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EC number: 279-075-9 | CAS number: 79135-28-7
- 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:
- key study
- Study period:
- From March 30th to May 25th, 2020
- 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:
- 2020
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:
- 2018
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Acid Brown 303
- IUPAC Name:
- Acid Brown 303
- Test material form:
- solid: particulate/powder
Constituent 1
In vitro test system
- Details on the study design:
- FORMULATION
First, the test item was tried to be formulated in sodium chloride solution (saline). At the concentration of 100 mg/mL a total dissolution of the test item was observed with approximately 3 minutes of vortexing and 3 minutes of sonication and a rust color stable suspension was formed.
Then DMSO was tried at 500 mg/mL, but at this concentration only partial dissolution of the test item was observed after approximately 3 minutes of vortexing and sonication and undissolved particles were sunk to the bottom of the test tube. With additional amount of DMSO, the test item was tried to be dissolved at 250 mg/mL, however still no total dissolution could be observed. By going down another 2-fold, at 125 mg/mL the same result as before was obtained. No further dilution of the test item was attempted.
Since the formulation with saline was suitable for the study and the obtained concentration was higher than any concentration that could be gained with DMSO, moreover saline is the first preferred solvent for the test item, it was chosen as the appropriate solvent of the test item.
POSITIVE CONTROL
1-Chloro-2,4-dinitrobenzene
CELL / TEST SYSTEM
- Cells: THP-1 cell line, Human Acute Monocytic Leukemia
- Supplier: ATCC. The original cells (TIB-202) were subcultured into prepared cell lines (master cultures-MCs) in the testing laboratory.
- Maintance: RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.05 mM L-glutamine solution and 0.05 mM 2-mercaptoethanol.
- Preparation of the cells for the test: For testing, THP-1 cells were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × 106 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 μL cell suspension / well (1 × 106 cells/well).
PRELIMINARY TESTS
Preparation master and working solutions
Preparation master and working solution were prepared with saline as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution, by two-fold serial dilutions using saline. In order to be able to determine CV75 value more precisely the concentration range was lowered in the second preliminary run. These master solutions were then further diluted 50-fold into culture medium to obtain the working solutions (WS).
The working solutions were finally used for exposure by adding an equal volume of working solution (500 μL) to the volume of THP-1 cell suspension (500 μL) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item.
Solvent/vehicle control
The solvent/vehicle control used for the test item and the positive control were culture medium and 0.2 % DMSO.
Test item exposure
The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for 24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item.
PI Staining
After 24 ± 0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample.
Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
MAIN TEST
Concentrations: 71.3, 59.4, 49.5, 41.3, 34.4, 28.7, 23.9 and 19.9 μg/ml
Test item dilutions
Saline was used to dissolve the test item for stock solution (SS) in the main tests, as well. The test item was first diluted to the concentration corresponding to the CV75 × 1.2 value (71.3 μg/mL) determined in dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using saline (eight concentrations). The master solutions were then further diluted 50-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.
Solvent/vehicle controls
Culture medium was used as negative control for the test item and to assess the impact of DMSO. DMSO was tested as a solvent control for the positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions.
Positive control
DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.
Application of test item and control substances
Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours.
Fluorescein Isothiocyanate (FITC) staining
After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at approximately 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes.
After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4° C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.
Flow cytometry measurement and relative fluorescence intensity (RFI) determination
The expression of CD86 and CD54 is analysed with flow cytometry with the acquisition channel FL-1. Cytotoxicity was checked based on the PI uptake as in the preliminary tests and was analysed with the acquisition channel FL-3.
A total of 10,000 living cells (PI negative) are acquired (when the cell viability was low, up to 30,000 cells including dead cells could be acquired). Alternatively, data could be acquired for one minute after the initiation of the analysis. The cell viability was automatically calculated by the cytometer analysis program.
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 marked positive control (ctrl) and chemical-treated cells were calculated.
ACCEPTANCE CRITERIA
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90 %.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and cell viability should be more than 50 %.
- In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 % and CD54 > 200 %).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %.
Abnormal values
- RFI values cannot be less than zero. Regardless of the reason, such values should be omitted from the prediction.
- If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section.
Requirement for data acceptance
- For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
- For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90 %. When the test item is tested at 5000 μg/mL in saline (maintenance medium alternatively) or 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration, a negative result is acceptable even if the cell viability is above 90 %.
