4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05.10-2017.-26-01-2018

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 guideline 442E (In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In accordance with Annex VII of REACH regulation

Test material

Test material information

Constituent 1

Reference substance name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

EC Number:

287-722-1

EC Name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Cas Number:

85567-10-8

Molecular formula:

C18H13N4Na3O12S3

IUPAC Name:

trisodium 5-oxo-4-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-1-(4-sulfonatophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: stable

In vitro test system

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line (ATCC TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentration dose finding assay 1: 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/mL
- Concentration dose finding assay 2: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL
- Concentrations: experiment 1 and 2: 5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41 µg/mL.
- Vehicle: 0.9% NaCl

CONTROLS
- Solvent control: 0.2% DMSO (v/v) in cell culture medium
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB) at a final conc. of 4.0 μg/mL

MEDIUM
- Culture medium: RPMI 1640: with 2 mM L-glutamine, 25mM HEPES + 10% FBS + 100 U/ml Penicillin/100 µg/mL Streptomycin + 0.05 mM 2-mercaptoethanol
- FACS Buffer: Phosphate Buffered Saline (DPBS) + 0.1% BSA
- Blocking buffer: FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction)
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 2
- Exposure period: 24 ± 0.5 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin and 2 -mercaptethanol

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 ± 0.5 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 vehicle control cells - MFI of vehicle isotype control cells) * 100

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 (cell vialbility ≥ 50%)
- Negative result: 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%.

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%.

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 (415% experiment 1; 376% experiment 2) and 200% for CD54 (312% experiment 1; 476% experiment 2) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: experiment 1

Parameter:

other: CD86 expression (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: experiment 2

Parameter:

other: CD86 expression (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: experiment 1

Parameter:

other: CD54 expression (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: experiment 2

Parameter:

other: CD54 expression (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The controls confirmed the validity of the study.
The viability of the solvent control was > 90% (96.3-98.3% experiment 1; 97.1-97.69% experiment 2).
The number of tested test item concentrations with cell viability > 50% was ≥ 4 (8 in experiment 1 and 8 in experiment 2).
The RFI for CD86 and CD54 of cells treated with the solvent DMSO was < 150% (131% experiment 1; 117% experiment 2) and ≤ 200% (113% experiment 1; 88% experiment 2).
The MFI ratio of the medium control and isotype IgG1control was ≥ 105% for CD86 (149% experiment 1; 206% experiment 2) and CD54 (129% experiment 1; 171% experiment 2).
The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (173% experiment 1; 2256% experiment 2) and CD54 (137% experiment 1; 163% experiment 2).

Any other information on results incl. tables

CD54 and CD86 Expression Experiment 1

Sample	Conc (mg/ml)	Cell Viability (%) CD86	Cell Viability (%) CD54	RFI (%) CD 86	RFI (%) CD54
Medium control		98.3	97.7	100	100
Solvent Control	0.20%	96.4	96.3	131	113
DNCB	4.0	82.0	80.4	404	476
Test item	5000	92.3	93.1	-7	142
	4166.67	93.4	92.7	2	163
	3472.22	93.0	93.5	13	168
	2893.52	93.5	92.1	12	175
	2411.27	93.3	93.9	18	119
	2009.39	94.5	93.3	21	99
	1674.49	94.4	95.0	22	88
	1395.41	93.8	93.5	32	92

CD54 and CD86 Expression Experiment 2

Sample	Conc (mg/ml)	Cell Viability (%) CD86	Cell Viability (%) CD54	RFI (%) CD 86	RFI (%) CD54
Medium control		97.2	97.4	100	100
Solvent Control	0.20%	97.6	97.4	117	88
DNCB	4.0	80.2	81.1	317	410
test item	5000	92.1	90.7	-4	55
	4166.67	93.2	92.5	3	96
	3472.22	93.1	92.6	7	105
	2893.52	93.1	93.3	18	95
	2411.27	94.6	95.0	11	69
	2009.39	95.0	94.7	-2	24
	1674.49	95.2	94.6	28	65
	1395.41	95.4	95.3	28	60

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:

The test item did not upregulate the cell surface marker in at least two independant runs.
Therefore, the test item might be considered as non-sensitiser.

Executive summary:

In the current study the skin sensitisation potential of the test item was assessed according to the new OECD 442E TG and in compliance to GLP.

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.

The test item was dissolved in 0.9% NaCl. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No CV75 was derived in the dose finding assay.

The main experiment was performed covering the following concentration steps:

5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 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.

No cytotoxicity effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 92.3% (CD86), 93.1% (CD54) and 92.1% (isotype IgG1 control) in the first experiment. and to 92.1% (CD86), 90.7% (CD54) and 89.8% (isotype IgG1 control).

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 any of the experiments. Therefore, the test item might be considered as non-sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (404% experiment 1; 317% experiment 2) and 200% for CD54 (476% experiment 1; 410% experiment 2) were clearly exceeded.

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.

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.