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Toxicological information

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:
January - April 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:
other: OECD guideline 442E h-CLAT
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: Dose range finder: 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 µg/mL; Main experiment: 97.71, 81.42, 67.85, 56.54, 47.12, 39.27, 32.72, 27.27 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Solvent: DMSO

CONTROLS
- Negative control (NC):Medium
- Solvent control (SC): 0.2% DMSO
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25 mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III with DPBS
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 3
- Exposure period: 24 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2 -mercaptoethanol (30 > passage >= 5)

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 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 solvent control cells - MFI of solvent isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.

ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity) values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86 =150% and CD54 =200%) and cell viability should be =50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be =105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.

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.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).

Results and discussion

Positive control results:
Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3).

In vitro / in chemico

Results
Key result
Parameter:
other: RFI CD54 mean (%) and RFI CD54 mean (%)
Run / experiment:
Please see 'Any other information on results incl. tables'.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

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

-

94.7

94.7

94.3

2943

1205

613

2330

592

101

111

480

197

Solvent Control

0.20%

94.8

95.2

94.8

2988

1214

679

2309

535

100

100

440

179

DNCB

4.00

80.9

80.5

80.0

6868

2013

779

6089

1234

264

231

882

258

ISOBUTYL SALICYLATE

97.71

12.4

11.9

12.3

4427

3822

942

3485

2880

151

538

470

406

81.43

57.3

58.4

57.4

3480

1781

726

2754

1055

119

197

479

245

67.85

83.9

83.4

84.1

3116

1540

673

2443

867

106

162

463

229

56.55

88.9

89.3

89.5

3066

1380

658

2408

722

104

135

466

210

47.12

93.2

93.5

93.6

2778

1235

669

2109

566

91

106

415

185

39.27

94.5

94.3

93.9

2801

1230

686

2115

544

92

102

408

179

32.72

94.4

94.8

94.9

2996

1287

660

2336

627

101

117

454

195

27.27

94.7

94.6

94.7

3063

1276

669

2394

607

104

113

458

191

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

-

94.7

94.8

93.9

4619

1554

646

3973

908

102

95

715

241

Solvent Control

0.20%

95.0

95.1

94.9

4571

1620

669

3902

951

100

100

683

242

DNCB

4.0

81.4

80.5

81.2

13735

3370

633

13102

2737

336

288

2170

532

ISOBUTYL SALICYLATE

97.71

13.2

12.8

12.4

7467

5420

935

6532

4485

167

472

799

580

81.43

45.9

45.5

45.7

6028

3615

1213

4815

2402

123

253

497

298

67.85

79.3

79.0

79.7

5291

3011

782

4509

2229

116

234

677

385

56.55

90.0

90.8

89.7

4710

2761

874

3836

1887

98

198

539

316

47.12

93.5

93.5

92.7

4543

2413

735

3808

1678

98

176

618

328

39.27

93.4

92.5

92.8

4587

2304

746

3841

1558

98

164

615

309

32.72

94.6

94.1

94.2

4335

2049

756

3579

1293

92

136

573

271

27.27

95.1

95.0

94.9

4489

2013

790

3699

1223

95

129

568

255

CD54 and CD86 Expression Experiment 3

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

-

95.2

94.5

94.3

3732

1289

640

3092

649

88

87

583

201

Solvent Control

0.20%

94.7

94.5

94.5

4176

1395

649

3527

746

100

100

643

215

DNCB

4.0

81.9

81.1

82.4

11303

2502

583

10720

1919

304

257

1939

429

ISOBUTYL SALICYLATE

97.71

40.5

38.6

39.3

4938

2769

816

4122

1953

117

262

605

339

81.43

64.4

64.9

64.8

4625

2417

806

3819

1611

108

216

574

300

67.85

86.4

85.4

86.3

4465

2304

739

3726

1565

106

210

604

312

56.55

93.0

91.8

93.0

4049

1925

742

3307

1183

94

159

546

259

47.12

94.0

93.8

94.4

3792

1728

740

3052

988

87

132

512

234

39.27

94.5

94.1

93.9

3707

1636

730

2977

906

84

121

508

224

32.72

95.0

94.9

95.2

3538

1580

735

2803

845

79

113

481

215

27.27

95.2

95.6

95.0

4119

1701

811

3308

890

94

119

508

210

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results, it is concluded that the test substance induces dendritic cell activation (CD54 expression increased =200% in at least one assay).

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

 

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at one concentration with a cell viability ≥50% in at least two independent runs.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

 

In summary, it has to be concluded that the test substance induces dendritic cell activation.