
475.86 | 94.7 | 95 | 72 | 233 |
Applicant's summary and conclusion
- Interpretation of results:
- other: The study alone cannot be used for the classification purpose but in a WoE approach
- Conclusions:
- In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs.
- Executive summary:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers 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 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 100 mg/mL to 0.78 mg/mL (dose finding experiment 1) and from 500 mg/mL to 3.91 mg/mL (dose finding experiment 2) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 1705.09 μg/mL was derived in the dose finding assay
Based on the CV75, the main experiment was performed covering the following concentration steps: 1705.1, 1420.9, 1184.1, 986.7, 822.3, 685.2, 571.0, 475.9 μg/mL. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
Clear cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 13.7% (CD86), 11.4% (CD54) and 14.0% (isotype IgG1 control) in the first experiment, to 56.5% (CD86), 57.1% (CD54) and 54.1% (isotype IgG1 control) in the second experiment and to 52.4% (CD86), 50.3% (CD54) and 48.7% (isotype IgG1 control) in the third experiment.
The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in the first experiment. In the second experiment the expression of the cell surface marker CD54 was upregulated to 291%. The upregulation above the threshold of 200% was observed at a concentration of 986.74 μg/mL. To verify the results a third experiment was performed. In this third experiment the expression of the cell surface marker CD54 was upregulated to 335%. The upregulation above the threshold of 200% was observed at a concentration of 685.24 μg/mL.
Since one of the cell surface marker clearly exceeded the threshold in two independent experiments the test item considered to have a skin sensitising potential. The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.
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