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- other: 2 out of 3 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
- Run / experiment:
- other: 2 out of 3 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
Any other information on results incl. tables
MFI, RFI AND VIABILITY VALUES OF CD86 AND CD54 MEASUREMENTS
|
MFI (geom mean) |
Corrected MFI |
RFI (CD86) |
RFI (CD54) |
viability |
MFI > 105 % to IgG |
|||||||
CD 86 |
CD54 |
isotype |
CD86 |
CD54 |
|
|
IgG |
CD86 |
CD54 |
||||
1st run |
|||||||||||||
Control |
5593 |
4344 |
3790 |
1803 |
554 |
100 |
100 |
94.8 |
148% |
115% |
|||
DMSO 0.2 % |
5084 |
4421 |
3497 |
1587 |
924 |
88 |
167 |
94.3 |
145% |
126% |
|||
DNCB 4.0 µg/ml |
13483 |
7522 |
4094 |
9389 |
3428 |
592 |
371 |
78.4 |
|
||||
Test item 71.0μg/ml |
3564 |
3974 |
3257 |
307 |
717 |
17 |
129 |
77.6 |
|||||
Test item 59.2 μg/ml |
3987 |
4112 |
3331 |
656 |
781 |
36 |
141 |
78.9 |
|||||
Test item 49.3 μg/ml |
3852 |
4273 |
3478 |
374 |
795 |
21 |
144 |
78.8 |
|||||
Test item 41.1 μg/ml |
4124 |
4116 |
3057 |
1067 |
1059 |
59 |
191 |
77.7 |
|||||
Test item 34.2 μg/ml |
4617 |
4329 |
3640 |
977 |
689 |
54 |
124 |
83.2 |
|||||
Test item 28.5 μg/ml |
4365 |
4362 |
3535 |
830 |
827 |
46 |
149 |
86.8 |
|||||
Test item 23.8 μg/ml |
4496 |
4222 |
3623 |
873 |
599 |
48 |
108 |
87.6 |
|||||
Test item 19.8 μg/ml |
4656 |
4210 |
3795 |
861 |
415 |
48 |
75 |
87.9 |
|||||
2nd run |
|||||||||||||
Control |
4456 |
3854 |
3101 |
1355 |
753 |
100 |
100 |
93.7 |
144% |
124% |
|||
DMSO 0.2 % |
5143 |
3583 |
2852 |
2291 |
731 |
169 |
97 |
94.2 |
180% |
126% |
|||
DNCB 4.0 µg/ml |
11890 |
6956 |
3044 |
8846 |
3912 |
386 |
535 |
75.2 |
|
||||
Test item 72.0 μg/ml |
3760 |
3756 |
3096 |
664 |
660 |
49 |
88 |
76.8 |
|||||
Test item 60.0 μg/ml |
3503 |
3699 |
3002 |
501 |
697 |
37 |
93 |
74.2 |
|||||
Test item 50.0 μg/ml |
4124 |
3738 |
2968 |
1156 |
770 |
85 |
102 |
79.9 |
|||||
Test item 41.7 μg/ml |
4038 |
3803 |
2861 |
1177 |
942 |
87 |
125 |
81.9 |
|||||
Test item 34.7 μg/ml |
3805 |
3746 |
3023 |
782 |
723 |
58 |
96 |
82.6 |
|||||
Test item 28.9 μg/ml |
3694 |
3752 |
2959 |
735 |
793 |
54 |
105 |
85.8 |
|||||
Test item 24.1 μg/ml |
4062 |
3707 |
3041 |
1021 |
666 |
75 |
88 |
87.3 |
|||||
Test item 20.1 μg/ml |
4308 |
3768 |
2951 |
1357 |
817 |
100 |
109 |
88.3 |
|||||
3rd run |
|||||||||||||
Control |
5705 |
4128 |
3385 |
2320 |
743 |
100 |
100 |
93.3 |
169% |
122% |
|||
DMSO 0.2 % |
5356 |
4117 |
3361 |
1995 |
756 |
86 |
102 |
92.3 |
159% |
122% |
|||
DNCB 4.0 µg/ml |
9058 |
5748 |
3305 |
5753 |
2443 |
288 |
323 |
80.6 |
|
||||
Test item 71.0 μg/ml |
4433 |
4237 |
3330 |
1103 |
907 |
48 |
122 |
80.5 |
|||||
Test item 59.2 μg/ml |
4768 |
4191 |
3268 |
1500 |
923 |
65 |
124 |
81.3 |
|||||
Test item 49.3 μg/ml |
4477 |
4302 |
3283 |
1194 |
1019 |
51 |
137 |
82.5 |
|||||
Test item 41.1 μg/ml |
4736 |
4310 |
3294 |
1442 |
1016 |
62 |
137 |
79.5 |
|||||
Test item 34.2 μg/ml |
4442 |
4296 |
3312 |
1130 |
984 |
49 |
132 |
85.0 |
|||||
Test item 28.5 μg/ml |
4505 |
4043 |
3210 |
1295 |
833 |
56 |
112 |
87.3 |
|||||
Test item 23.8 μg/ml |
4809 |
4107 |
3144 |
1665 |
963 |
72 |
130 |
87.0 |
|||||
Test item 19.8 μg/ml |
5487 |
4110 |
3213 |
2274 |
897 |
98 |
121 |
88.4 |
DOSE FINDING TEST RESULTS
Test dose (µg/ml) | 7.8 | 15.6 | 31.3 | 62.5 | 125.0 | 250.0 | 500.0 | 1000.0 |
Viability (%) | 92.9 | 90.8 | 83.0 | 78.9 | 59.5 | 40.7 | 37.1 | 29.1 |
Test dose (µg/ml) | 2.0 | 3.9 | 7.9 | 15.8 | 31.5 | 63.0 | 126.0 | 252.0 |
Viability (%) |
96.2 | 95.6 | 94.3 | 90.6 | 85.8 | 67.1 | 54.2 | 41.4 |
Applicant's summary and conclusion
- Interpretation of results:
- other: not sensitising
- Conclusions:
- The test item demonstrated an in vitro non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.
- Executive summary:
The skin sensitization potential of the substance was studied in in vitro skin sensitisation human cell line activation test, according to OECD 442E.
The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. Based on their result, eight doses between 72.0 μg/mL – 19.8 μg/mL were used for the main test in three independent runs out of which two runs were concluded valid.
The increase in CD86 marker expression (RFI) was lower than 150 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the two valid negative results out of two valid runs, CD86 marker expression was concluded to be negative. Therefore, effective concentration for CD86 expression (EC150) was not determined.
The increase in CD54 marker expression (RFI) was lower than 200 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the two valid negative results out of two valid runs, CD54 marker expression was concluded to be negative. Therefore, effective concentration for CD54 expression (EC200) was not determined.
Since the CD86 and CD54 markers gave negative results in both valid runs, the overall h-CLAT prediction was concluded to be negative, as well.
Conclusion
Based on these results and the h-CLAT prediction model, the test item demonstrated an in vitro non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.
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