Registration Dossier

Administrative data

Description of key information

in vitro data:

OECD 442C:

In preparation of the OECD 442 assay, solubility testing in the solvents used for the OECD 422 C assay was performed. The test item solubility was studied in acetonitrile, water, isopropanol, methanol, and ethanol. In the solubility testing no test item solvent compatible with the DPRA was found. Therefore, the study cannot be performed.

OECD 442D: positive

OECD 442E:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser. Due to the high logP of the substance (> 6.5) the negative result should not be considered (cf. OECD 442E).

in vivo data:

OECD 429:

Test material formulations in DMF at concentrations between 25 and 0.5 % showed systemic and local effects including body weight loss, reduced activity, ear swelling, increased ear weight, scaly skin and eschar formation. The local effects were present even at a low dose of 0.5 %. Therefore, the main test was cancelled due to these technical difficulties and no conclusion on skin sensitisation potential can be derived

QSAR:

negative no structural alerts. no unknown structural features

Exposure

Below dermal sensitisation threshold. Margin of safety for skin sensitisation: 10000 (non reactive), 384 (reactive)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
Expert Judgement
Type of information:
other: Expert Judgement
Adequacy of study:
other information
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Please refer to the justification in the field "attached full study report".
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
A weight of evidence approach was used to obtain sufficient information on the skin sensitisation potential of this compound. For this compound evidence was accumulated from exposure assessment, from the chemical reactivity analysis of the molecule and from responses obtained in in vitro or in vivo assays addressing key events of the biological mechanism associated with sensitisation.
GLP compliance:
no
Type of study:
other: Expert Judgement
Reading:
other: overall
No. with + reactions:
0
Interpretation of results:
GHS criteria not met
Conclusions:
The absence of structural alerts, the in vitro battery results and the exposure assessment provide sufficient evidence to conclude that for the test material there is no safety concern for skin sensitisation.
Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ECHA Guidance QSAR
GLP compliance:
no
Parameter:
other:
Run / experiment:
1
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
other: The result must be considered in the context of an integrated approach.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-02-10 to 2017-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.83 in experiment 1; 2.67 in experiment 2).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
13.17
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
208.2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Value:
2.41
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
9.01
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
143.9
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Value:
2.56
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Table1: Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

100.4

111.4

105.9

7.8

8.00

102.2

109.0

105.6

4.9

16.00

116.3

109.7

113.0

4.6

32.00

117.7

115.0

116.3

1.8

64.00

129.4

123.2

126.3

4.4

Test Item

0.98

143.4

109.1

126.2

24.3

1.95

153.7

113.5

133.6

28.4

3.91

202.9

139.1

171.0

45.1

7.81

206.8

136.7

171.7

49.6

15.63

203.6

141.3

172.5

44.1

31.25

208.2

137.5

172.8

50.0

62.50

205.9

140.7

173.3

46.1

125.00

217.7

143.9

180.8

52.2

250.00

208.3

143.4

175.9

45.9

500.00

210.1

144.3

177.2

46.6

1000.00

191.9

138.6

165.2

37.7

2000.00

198.0

129.2

163.6

48.7

 

Table2: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.20

1.20

1.22

1.21

0.01

 

8.00

1.20

1.23

1.29

1.24

0.05

 

16.00

1.30

1.25

1.56

1.37

0.17

 

32.00

1.86

1.58

2.28

1.90

0.35

*

64.00

2.86

2.81

2.81

2.83

0.03

*

Test Item

0.98

1.16

1.14

1.12

1.14

0.02

 

1.95

1.20

0.98

1.22

1.14

0.14

 

3.91

2.38

2.73

2.99

2.70

0.31

*

7.81

11.91

8.86

11.80

10.86

1.73

*

15.63

13.08

10.89

12.28

12.08

1.11

*

31.25

14.54

11.62

13.36

13.17

1.47

*

62.50

12.97

11.71

13.08

12.58

0.76

*

125.00

14.70

10.24

13.51

12.82

2.31

*

250.00

11.87

9.37

11.35

10.86

1.32

*

500.00

10.34

9.06

11.72

10.37

1.33

*

1000.00

9.14

7.65

9.33

8.71

0.92

*

2000.00

4.97

5.52

7.25

5.91

1.19

*

* = significant induction according to Student’s t-test, p<0.05

Table3: Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.00

1.19

1.44

1.21

0.22

 

