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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:
weight of evidence
Study period:
28 Dec 2017 - 08 Mar 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:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”
Version / remarks:
adopted 09 October 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
Version / remarks:
July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
- Details of the test procedure used: For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks and then harvested, afterwards the cell suspension were seeded into a 24 well flat-bottom plate at a final density of 1 x 10E6 cells/well.
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution (propidium iodide) was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625µg/mL). Finally the expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI.

- Cell line used: THP-1 cells (ATCC® TIB-202TM).

- Doses of test chemical and control substances used:
Test Item:
Dose finding assay 1 and 2: 1000, 500.0, 250.0, 125.0, 62.50, 31.25, 15.63 and 7.81 µg/mL
Main experiment 1 and 2: 21.09, 17.58, 14.65, 12.21, 10.17, 8.48, 7.06, 5.89 µg/mL
Medium Control: cell culture medium
Solvent Control: cell culture medium
Positive Control: 4 µg/mL DNCB (2, 4-dinitrochlorobenzene)

- Duration and temperature of exposure: 24 h ± 0.5 h at 37 °C± 1 °C and 5% CO2.

- Cell reactivity check and Dose finding assay: Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. DNCB (4 µg/mL) and nickel sulphate (100 µg/mL) served as positive control while lactic acid (1000 µg/mL) as negative control. Cells were accepted when both positive controls produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.
In dose finding assay, The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate (with the final density of 1 x 106 cells/well). The cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. The CV75 value (75% cell survival) was calculated and used to calculate the concentration range of the test item for the main experiment.

- Number of tissue replicates used per test chemical and controls: Two repeated Experiments respectively with one sample per treatment: test substance, positive and negative controls.

- 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 both, the medium and DMSO control, is >105%.

- Evaluation criteria:
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 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
- 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

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%. But a negative result for test items with a log Kow >3.5 should be considered as inconclusive.

Results and discussion

Positive control results:
The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (326% experiment 1; 309% experiment 2) and 200% for CD54 (572% experiment 1; 309% experiment 2) were clearly exceeded.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: RFI (relative fluorescence intensity)
Run / experiment:
CD86/Experiment 1
Value:
>= 122 - <= 220
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
The expression of the cell surface marker CD86 was upregulated above the threshold of 150 starting from a concentration of 21.09 µg/mL to 12.20 µg/mL
Key result
Parameter:
other: RFI (relative fluorescence intensity)
Run / experiment:
CD54/Experiment 1
Value:
>= 83 - <= 113
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The expression of CD54 was not upregulated above the threshold of 200%
Key result
Parameter:
other: RFI (relative fluorescence intensity)
Run / experiment:
CD86/Experiment 2
Value:
>= 116 - <= 219
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
The expression of CD86 was upregulated above the threshold of 150 starting from a concentration of 21.09 µg/mL to 17.58 µg/mL
Key result
Parameter:
other: RFI (relative fluorescence intensity)
Run / experiment:
CD54/Experiment 2
Value:
>= 101 - <= 119
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The expression of CD54 was not upregulated above the threshold of 200%
Other effects / acceptance of results:
Slight cytotoxic effects were observed for the cells treated with the test item in the highest concentrations. Relative cell viability at the highest test item concentration was reduced to 76.2% (CD86), 75.4% (CD54) and 74.9% (isotype IgG1 control) in the first experiment and to 78.5% (CD86), 78.0% (CD54) and 77.8% (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was upregulated above the threshold of 150% starting from a concentration of 21.09 µg/mL to 12.20 µg/mL in the first experiment and of 21.09 µg/mL to 17.58 µg/mL in the second experiment. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Since one of the cell surface markers (CD86) clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.
All the controls confirmed the validity of the study. The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (332% experiment 1; 272% experiment 2) and 200% for CD54 (304% experiment 1; 544% experiment 2) were clearly exceeded.

Any other information on results incl. tables

Table 1: 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

-

97.1

96.3

96.0

2863

1435

657

2206

778

100

100

436

218

Solvent Control

0.20%

96.6

94.9

95.7

3156

1228

668

2488

560

113

72

472

184

DNCB

4.00

81.3

80 .8

80.6

8942

2395

693

8249

1702

332

304

1290

346

NEO-HITENOL LM-20 (Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts)

21.09

76.2

75.4

74.9

5612

1631

750

4862

881

220

113

748

217

17.58

87.7

88.4

87.7

4980

1521

714

4266

807

193

104

697

213

14.65

91.1

91.2

91.6

4390

1492

726

3664

766

166

98

605

206

12.20

93.9

93.4

93.2

4160

1494

737

3423

757

155

97

564

203

10.17

94.3

94.2

94.1

3990

1591

786

3204

805

145

103

508

202

8.48

94.7

95.0

94.8

3698

1515

871

2827

644

128

83

425

174

7.06

95.0

94.9

94.7

3692

1453

774

2918

679

132

87

477

188

5.89

95.4

95.6

95.0

3398

1507

713

2685

794

122

102

477

211

Table 2: 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

-

96.9

96.8

96.5

2594

1130

618

1976

512

100

100

420

183

Solvent Control

0.20%

96.6

95.8

96.6

2483

1056

619

1864

437

94

85

401

171

DNCB

4.0

84.1

84.5

83.4

5810

3109

732

5078

2377

272

544

794

425

NEO-HITENOL LM-20 (Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts)

21.09

78.5

78.0

77.8

4943

1222

615

4328

607

219

119

804

199

17.58

92.7

92.9

92.5

3601

1166

578

3023

588

153

115

623

202

14.65

94.1

94.4

94.4

3209

1191

600

2609

591

132

115

535

199

12.20

95.4

95.3

95.4

3301

1168

636

2665

532

135

104

519

184

10.17

95.6

95.4

95.9

3395

1162

584

2811

578

142

113

581

199

8.48

96.1

96.2

96.1

3309

1146

611

2698

535

137

104

542

188

7.06

95.6

96.1

95.7

2893

1123

604

2289

519

116

101

479

186

5.89

94.7

95.5

95.6

3007

1103

584

2423

519

123

101

515

189

Table3: Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

94.9

-

97 .1

pass

95.8

-

96.9

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

Pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

RFI of positive control of CD86

≥150

332

pass

272

pass

RFI of positive control of CD54

≥200

304

pass

544

pass

RFI of solvent control of CD86

<150

113

pass

94

pass

RFI of solvent control of CD54

<200

72

pass

85

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

436

pass

420

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

472

pass

401

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

218

pass

183

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

184

pass

171

pass

Applicant's summary and conclusion

Interpretation of results:
other: skin sensitising based on the key event “activation of dendritic cells”
Conclusions:
In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at least two independent experiment runs. Therefore, based on the observed results it was concluded that the test substance induces dendritic cell activation. The result alone does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required. Based on the results of all three key events the test substance has to be classified as skin sensitiser.