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EC number: 214-333-6 | CAS number: 1121-60-4
- 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:
- 2018-01-08 to 2018-03-29
- 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 guidelineopen allclose all
- 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)”
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
- Version / remarks:
- 2015
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- This test method is able to detect chemicals that have sensitisation potential by addressing the third molecular key event of the adverse outcome pathway, namely dendritic celll activation, and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as Integrated Approach to Testing and Assessment (IATA), combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the adverse outcome pathway.
Test material
- Reference substance name:
- Pyridine-2-carbaldehyde
- EC Number:
- 214-333-6
- EC Name:
- Pyridine-2-carbaldehyde
- Cas Number:
- 1121-60-4
- Molecular formula:
- C6H5NO
- IUPAC Name:
- pyridine-2-carbaldehyde
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- Two batches of test item were used for this assay. The preliminary solubility test and dose range finding assays 1, 2 and 3 were performed using batch 2071700008 of the test item. Dose range finding assay 4 and the main experiments used batch 2901700033.
In vitro test system
- Details on the study design:
- TEST SYSTEM:
Cell line: The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for dendritic cells (DC). Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10^6 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) RPMI-1640, supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37 ¿ 1°C and 5% CO2.
Stock: The test item was dissolved in 0.9% NaCl solution at a concentration of 100 mg/mL. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. No precipitation, turbidity or phase separation was observed when diluted 1:50 in cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.
CD54 and CD86 Expression:
THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL.
500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2.
Blocking solution: 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min.
Staining: 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement (final concentration of PI was 0.625 µg/mL).
Expression level and cell viability: The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of ¿ = 488 nm and an emission wavelength of ¿ = 530 nm ± 15 nm for FITC and ¿ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated
Concentrations:
Test item doses in experiments 1, 2 and 3: 237.58, 197.98, 164.98, 137.49, 114.57, 95.48, 79.56, 66.30 µg/mL.
Controls: A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.
Medium Control: A medium control was included in the test. Since the test item was solubilized in either cell culture medium or 0.9% NaCl, the medium control served as a solvent control.
Positive Control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL.
Negative control: Lactic acid at a final concentration of 1000 µg/mL.
Results and discussion
- Positive control results:
- Please refer to 'any other information on results incl. tables'.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1,2 and 3
- Parameter:
- other: RFI CD86 [%]
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: 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
- Key result
- Run / experiment:
- other: 1,2 and 3
- Parameter:
- other: RFI CD54 [%]
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: 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
Any other information on results incl. tables
Results of the Cell Batch Activation Test
Sample |
Concentration |
CD86 |
CD54 |
Activated |
Pass /Fail |
||||
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
yes/no |
|||
DNCB |
4 µg/mL |
81.1 |
347 |
>150 |
79.7 |
269 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
82.1 |
347 |
>150 |
