Sodium 2,4,5-trichlorobenzenesulphonate

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:

Sodium 2,4,5-trichlorobenzenesulphonate

EC Number:

258-548-3

EC Name:

Sodium 2,4,5-trichlorobenzenesulphonate

Cas Number:

53423-65-7

Molecular formula:

C6H2Cl3O3S.Na

IUPAC Name:

sodium 2,4,5-trichlorobenzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: STB-FR
Batch No.: 1710-06
Chemical name: Benzenesulfonic acid, 2,4,5-trichloro-, sodium salt
CAS No: 53423-65-7
Composition: 90.5% STB; 9.5% other
Molecular Weight: 283.494 g/mol
Purity: 90.5%
Physical State: solid powder
Colour: white
Log KOW: not specified by the sponsor
Stability: stable
Storage Conditions: at room temperature
Expiry Date: October 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:

81

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:

95

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:

103

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:

114

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 92.4% (CD86), 91.8% (CD54) and 91.5% (isotype IgG1 control) in the first experiment and to 87.6% (CD86), 87.0% (CD54) and 85.4% (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 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	-	94.8	94.2	94.2	4504	1496	854	3650	642	89	86	527	175
Solvent Control	0.20%	93.4	93.6	93.6	4829	1457	712	4117	745	100	100	678	205
DNCB	4.00	82.0	82.8	82.3	11458	2405	702	10756	1703	261	229	1632	343
STB-FR	1000	92.4	91.8	91.5	4088	1599	1022	3066	577	74	77	400	156
	833.33	92.3	92.5	92.4	3846	1601	945	2901	656	70	88	407	169
	694.44	89.0	88.7	89.3	4176	1692	924	3252	768	79	103	452	183
	578.70	90.8	91.3	91.9	4196	1700	958	3238	742	79	100	438	177
	482.25	91.1	91.5	90.4	4073	1569	924	3149	645	76	87	441	170
	401.88	91.9	91.9	92.0	4052	1567	904	3148	663	76	89	448	173
	334.90	92.6	93.5	93.3	3854	1535	923	2931	612	71	82	418	166
	279.08	92.6	93.4	92.7	4255	1596	924	3331	672	81	90	460	173

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	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.6	94.8	94.8	3501	1395	752	2749	643	90	110	466	186
Solvent Control	0.20%	94.0	94.4	95.0	3793	1314	731	3062	583	100	100	519	180
DNCB	4.0	83.1	83.1	82.1	9665	2532	1064	8601	1468	281	252	908	238
STB-FR	1000.00	87.6	87.0	85.4	3545	1433	924	2621	509	86	87	384	155
	833.33	90.2	88.8	89.0	3429	1570	993	2436	577	80	99	345	158
	694.44	91.1	91.0	91.1	3726	1476	892	2834	584	93	100	418	165
	578.70	91.2	92.5	92.6	3756	1432	853	2903	579	95	99	440	168
	482.25	94.3	92.4	93.3	3695	1574	907	2788	667	91	114	407	174
	401.88	93.7	93.5	93.4	3707	1441	849	2858	592	93	102	437	170
	334.90	93.4	93.7	93.3	3723	1405	835	2888	570	94	98	446	168
	279.08	94.0	94.5	93.5	3430	1344	839	2591	505	85	87	409	160

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.