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Skin sensitisation

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD 442E (In VItro Skin Sensitisation h-CLAT)
Deviations:
no
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

Constituent 1
Chemical structure
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-trichlorobenzene-1-sulfonate
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

Resultsopen allclose all
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
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
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
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.