8.00

1.32

1.22

1.28

1.27

0.05

 

16.00

1.34

1.62

1.34

1.43

0.16

 

32.00

1.61

2.16

1.56

1.78

0.33

 

64.00

3.06

2.85

2.11

2.67

0.50

*

Test Item

0.98

1.07

1.06

1.16

1.10

0.05

 

1.95

1.24

1.12

1.16

1.17

0.06

 

3.91

1.91

2.74

2.01

2.22

0.45

*

7.81

7.92

8.46

7.20

7.86

0.63

*

15.63

8.14

8.75

7.57

8.15

0.59

*

31.25

8.72

9.63

8.39

8.92

0.64

*

62.50

9.36

9.19

8.14

8.90

0.66

*

125.00

9.41

9.58

8.05

9.01

0.84

*

250.00

8.17

7.68

6.62

7.49

0.79

*

500.00

8.26

6.90

6.52

7.23

0.92

*

1000.00

6.67

4.98

4.93

5.53

0.99

*

2000.00

4.98

3.57

5.50

4.68

1.00

*

* = significant induction according to Student’s t-test, p<0.05

Table4: Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.21

1.21

1.21

0.00

 

8.00

1.24

1.27

1.26

0.02

 

16.00

1.37

1.43

1.40

0.05

 

32.00

1.90

1.78

1.84

0.09

*

64.00

2.83

2.67

2.75

0.11

*

Test Item

0.98

1.14

1.10

1.12

0.03

 

1.95

1.14

1.17

1.15

0.03

 

3.91

2.70

2.22

2.46

0.34

*

7.81

10.86

7.86

9.36

2.12

*

15.63

12.08

8.15

10.12

2.78

*

31.25

13.17

8.92

11.05

3.01

*

62.50

12.58

8.90

10.74

2.61

*

125.00

12.82

9.01

10.92

2.69

*

250.00

10.86

7.49

9.18

2.38

*

500.00

10.37

7.23

8.80

2.23

*

1000.00

8.71

5.53

7.12

2.25

 

2000.00

5.91

4.68

5.30

0.87

*

* = significant induction according to Student’s t-test, p<0.05

Interpretation of results:
other: The result must be considered in the context of an integrated approach.
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.
Executive summary:

In the present study the test material was dissolved in DMSO.

Based on a molecular weight of 395 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 13.17 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 208.2%. The lowest tested concentration with a significant luciferase induction >1.5 (2.70) was found to be 3.91 µM. The corresponding cell viability was >70% (202.9%). The calculated EC1.5was < 1000 µM (2.41 µM). Microscopically, signs of precipitates were observed at the 4 highest test item concentrations.This may be the reason for the decrease in luciferase activity at the highest test item concentrations.

In the second experiment, a max luciferase activity (Imax) induction of 9.01 was determined at a test item concentration of 125.0 µM. The corresponding cell viability was 143.9%. The lowest tested concentration with a significant luciferase induction >1.5 (2.22) was found to be 3.91 µM. The corresponding cell viability was >70% (139.1%). The calculated EC1.5was < 1000 µM (2.56 µM). Microscopically, signs of precipitates were observed at the 4 highest test item concentrations. This may be the reason for the decrease in luciferase activity at the highest test item concentrations.

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-09-15 to 2018-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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.
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 (352% experiment 1; 288% experiment 2) and 200% for CD54 (248% experiment 1; 286% experiment 2) were clearly exceeded
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1
Value:
143
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 334.90 µM
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1
Value:
95
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 334.90 µM
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2
Value:
149
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 833.33 µM
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2
Value:
93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 694.44 µM
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 mets the acceptance criteria.

