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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:
2018-04-09 to 2018-05-08
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: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
In vitro testing strategy

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Details on 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 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 sensitisers and non-sensitisers.

Results and discussion

Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (252% experiment 1; 303% experiment 2) and 200% for CD54 (201% experiment 1; 226% experiment 2) were clearly exceeded.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1
Value:
116
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 11979.17 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1
Value:
122
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration:14375.00 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2
Value:
119
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 17250.00 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2
Value:
113
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 17250.00µg/mL
Other effects / acceptance of results:
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 the medium and DMSO control, is >105%.

The test met the acceptance criteria.

Any other information on results incl. tables

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

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 79.4% (CD86), 79.4% (CD54) and 79.5% (isotype IgG1 control) in the first experiment and to 87.5% (CD86), 86.1% (CD54) and 86.8% (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 considered to be no skin sensitiser.

The controls confirmed the validity of the study for all experiments.

Results of the Cell Batch Activation Test (Batch 19)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

81.1

347

>150

79.7

269

>200

yes

pass

NiSO4

100 µg/mL

82.1

347

>150

82.5

391

>200

yes

pass

LA

1000 µg/mL

96.7

89

150

96.4

109

200

no

pass

 

Results of the Cell Batch Activation Test (Batch 20)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

89.2

266

>150

88.4

206

>200

yes

pass

NiSO4

100 µg/mL

82.3

220

>150

80.6

283

>200

yes

pass

LA

1000 µg/mL

96.2

79

150

96.9

101

200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Concentration applied [µg/mL]

Cell Viability [%]

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

93.10

--

95.70

Solvent Control

NaCl

--

92.60

--

94.70

Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine

C8

26.95

95.10

134.77

94.90

C7

53.91

95.40

269.53

95.40

C6

107.81

95.40

539.06

94.80

C5

215.63

95.30

1078.13

95.00

C4

431.25

95.00

2156.25

95.40

C3

862.50

95.00

4312.50

94.60

C2

1725.00

95.00

8625.00

92.20

C1

3450.00

93.30

17250.00

84.90

Calculated CV75 [µg/mL]

No CV75

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

No SD

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

-

93.6

93.2

94.0

5539

1543

723

4816

820

100

100

766

213

Solvent Control

0.20%

92.7

92.2

92.8

5225

1464

712

4513

752

94

92

734

206

DNCB

4.00

83.8

84.4

84.5

12159

2295

786

11373

1509

252

201

1547

292

Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine

17250

79.4

79.4

79.5

6232

1753

820

5412

933

112

114

760

214

14375.00

82.0

81.4

81.7

6213

1893

896

5317

997

110

122

693

211

11979.17

84.6

84.6

84.1

6359

1619

787

5572

832

116

101

808

206

9982.64

87.5

88.3

87.9

5801

1697

779

5022

918

104

112

745

218

8318.87

89.6

90.3

89.3

5688

1620

767

4921

853

102

104

742

211

6932.39

91.5

91.7

92.0

5302

1523

781

4521

742

94

90

679

195

5776.99

91.5

91.1

90.9

5600

1518

788

4812

730

100

89

711

193

4814.16

91.3

92.2

91.7

5326

1491

779

4547

712

94

87

684

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

CD86

CD54

Medium Control

-

94.6

94.3

94.1

5423

1785

731

4692

1054

100

100

742

244

Solvent Control

0.20%

94.7

95.1

94.5

5772

1783

704

5068

1079

108

102

820

253

DNCB

4.0

81.4

82.1

82.3

16087

3161

726

15361

2435

303

226

2216

435

Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine

17250.00

87.5

86.1

86.8

6383

1991

801

5582

1190

119

113

797

249

14375.00

87.2

88.3

88.4

5982

1863

999

4983

864

106

82

599

186

11979.17

89.6

89.6

89.8

5988

1944

970

5018

974

107

92

617

200

9982.64

91.0

90.9

90.9

6024

1830

813

5211

1017

111

96

741

225

8318.87

92.5

92.2

92.4

5436

1761

819

4617

942

98

89

664

215

6932.39

92.7

92.3

92.8

5946

1769

807

5139

962

110

91

737

219

5776.99

93.9

93.4

93.9

5103

1761

799

4304

962

92

91

639

220

4814.16

93.5

93.6

94.1

5217

1671

825

4392

846

94

80

632

203

Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

92.2

-

94.0

pass

94.1

-

95.1

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

252

pass

303

pass

RFI of positive control of CD54

≥200

201

pass

226

pass

RFI of solvent control of CD86

<150

94

pass

108

pass

RFI of solvent control of CD54

<200

92

pass

102

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

766

pass

742

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

734

pass

820

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

213

pass

244

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

206

pass

253

pass

 

Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

97.0

1.3

672

number of test doses with viability >50%

-

-

1786

RFI of positive control of CD86

401.0

146.8

112

RFI of positive control of CD54

576.6

312.0

112

RFI of solvent control of CD86

115.0

15.1

112

RFI of solvent control of CD54

118.8

25.5

112

MFI ratio IgG1/CD86 for medium control [%]

202.4

50.0

112

MFI ratio IgG1/CD86 for DMSO control [%]

221.6

58.5

112

MFI ratio IgG1/CD54 for medium control [%]

141.0

24.7

112

MFI ratio IgG1/CD54 for DMSO control [%]

147.7

25.6

112

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore the test item might be considered as non-sensitiser.
Executive summary:

In the present study the test item was dissolved in 0.9% NaCl. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps:

5000.00, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41µg/mL

This corresponds to the following weights, since a correction factor of 3.45 was applied to correct for the active component of the test item: 17250, 14375, 11979.17, 9982.64, 8318.87, 6932.39, 5776.99, 4814.16 µg/mL.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

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 79.4% (CD86), 79.4% (CD54) and 79.5% (isotype IgG1 control) in the first experiment and to 87.5% (CD86), 86.1% (CD54) and 86.8% (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 considered to be no skin sensitiser.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (252% experiment 1; 303% experiment 2) and 200% for CD54 (201% experiment 1; 226% experiment 2) were clearly exceeded.