Reaction mass of dipotassium 3,3'-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2018 - 09 May 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 442E (In VItro Skin Sensitisation h-CLAT)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol n°158 (Skin Sensitisation h-CLAT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

Current REACH protocol requires In vitro / In chemico assessment before in vivo.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dipotassium 3,3'-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate

EC Number:

915-932-1

Molecular formula:

Reaction mass of C12H8O8S3.2K and C12H9O5S2.K

IUPAC Name:

Reaction mass of dipotassium 3,3'-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: KSS-FR
Chemical Name: Reaction mass of dipotassium 3,3’-sulphonylbis(benzenesulphonate) and potassium 3-(phenylsulphonyl)benzenesulphonate
Batch No.: KSS-1711-02
EC#: 915-932-1
Composition: 71.5% KSS (CAS No.: 63316-43-8): 264 g/mol
21.1% Di KSS (CAS No.: 63316-33-6): 334 g/mol
7.4% other
Calculated Molecular Weight: 259.23 g/mol
Purity: 92.6%
Physical State: crystalline powder
Colour: White
Log KOW: not specified by the sponsor
Storage Conditions: at room temperature
Expiry Date: November 2019

In vitro test system

Details on the study design:

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 (DC) 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 this study, the test substance was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500.00 mg/mL to 3.91 mg/mL 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.

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%.

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 would 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%.

Results and discussion

Positive control results:

The acceptance criteria for the positive control were fulfilled.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: CD86 Relative Flourescence Intensity (RFI, %)

Value:

75

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: 2

Parameter:

other: CD86 Relative Flourescence Intensity (RFI, %)

Value:

93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: 1

Parameter:

other: CD54 Relative Flourescence Intensity (RFI, %)

Value:

80

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: 2

Parameter:

other: CD54 Relative Flourescence Intensity (RFI, %)

Value:

82

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:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 90.7% (CD86), 90.9% (CD54) and 90.9% (isotype IgG1 control) in the first experiment and to 90.7% (CD86), 90.1% (CD54) and 90.0% (isotype IgG1 control) in the second 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 the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is not considered to be a skin sensitiser. The controls confirmed the validity of the study for all experiments.

Any other information on results incl. tables

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	-	94.2	94.0	94.1	5299	1695	716	4583	979	94	96	740	237
Solvent Control	0.20%	93.6	94.4	94.6	5551	1696	680	4871	1016	100	100	816	249
DNCB	4.00	79.8	79.6	80.3	15356	3192	653	14703	2539	302	250	2352	489
KSS-FR	1000	90.7	90.9	90.9	3023	1395	860	2163	535	44	53	352	162
	833.33	92.0	92.0	91.6	3116	1431	861	2255	570	46	56	362	166
	694.44	93.1	93.5	92.8	3297	1557	816	2481	741	51	73	404	191
	578.70	93.3	93.1	92.7	3794	1481	799	2995	682	61	67	475	185
	482.25	93.7	94.1	93.5	3809	1556	802	3007	754	62	74	475	194
	401.88	93.2	93.1	92.8	4147	1564	783	3364	781	69	77	530	200
	334.90	93.3	93.6	93.1	4010	1575	770	3240	805	67	79	521	205
	279.08	93.8	93.8	93.6	4432	1610	799	3633	811	75	80	555	202

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	-	96.0	95.5	94.9	4314	1593	708	3606	885	98	107	609	225
Solvent Control	0.20%	95.4	94.8	94.0	4376	1508	684	3692	824	100	100	640	220
DNCB	4.0	84.2	85.3	84.3	10982	2461	722	10260	1739	278	211	1521	341
KSS-FR	1000.00	90.7	90.1	90.0	2957	1377	958	1999	419	54	51	309	144
	833.33	92.8	92.7	91.8	2923	1428	984	1939	444	53	54	297	145
	694.44	92.2	92.5	93.2	2962	1374	869	2093	505	57	61	341	158
	578.70	93.3	93.2	93.0	3213	1404	835	2378	569	64	69	385	168
	482.25	93.3	92.2	92.5	3488	1394	1076	2412	318	65	39	324	130
	401.88	93.9	94.0	93.4	3554	1408	925	2629	483	71	59	384	152
	334.90	94.3	94.0	93.9	3665	1408	887	2778	521	75	63	413	159
	279.08	93.6	92.8	94.0	4238	1500	823	3415	677	93	82	515	182

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was non-sensitising in the in vitro human cell line activation test (h-CLAT) assay.

Executive summary:

In this GLP guideline study conducted according to OECD 442E "In vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation" human cell line activation test (h-CLAT) to assess a key event associated with skin sensitisation, namely dendritic cell activation, the test substance did not upregulate the expression of the cell surface marker in at least two independent experimental runs and thus can be regarded as not a skin sensitiser in the h-CLAT assay.