82.5 |
391 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
96.7 |
89 |
</=150 |
96.4 |
109 |
</=200 |
no |
pass |
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.
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 |
- |
96.5 |
96.2 |
95.2 |
3671 |
1717 |
662 |
3009 |
1055 |
100 |
100 |
555 |
259 |
Solvent Control |
0.20% |
95.5 |
95.7 |
95.5 |
3573 |
1670 |
639 |
2934 |
1031 |
98 |
98 |
559 |
261 |
DNCB |
4.00 |
78.8 |
77.5 |
78.5 |
11590 |
3661 |
639 |
10951 |
3022 |
373 |
293 |
1814 |
573 |
Pyridine-2-Aldehyde |
237.58 |
64.0 |
62.7 |
62.0 |
3560 |
1209 |
839 |
2721 |
370 |
90 |
35 |
424 |
144 |
197.98 |
71.5 |
71.3 |
70.7 |
3164 |
1471 |
731 |
2433 |
740 |
81 |
70 |
433 |
201 |
|
164.99 |
73.9 |
73.7 |
72.8 |
4367 |
2924 |
718 |
3649 |
2206 |
121 |
209 |
608 |
407 |
|
137.49 |
74.3 |
73.5 |
74.3 |
5228 |
4633 |
733 |
4495 |
3900 |
149 |
370 |
713 |
632 |
|
114.57 |
78.1 |
77.8 |
77.9 |
7672 |
6418 |
719 |
6953 |
5699 |
231 |
540 |
1067 |
893 |
|
95.48 |
79.9 |
79.7 |
79.0 |
7983 |
8078 |
811 |
7172 |
7267 |
238 |
689 |
984 |
996 |
|
79.57 |
82.1 |
82.9 |
81.5 |
7170 |
6649 |
731 |
6439 |
5918 |
214 |
561 |
981 |
910 |
|
66.30 |
83.4 |
83.0 |
83.4 |
6231 |
4588 |
729 |
5502 |
3859 |
183 |
366 |
855 |
629 |
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 |
- |
95.5 |
95.6 |
95.5 |
2692 |
1373 |
636 |
2056 |
737 |
100 |
100 |
423 |
216 |
Solvent Control |
0.20% |
95.9 |
95.6 |
95.4 |
2792 |
1412 |
610 |
2182 |
802 |
106 |
109 |
458 |
231 |
DNCB |
4.0 |
82.2 |
81.9 |
82.9 |
7243 |
2334 |
639 |
6604 |
1695 |
303 |
211 |
1133 |
365 |
Pyridine-2-Aldehyde |
237.58 |
55.6 |
54.0 |
54.1 |
2991 |
1140 |
784 |
2207 |
356 |
107 |
48 |
382 |
145 |
197.98 |
66.7 |
67.4 |
66.1 |
2620 |
1188 |
701 |
1919 |
487 |
93 |
66 |
374 |
169 |
|
164.99 |
71.2 |
72.4 |
71.4 |
3189 |
1857 |
666 |
2523 |
1191 |
123 |
162 |
479 |
279 |
|
137.49 |
70.9 |
72.9 |
73.2 |
4251 |
2620 |
685 |
3566 |
1935 |
173 |
263 |
621 |
382 |
|
114.57 |
76.3 |
76.4 |
75.9 |
5954 |
3605 |
840 |
5114 |
2765 |
249 |
375 |
709 |
429 |
|
95.48 |
79.5 |
78.8 |
79.4 |
6403 |
4120 |
792 |
5611 |
3328 |
273 |
452 |
808 |
520 |
|
79.57 |
80.2 |
80.8 |
80.1 |
6159 |
3630 |
829 |
5330 |
2801 |
259 |
380 |
743 |
438 |
|
66.30 |
82.5 |
82.9 |
83.3 |
5222 |
2634 |
1042 |
4180 |
1592 |
203 |
216 |
501 |
253 |
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 |
- |
96.2 |
96.2 |
95.5 |
2816 |
1265 |
613 |
2203 |
652 |
100 |
100 |
459 |
206 |
Solvent Control |
0.20% |
95.0 |
95.6 |
95.3 |
3107 |
1268 |
609 |
2498 |
659 |
113 |
101 |
510 |
208 |
DNCB |
4.0 |
81.3 |
81.7 |
81.8 |
6285 |
2166 |
677 |
5608 |
1489 |
225 |
226 |
928 |
320 |
Pyridine-2-Aldehyde |
237.58 |
51.0 |
52.4 |
51.6 |
3212 |
1259 |
923 |
2289 |
336 |
104 |
52 |
348 |
136 |
197.98 |
60.6 |
59.4 |
59.7 |
2840 |
1218 |
769 |
2071 |
449 |
94 |
69 |
369 |
158 |
|
164.99 |
65.5 |
66.2 |
65.7 |
2934 |
1784 |
838 |
2096 |
946 |
95 |
145 |
350 |
213 |
|
137.49 |
69.2 |
68.3 |
67.5 |
4302 |
3135 |
765 |
3537 |
2370 |
161 |
364 |
562 |
410 |
|
114.57 |
72.5 |
71.1 |
71.0 |
5636 |
3920 |
749 |
4887 |
3171 |
222 |
486 |
752 |
523 |
|
95.48 |
75.2 |
76.2 |
75.4 |
6429 |
4086 |
828 |
5601 |
3258 |
254 |
500 |
776 |
493 |
|
79.57 |
76.7 |
78.3 |
75.4 |
6553 |
3684 |
832 |
5721 |
2852 |
260 |
437 |
788 |
443 |
|
66.30 |
75.4 |
77.6 |
76.1 |
5978 |
2979 |
966 |
5012 |
2013 |
228 |
309 |
619 |
308 |
Acceptance Criteria
Acceptance Criterion |
range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
Experiment 3 |
pass/fail |
||||||
cell viability medium and solvent control [%] |
>90 |
95.2 |
- |
96.5 |
pass |
95.4 |
- |
95.9 |
pass |
95.0 |
- |
96.2 |
pass |
number of test dosed with viability >50% CD86 |
=4 |
8 |
pass |
8 |
pass |
7 |
pass |
||||||
number of test dosed with viability >50% CD54 |
=4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
number of test dosed with viability >50% IgG1 |
=4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
RFI of positive control of CD86 |
=150 |
