Pyridine-2-carbaldehyde

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material information

Constituent 1

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

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

Results

Results 1

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

Results 2

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 [µg/mL]	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. [µg/mL]	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. [µg/mL]	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. [µg/mL]	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.