2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-05 to 2017-09-26

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

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)"

Version / remarks:

July 29, 2016

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

EC Number:

287-719-5

EC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Cas Number:

85567-07-3

Molecular formula:

C36H42Cl2KN6NaO28S8

IUPAC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Test material form:

solid

Details on test material:

Batch: 1283067001

Specific details on test material used for the study:

purity: 97.9%

In vitro test system

Details on the study design:

- Test system: human cell line

- Source species: human

- Cell type: human monocytic leukemic cell line

- Cell source: other: THP-1 cells (ATCC® TIB-202TM)

- Justification for test system used:
The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.

- Vehicle: 0.9% NaCl

- Details on test system: CD54 and CD86 Expression:
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.

FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.

- Evaluation of the Results:
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if 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 or if 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 or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs.
In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 μg/mL for 0.9% NaCl solution; 1000 μg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

- Acceptance criteria:
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

- Deviations:
deviations did not influence the quality or integrity of the present study

- Control samples:
-- solvent control: 0.9% NaCl
-- solvent control for the positive control: DMSO
-- positive control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL

- Amount/concentration applied:
-- dose finding assay 1: 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81 μg/mL
-- dose finding assay 2: 5000, 2500, 1250, 625.0, 312.5, 156.25, 78.13, 39.06 μg/mL
-- main experiment 1, 2 and 3: 1705.1, 1420.9, 1184.1, 986.7, 822.3, 685.2, 571.0, 475.9 μg/mL

- Duration of treatment / exposure:
24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.

- Number of replicates:
2 replicates per independant run
3 independent experiments

Results and discussion

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (335% experiment 1; 296% experiment 2, 278% experiment 3) and 200% for CD54 (447% experiment 1; 290% experiment 2, 368% experiment 3) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Exp. 1 / CD86

Parameter:

other: Relative Fluorescence Intensity % (RFI %)

Value:

82

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Exp. 1 / CD54

Parameter:

other: Relative Fluorescence Intensity % (RFI %)

Value:

171

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: Exp. 2 / CD86

Parameter:

other: Relative Fluorescence Intensity % (RFI %)

Value:

72

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Run / experiment:

other: Exp. 2 / CD54

Parameter:

other: Relative Fluorescence Intensity % (RFI %)

Value:

291

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 5

Run / experiment:

other: Exp. 3 / CD86

Parameter:

other: Relative Fluorescence Intensity % (RFI %)

Value:

72

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 6

Run / experiment:

other: Exp. 3 / CD54

Parameter:

other: Relative cell viability (%)

Value:

335

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria were met

Any other information on results incl. tables

CD54 and CD86 Expression Experiment 1:

Sample	Concentration (μg/mL)	Cell viability (%)		Relative Fluorescence Intensity % (RFI %)
		CD86	CD54	CD86	CD54
Medium Control		97.2	97.4	100	100
Solvent Control		97.6	97.5	108	85
DNCB	4	80.4	81.8	335	447
Test Item	1705.09	13.7	11.4	31	32
	1420.91	40.5	42.3	14	26
	1184.09	64.1	63.2	5	28
	986.74	83.9	81.5	4	52
	822.28	89.3	89.1	2	75
	685.24	94	94.7	13	131
	571.03	96	95.6	48	171
	475.86	95.2	95.7	82	137

CD54 and CD86 Expression Experiment 2:

Sample	Concentration (μg/mL)	Cell viability (%)		Relative Fluorescence Intensity % (RFI %)
		CD86	CD54	CD86	CD54
Medium Control		93.8	94	100	100
Solvent Control		92.2	92.1	104	105
DNCB	4	79.2	76.2	296	290
Test Item	1705.09	56.5	57.1	-3	52
	1420.91	80.2	79.9	4	102
	1184.09	88.9	86.9	9	261
	986.74	90.9	91	10	291
	822.28	93	91.8	38	228
	685.24	92.3	92.9	52	142
	571.03	91.5	92.4	72	108
	475.86	93.9	91.8	65	110

The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in the first experiment. In the second experiment the expression of the cell surface marker CD54 was upregulated to 291%. The upregulation above the threshold of 200% was observed at a concentration of 986.74 μg/mL. To verify the results a third experiment was performed.

CD54 and CD86 Expression Experiment 3:

Sample	Concentration (μg/mL)	Cell viability (%)		Relative Fluorescence Intensity % (RFI %)
		CD86	CD54	CD86	CD54
Medium Control		97.5	95	100	100
Solvent Control		96	96.2	129	112
DNCB	4	85.5	84.2	278	368
Test Item	1705.09	52.4	50.3	5	27
	1420.91	72.5	69.9	1	27
	1184.09	72.4	74.4	7	63
	986.74	82.8	84.5	38	226
	822.28	91.3	91.3	28	245
	685.24	94.8	94.1	37	335
	571.03	95.1	94.1	-49	102
	475.86	94.7	95	72	233

Applicant's summary and conclusion

Interpretation of results:

other: The study alone cannot be used for the classification purpose but in a WoE approach

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs.

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 sensitisers and non-sensitisers.

In the present study, the test item 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 (dose finding experiment 1) and from 500 mg/mL to 3.91 mg/mL (dose finding experiment 2) 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 1705.09 μg/mL was derived in the dose finding assay

Based on the CV75, the main experiment was performed covering the following concentration steps: 1705.1, 1420.9, 1184.1, 986.7, 822.3, 685.2, 571.0, 475.9 μg/mL. 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.

Clear cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 13.7% (CD86), 11.4% (CD54) and 14.0% (isotype IgG1 control) in the first experiment, to 56.5% (CD86), 57.1% (CD54) and 54.1% (isotype IgG1 control) in the second experiment and to 52.4% (CD86), 50.3% (CD54) and 48.7% (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in the first experiment. In the second experiment the expression of the cell surface marker CD54 was upregulated to 291%. The upregulation above the threshold of 200% was observed at a concentration of 986.74 μg/mL. To verify the results a third experiment was performed. In this third experiment the expression of the cell surface marker CD54 was upregulated to 335%. The upregulation above the threshold of 200% was observed at a concentration of 685.24 μg/mL.

Since one of the cell surface marker clearly exceeded the threshold in two independent experiments the test item considered to have a skin sensitising potential. The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.