373 |
pass |
303 |
pass |
225 |
pass |
||||||
RFI of positive control of CD54 |
=200 |
293 |
pass |
211 |
pass |
226 |
pass |
||||||
RFI of solvent control of CD86 |
<150 |
98 |
pass |
106 |
pass |
113 |
pass |
||||||
RFI of solvent control of CD54 |
<200 |
98 |
pass |
109 |
pass |
101 |
pass |
||||||
MFI ratio IgG1/CD86 for medium control [%] |
>105 |
555 |
pass |
423 |
pass |
459 |
pass |
||||||
MFI ratio IgG1/CD86 for solvent control [%] |
>105 |
559 |
pass |
458 |
pass |
510 |
pass |
||||||
MFI ratio IgG1/CD54 for medium control [%] |
>105 |
259 |
pass |
216 |
pass |
206 |
pass |
||||||
MFI ratio IgG1/CD54 for solvent control [%] |
>105 |
261 |
pass |
231 |
pass |
208 |
pass |
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In this study under the given conditions the test item did upregulate the cell surface markers in at least three independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
In the context of the IATA, combining the results of the three available in vitro tests (OECD 442 D, C and E) the test item is classified as a Category 1 skin sensitiser (H317) according to CLP criteria (Regulation EC No 1272/2008). - 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 sensitiser and non-sensitisers.
Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.
In the present study Pyridine-2-Aldehyde 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 (experiment 1) and from 500 mg/mL to 3.91 mg/mL (experiment 2, 3 and 4) 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 197.98 ± 22.91 µg/mL was derived in the dose finding assay 1 -3 (batch 2071700008). Since the dose finding assay 1-3 were performed using an expired batch 2071700008 of the test item, a fourth dose finding was performed with the batch 2901700033, to verify the result. The CV75 derived from one independent run with batch 2901700033 was found to be 201.46 µg/mL which was in line with the CV75 derived from dose finding assays 1-3. Therefore, the concentration range for the main experiment that was calculated based on the CV75 of dose finding assay 2 and 3 were accepted for further testing.
Based on that CV75, the main experiment was performed covering the following concentration steps: 237.58, 197.98, 164.98, 137.49, 114.57, 95.48, 79.56, 66.30 µg/mL
In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.
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.
Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentrationwas reduced to 64.0%(CD86), 62.7% (CD54) and 62.0% (isotype IgG1 control) inthe first experiment, to 55.6% (CD86), 54.0% (CD54) and 54.1% (isotype IgG1 control) in thesecond experiment and to 51.0% (CD86), 52.4% (CD54) and 51.6% (isotype IgG1 control) in the third experiment.
In the first experiment, an increase in the expression of the cell surface marker CD86 was observed from 66.30 up to 114.57 µg/mL, with the highest increase of up to 238% observed at 95.48 µg/mL. In the second experiment, an increase in the expression of CD86 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 273% observed at 95.48 µg/mL. In the third experiment, an increase in the expression of CD86 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 260% observed at 79.57 µg/mL.
In the first experiment, an increase in the expression of CD54 was observed from 66.30 up to 164.99 µg/mL, with the highest increase of up to 689% observed at 95.48 µg/mL. In the second experiment, an increase in the expression of CD54 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 452% observed at 95.48 µg/mL. In the third experiment, an increase in the expression of CD54 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 500% observed at 95.48 µg/mL.
Since the expression of both cell surface markers clearly exceeded the threshold in three independent experiments the test item is considered to be a skin sensitiser.
The controls confirmed the validity of the study for all experiments.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.