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EC number: 284-748-5 | CAS number: 84962-50-5
- 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:
- From October 04 to November 13, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
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 Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)
- Version / remarks:
- 9 October 2017
- GLP compliance:
- yes
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Acid Yelow 236
- IUPAC Name:
- Acid Yelow 236
Constituent 1
In vitro test system
- Details on the study design:
- CELLS
- Source: THP-1 (monocytic leukeamia cell line) cells provided from American Type Culture Collection.
- Storage: cells were stored in liquid nitrogen and the assays were performed thanks to a master bank supplied by Biopredic International (Saint Gregoire - France).
- Culturing: cryopreserved cells have been thawed. Cells were cultured, at 37 °C under 5 % CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10 % Foetal Calf Serum, 100 units/ml penicillin and 100 pg/ml streptomycin.
- Seeding: THP-1 were routinely seeded every 3-4 days at the density of 0.15 to 0.2 x10^6 cells/ml.
- Preparation of test incolumun: for testing, THP-1 cells were seeded at a density of 0.2 x 10^6 and pre-cultured in culture flasks for 72 hours.
- Viability: the quality of each batch of THP-1 cells should be checked. Viability of the cells must be above 90 %.
MAIN EXPERIMENT
Test item
- Concentration range: 7.81 to 100 μg/ml. The maximum concentration tested was 100 μg/ml, depending on its poor solubility.
- Solvent: the test item was tested in a medium containing 0.2 % DMSO. (Dimethysulfoxide).
Procedures
Based on the cytotoxlcity assay the eight final test item concentrations were selected.
ln case of non-toxic concentration for the top dose used in preliminary test, the maximum concentration selected for activation test did not exceed 1000 µg/ml when the test item was dissolved or stably dispersed in Ethanol or DMSO, and 5000 µg/ml when the test item was dissolved in a saline vehicle.
Each experiment of activation test was performed on eight concentrations.
THP-1 cells were plated at 1 x 10^6 cells/ml/well in 24 well plates and treated for 24:05 hours with selected test item concentrations. After treatment cells were washed twice with FACS buffer. Then cells were stained for 30 min at 4 °C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse lgG1. Using the manufacturer's recommended dilutions, cells were incubated with above mAbs at 6 µl/3 x 10^5 cells /50 µl for the anti-human CD86 mAb, and 3 µl/3 x 10^§5 cells /50 µl for the anti-human CD54 mAb. FITC labelled-mouse lgG1 was used as an isotype control at a dilution of 3 µl/3 x 10^5 cells /50 µl. Then, the cells were stained also with 7-AAD for 30 min at 4 °C. After washing and resuspension with FACS buffer, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA and InCyte software, on 10000 living cells.
CONTROLS
Vehicle control: as the maximal dose for test item was obtained in the DMSO, the vehicle control for all of the assays in the study was the DMSO.
Negative control: Lactic Acid (LA) produced a negative response for both markers.
Reference controls: 2,4-dinitrochlorobenzene (DNCB), nickel sulfate (NiSO4) produced a positive response for both CD86 and CD54.
PRELIMINARY CYTOTOXICITY
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50 %, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.
Eight concentrations of test item have been prepared by a two-fold serial dilution from a maximum final concentration of 1000 µg/ml or lower, depending on its solubility limit.
In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at 2 x 10^6 cells/ml. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µl (1 x 10^6 cells/well) with various concentrations of test item (1:1 ratio) for 24±0.5 hours at 37 “C under 5 % CO2. After treatment cells were washed twice with phosphate-buffered containing 0.1 % (w/v) bovine serum albumin identified as FACS buffer. The cells were stained with 7-AAD (5 µg/ml final concentration). Then cells were analysed with flow cytometry using GUAVA and InCyte software to measure cell viability. The living cells (7-AAD-) gate was set in the 7-AAD negative area. 10^4 7-AAD- cells were counted as the living population.
According to the results the dose levels for the main study were selected.
VALIDITY OF THE STUDY
Acceptability criteria for evaluating results induced by the positive control, the vehicles controls and the test item, were based on previous studies cited above and the guideline for h-CLAT (OECD 442E, 2016).
The study is considered valid if the following criteria are fully met:
- in the positive control (DNCB) RFl values of both CD86 and CD54 should be over the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %).
- in the positive control the cell viability should be more than 50 %
- in the vehicle control cell viability should be more than 90 %
- in the vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) compared to the medium control
- in the vehicle control the MFI ratio of both CD86 and CD54 to isotype control should be > 105 %
- in the test item cell viability should be more than 50 % in at least four tested concentrations in each run.
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- other: both two runs
- Parameter:
- other: % RFI of CD86
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- cell viability > 50 %
- Run / experiment:
- other: both two runs
- Parameter:
- other: % RFI of CD54
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- cell viability > 50 %
- Other effects / acceptance of results:
- No "increase" of the CD54/86 expression compared with the negative control was noticed, for any dose of test item.
