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EC number: 201-730-4 | CAS number: 87-20-7
- 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:
- January - April 2018
- 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 guideline 442E h-CLAT
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Isobutyl salicylate
- EC Number:
- 201-729-9
- EC Name:
- Isobutyl salicylate
- Cas Number:
- 87-19-4
- Molecular formula:
- C11H14O3
- IUPAC Name:
- Isobutyl salicylate
Constituent 1
In vitro test system
- Details on the study design:
- TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)
TEST-SUBSTANCE PREPARATION
- Concentrations: Dose range finder: 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 µg/mL; Main experiment: 97.71, 81.42, 67.85, 56.54, 47.12, 39.27, 32.72, 27.27 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Solvent: DMSO
CONTROLS
- Negative control (NC):Medium
- Solvent control (SC): 0.2% DMSO
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL
MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25 mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III with DPBS
- Reagent for cytotoxicity test: Propidium iodide (Sigma)
EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 3
- Exposure period: 24 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2 -mercaptoethanol (30 > passage >= 5)
ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 hours after exposure
DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of aquired cells) * 100
- Relative fluorescence intensity: RFI (%) = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of solvent control cells - MFI of solvent isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.
ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity) values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86 =150% and CD54 =200%) and cell viability should be =50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be =105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.
EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased =150% and/or CD54 expression increased =200% in relation to vehicle control in at least 2 independent experiments.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).
Results and discussion
- Positive control results:
- Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3).
In vitro / in chemico
Results
- Key result
- Run / experiment:
- other: Please see 'Any other information on results incl. tables'.
- Parameter:
- other: RFI CD54 mean (%) and RFI CD54 mean (%)
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
Any other information on results incl. tables
CD54 and CD86 Expression Experiment 1
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
94.7 |
94.7 |
94.3 |
2943 |
1205 |
613 |
2330 |
592 |
101 |
111 |
480 |
197 |
Solvent Control |
0.20% |
94.8 |
95.2 |
94.8 |
2988 |
1214 |
679 |
2309 |
535 |
100 |
100 |
440 |
179 |
DNCB |
4.00 |
80.9 |
80.5 |
80.0 |
6868 |
2013 |
779 |
6089 |
1234 |
264 |
231 |
882 |
258 |
ISOBUTYL SALICYLATE |
97.71 |
12.4 |
11.9 |
12.3 |
4427 |
3822 |
942 |
3485 |
2880 |
151 |
538 |
470 |
406 |
81.43 |
57.3 |
58.4 |
57.4 |
3480 |
1781 |
726 |
2754 |
1055 |
119 |
197 |
479 |
245 |
|
67.85 |
83.9 |
83.4 |
84.1 |
3116 |
1540 |
673 |
2443 |
867 |
106 |
162 |
463 |
229 |
|
56.55 |
88.9 |
89.3 |
89.5 |
3066 |
1380 |
658 |
2408 |