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/ml]

Cell Viability [%]

Medium Control

0.00

95.50

Solvent Control (0.2% THF (v/v))

0.00

95.30

Test Item

7.81

96.00

15.63

96.30

31.25

95.80

62.50

95.90

125.00

94.90

250.00

95.50

500.00

94.70

1000.00

93.00

Calculated CV75 [µg/mL]

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

-

97.3

97.5

97.1

1173

892

571

602

321

99

90

205

156

Solvent Control THF

0.20%

97.1

97.2

97.3

1117.0

893.0

562.0

555

331

100

100

199

159

Solvent Control DMSO

0.20%

97.2

97.1

97.4

1181

930

575

606

355

100

100

205

162

DNCB

4.00

87.1

86.5

85.6

2736

1481

600

2136

881

352

248

456

247

Test Item

1000

95.7

94.7

94.9

1280

811

547

733

264

132

80

234

148

833.33

95.6

95.6

95.3

1280

818

545

735

273

132

82

235

150

694.44

96.3

96.3

96.8

1237

804

532

705

272

127

82

233

151

578.70

96.1

96.5

96.5

1280

807

550

730

257

132

78

233

147

482.25

96.4

96.1

95.9

1225

794

559

666

235

120

71

219

142

401.88

97.0

97.0

96.9

1114

840

536

578

304

104

92

208

157

334.90

97.0

96.8

96.8

1304

824

510

794

314

143

95

256

162

279.08

96.9

96.4

97.0

1178

798

527

651

271

117

82

224

151

 

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

C86

CD54

Medium Control

-

98.1

97.7

97.6

2058

1077

672

1386

405

77

81

306

160

Solvent Control 2

0.20%

97.6

97.6

97.3

2026

1078

652

1374

426

100

100

311

165

Solvent Control 1

0.20%

97.4

97.8

97.4

2490

1179

682

1808

497

100

100

365

173

DNCB

4.0

86.0

85.3

85.7

5888

2108

688

5200

1420

288

286

856

306

Test item

1000.00

97.0

96.9

96.9

2471

917

618

1853

299

135

70

400

148

833.33

96.9

96.9

97.0

2667

976

621

2046

355

149

83

429

157

694.44

97.2

96.9

97.0

2431

1039

644

1787

395

130

93

377

161

578.70

97.9

97.7

97.9

2276

1030

662

1614

368

117

86

344

156

482.25

97.7

97.6

97.8

2347

1036

639

1708

397

124

93

367

162

401.88

97.4

97.0

96.5

2374

1032

658

1716

374

125

88

361

157

334.90

97.7

97.1

97.2

2172

1036

691

1481

345

108

81

314

150

279.08

97.7

98.0

97.5

2273

1010

679

1594

331

116

78

335

149

 

Acceptance Criteria

Acceptance Criterion

range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability medium and solvent controls [%]

>90

97.1

-

97.5

pass

97.3

-

98.1

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

352

pass

288

pass

RFI of positive control of CD54

≥200

248

pass

286

pass

RFI of solvent control of CD86

<150

101

pass

130

pass

RFI of solvent control of CD54

<200

111

pass

123

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

205

pass

306

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

205

pass

365

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

156

pass

160

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

162

pass

173

pass

Interpretation of results:
other: The result must be considered in the context of an integrated approach.
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser. Due to the high logP of the substance (> 6.5) the negative result should not be considered (cf. OECD 442E).

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

In the present study the test material was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 500 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.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps:

1000; 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In both experiments precipitation of the test item was observed when mixing the test item stock solutions (prepared in THF) 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 95.7% (CD86), 94.7% (CD54) and 94.9% (isotype IgG1 control) in the first experiment and to 97.00% (CD86), 96.90% (CD54) and 96.90% (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 the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (352% experiment 1; 288% experiment 2) and 200% for CD54 (248% experiment 1; 286% experiment 2) were clearly exceeded.

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser. Due to the high logP of the substance (> 6.5) the negative result should not be considered (cf. OECD 442E).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.