In both experiments, an absence of any increase of the CD54/86 expression was observed with the test item. None of the tested doses induced a 1.5 or 2 fold increase of CD86 and CD54 expression respectively compared to the negative control.
No contamination was noticed during the study.
PRELIMINARY CYTOTOXICITY
Based on the solubility test, the doses range for cytotoxicity test was the following (with two fold dilution factor): from 0.78 to 100 µg/ml.
VALIDITY OF THE STUDY
Test system was validated as all criteria were fully accepted.
Negative controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria.
Positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.
These results validated the experimental conditions.
Any other information on results incl. tables
Concentration range* | Viability | CD54 | CD86 | MIT (μg/ml) | |
Run 1 | 7.81 to 100 μg/ml | > 50 % | RFI < 200 | RFI < 150 | - |
Run 2 | > 50 % | RFI < 200 | RFI < 150 |
RFI of CD54/86 expression
Sample | Experiment 1 | Experiment 2 | ||||
CD54 | CD86 | CV (%) | CD54 | CD86 | CV (%) | |
RPMI | 100 | 100 | 98 | 100 | 100 | 98 |
DMSO | 108 | 118 | 98 | 106 | 105 | 98 |
DNCB, 4 µg/ml | 1301 | 517 | 77 | 914 | 410 | 81 |
Test iten (vehicle 0.2 % DMSO), 0.78 µg/ml | 98 | 138 | 97 | 98 | 134 | 97 |
Test iten (vehicle 0.2 % DMSO), 1.56 µg/ml | 88 | 132 | 97 | 91 | 126 | 97 |
Test iten (vehicle 0.2 % DMSO), 3.13 µg/ml | 90 | 144 | 97 | 85 | 137 | 97 |
Test iten (vehicle 0.2 % DMSO), 6.25 µg/ml | 88 | 122 | 97 | 90 | 145 | 97 |
Test iten (vehicle 0.2 % DMSO), 12.5 µg/ml | 89 | 135 | 97 | 40 | 105 | 98 |
Test iten (vehicle 0.2 % DMSO), 25 µg/ml | 74 | 118 | 98 | 50 | 109 | 98 |
Test iten (vehicle 0.2 % DMSO), 50 µg/ml | 79 | 123 | 97 | 51 | 90 | 98 |
Test iten (vehicle 0.2 % DMSO), 100 µg/ml | 58 | 89 | 97 | 53 | 71 | 98 |
PRELIMINARY CYTOTOXICITY
Dose level µg/ml | % cell viability |
100 | 97 |
50.0 | 97 |
25.0 | 97 |
12.5 | 98 |
6.25 | 98 |
3.13 | 98 |
1.56 | 98 |
0.78 | 98 |
QUALITY CONTROL OF THE TEST SYSTEM
CV and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls
CV (%) | Ratio CD54/IgG | Ratio CD86/IgG | ||||
Sample | Criteria | Result | Criteria | Result | Criteria | Result |
RPMI | > 90 % | 98 | > 105 | 124 | > 105 | 183 |
0.9 % NaCl | > 90 % | 98 | > 105 | 124 | > 105 | 194 |
DMSO | > 90 % | 98 | > 105 | 128 | > 105 | 206 |
NiSO4 | > 50 % | 81 | ||||
DNCB | > 50 % | 77 | ||||
LA | > 50 % | 98 |
RFI values obtained for vehicles and controls
Sample | RFI | ||
Criteria | Result CD54 | Result CD86 | |
0.9 % NaCl | CD54 ≤ 200 and CD86≤150 % | 100 | 110 |
DMSO | CD54 ≤ 200 and CD86≤150 % | 108 | 118 |
NiSO4 | CD54 ≥ 200 % and CD86 2≥ 150 % | 4984 | 365 |
DNCB | CD54 ≥ 200 % and CD86 2≥ 150 % | 1301 | 517 |
LA | CD54 ≤ 200 and CD86≤150 % | 193 | 116 |
Applicant's summary and conclusion
- Interpretation of results:
- other: the test, quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, resulted to be negative
- Conclusions:
- The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell lineresulted to be negative.
- Executive summary:
The substance was tested for skin sensitisation potential, following the method and procedures described into the OECD Guideline 442E. DMSO (Dimethysulfoxide) was used as an intermediate solvent; the maximum concentration tested was 100 μg/ml, depending on its poor solubility. Experiment was conducted in duplicate.
The RFI of CD86 resulted to be lower than 150 % at any tested concentration (with cell viability ≥ 50 %), in both runs; the RFI of CD54 resulted to be lower than 200 % at any tested concentration (with cell viability ≥ 50 %), in both runs.
Therefore, under the test conditions of the experimental human Cell Line Activation Test, the test item has been judged to be a non-sensitizer.
Conclusion
The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell lineresulted to be negative.
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