722 |
104 |
135 |
466 |
210 |
|
47.12 |
93.2 |
93.5 |
93.6 |
2778 |
1235 |
669 |
2109 |
566 |
91 |
106 |
415 |
185 |
|
39.27 |
94.5 |
94.3 |
93.9 |
2801 |
1230 |
686 |
2115 |
544 |
92 |
102 |
408 |
179 |
|
32.72 |
94.4 |
94.8 |
94.9 |
2996 |
1287 |
660 |
2336 |
627 |
101 |
117 |
454 |
195 |
|
27.27 |
94.7 |
94.6 |
94.7 |
3063 |
1276 |
669 |
2394 |
607 |
104 |
113 |
458 |
191 |
CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
IgG Isotype |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
94.7 |
94.8 |
93.9 |
4619 |
1554 |
646 |
3973 |
908 |
102 |
95 |
715 |
241 |
Solvent Control |
0.20% |
95.0 |
95.1 |
94.9 |
4571 |
1620 |
669 |
3902 |
951 |
100 |
100 |
683 |
242 |
DNCB |
4.0 |
81.4 |
80.5 |
81.2 |
13735 |
3370 |
633 |
13102 |
2737 |
336 |
288 |
2170 |
532 |
ISOBUTYL SALICYLATE |
97.71 |
13.2 |
12.8 |
12.4 |
7467 |
5420 |
935 |
6532 |
4485 |
167 |
472 |
799 |
580 |
81.43 |
45.9 |
45.5 |
45.7 |
6028 |
3615 |
1213 |
4815 |
2402 |
123 |
253 |
497 |
298 |
|
67.85 |
79.3 |
79.0 |
79.7 |
5291 |
3011 |
782 |
4509 |
2229 |
116 |
234 |
677 |
385 |
|
56.55 |
90.0 |
90.8 |
89.7 |
4710 |
2761 |
874 |
3836 |
1887 |
98 |
198 |
539 |
316 |
|
47.12 |
93.5 |
93.5 |
92.7 |
4543 |
2413 |
735 |
3808 |
1678 |
98 |
176 |
618 |
328 |
|
39.27 |
93.4 |
92.5 |
92.8 |
4587 |
2304 |
746 |
3841 |
1558 |
98 |
164 |
615 |
309 |
|
32.72 |
94.6 |
94.1 |
94.2 |
4335 |
2049 |
756 |
3579 |
1293 |
92 |
136 |
573 |
271 |
|
27.27 |
95.1 |
95.0 |
94.9 |
4489 |
2013 |
790 |
3699 |
1223 |
95 |
129 |
568 |
255 |
CD54 and CD86 Expression Experiment 3
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
IgG Isotype |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
95.2 |
94.5 |
94.3 |
3732 |
1289 |
640 |
3092 |
649 |
88 |
87 |
583 |
201 |
Solvent Control |
0.20% |
94.7 |
94.5 |
94.5 |
4176 |
1395 |
649 |
3527 |
746 |
100 |
100 |
643 |
215 |
DNCB |
4.0 |
81.9 |
81.1 |
82.4 |
11303 |
2502 |
583 |
10720 |
1919 |
304 |
257 |
1939 |
429 |
ISOBUTYL SALICYLATE |
97.71 |
40.5 |
38.6 |
39.3 |
4938 |
2769 |
816 |
4122 |
1953 |
117 |
262 |
605 |
339 |
81.43 |
64.4 |
64.9 |
64.8 |
4625 |
2417 |
806 |
3819 |
1611 |
108 |
216 |
574 |
300 |
|
67.85 |
86.4 |
85.4 |
86.3 |
4465 |
2304 |
739 |
3726 |
1565 |
106 |
210 |
604 |
312 |
|
56.55 |
93.0 |
91.8 |
93.0 |
4049 |
1925 |
742 |
3307 |
1183 |
94 |
159 |
546 |
259 |
|
47.12 |
94.0 |
93.8 |
94.4 |
3792 |
1728 |
740 |
3052 |
988 |
87 |
132 |
512 |
234 |
|
39.27 |
94.5 |
94.1 |
93.9 |
3707 |
1636 |
730 |
2977 |
906 |
84 |
121 |
508 |
224 |
|
32.72 |
95.0 |
94.9 |
95.2 |
3538 |
1580 |
735 |
2803 |
845 |
79 |
113 |
481 |
215 |
|
27.27 |
95.2 |
95.6 |
95.0 |
4119 |
1701 |
811 |
3308 |
890 |
94 |
119 |
508 |
210 |
Applicant's summary and conclusion
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- Based on the results, it is concluded that the test substance induces dendritic cell activation (CD54 expression increased =200% in at least one assay).
- Executive summary:
The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.
In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at one concentration with a cell viability ≥50% in at least two independent runs.
The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.
In summary, it has to be concluded that the test substance induces dendritic cell activation.
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