Registration Dossier

Diss Factsheets

Administrative data

Description of key information

in vitro data:

OECD 442C: negative, but inconclusive due to precipitation

OECD 442D: positive

OECD 442E: negative, but inconclusive due to logP > 3.5

QSAR:

Negative no structural alerts. no unknown structural features

Exposure

Below dermal sensitisation threshold. Margin of safety for skin sensitisation: 2000 (non reactive), 142 (reactive)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-12 to 2019-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on the 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 (3.05 (experiment 1); 4.9 (experiment 2)).
Key result
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Cell viability:
150.4%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated
Key result
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Cell viability:
95.1%
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated
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.

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

106.2

99.1

102.6

5.0

8.00

101.1

98.7

99.9

1.7

16.00

101.0

97.0

99.0

2.8

32.00

108.5

90.8

99.6

12.5

64.00

107.1

101.4

104.2

4.1

Test Item

0.98

112.6

98.2

105.4

10.2

1.95

108.6

97.4

103.0

7.9

3.91

107.8

98.0

102.9

6.9

7.81

102.4

91.3

96.8

7.8

15.63

94.2

88.6

91.4

4.0

31.25

97.1

83.5

90.3

9.6

62.50

93.6

73.4

83.5

14.3

125.00

95.3

69.3

82.3

18.3

250.00

111.9

68.3

90.1

30.8

500.00

125.9

73.2

99.6

37.3

1000.00

150.4

78.3

114.4

50.9

2000.00

119.3

95.1

107.2

17.1

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

1.12

1.10

1.19

1.14

0.05

 

8.00

1.22

1.21

1.13

1.19

0.05

 

16.00

1.58

1.45

1.30

1.44

0.14

 

32.00

1.63

1.63

1.48

1.58

0.09

*

64.00

3.11

3.39

2.66

3.05

0.37

*

Test Item

0.98

1.15

1.09

1.10

1.11

0.03

 

1.95

1.20

0.98

1.12

1.10

0.11

 

3.91

1.10

0.95

1.18

1.08

0.12

 

7.81

0.93

0.95

0.93

0.94

0.01

 

15.63

1.00

0.89

1.03

0.97

0.08

 

31.25

1.24

1.04

0.93

1.07

0.16

 

62.50

1.02

1.06

1.01

1.03

0.02

 

125.00

1.13

1.02

0.96

1.04

0.09

 

250.00

1.19

1.11

0.97

1.09

0.11

 

500.00

1.18

1.17

1.13

1.16

0.03

 

1000.00

1.47

1.32

1.42

1.40

0.08

 

2000.00

1.00

0.91

0.81

0.91

0.10

 

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

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

1.11

1.41

1.14

1.22

0.17

 

8.00

1.17

1.59

1.24

1.34

0.23

 

16.00

1.58

1.58

1.64

1.60

0.04

*

32.00

2.05

2.33

2.01

2.13

0.17

*

64.00

4.85

4.51

5.35

4.90

0.42

*

Test Item

0.98

1.30

1.19

1.25

1.25

0.06

 

1.95

1.00

1.13

1.08

1.07

0.07

 

3.91

0.98

1.18

1.10

1.09

0.10

 

7.81

0.92

1.11

1.15

1.06

0.12

 

15.63

1.00

1.06

1.00

1.02

0.03

 

31.25

0.97

1.07

1.05

1.03

0.05

 

62.50

1.15

1.20

1.11

1.15

0.05

 

125.00

1.01

1.07

1.07

1.05

0.04

 

250.00

1.00

1.11

1.08

1.06

0.05

 

500.00

1.01

1.12

1.27

1.13

0.13

 

1000.00

0.99

1.34

1.16

1.17

0.18

 

2000.00

1.39

1.24

1.12

1.25

0.14

 

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

Induction of Luciferase Activity – Overall Induction

Overall Induction

Concentration

[µM]

Fold Induction

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

Positive Control

4.00

1.14

1.22

1.18

0.06

8.00

1.19

1.34

1.26

0.11

16.00

1.44

1.60

1.52

0.11

32.00

1.58

2.13

1.85

0.39

64.00

3.05

4.90

3.98

1.31

Test Item

0.98

1.11

1.25

1.18

0.09

1.95

1.10

1.07

1.08

0.02

3.91

1.08

1.09

1.09

0.01

7.81

0.94

1.06

1.00

0.09

15.63

0.97

1.02

1.00

0.03

31.25

1.07

1.03

1.05

0.03

62.50

1.03

1.15

1.09

0.08

125.00

1.04

1.05

1.04

0.01

250.00

1.09

1.06

1.08

0.02

500.00

1.16

1.13

1.14

0.02

1000.00

1.40

1.17

1.28

0.17

2000.00

0.91

1.25

1.08

0.24

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5

n.a.

n.a.

n.a.

n.a.

Imax

1.40

1.25

1.33

0.11

IC30

n.a.

336.7

336.7

n.a.

IC50

n.a.

n.a.

n.a.

n.a.

IC70

n.a.

n.a.

n.a.

n.a.

n.a. = not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control [%]

< 20%

5.1

pass

17.3

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

3.0

pass

EC1.5 PC [µM]

±2 x SD of historical mean

22.71

pass

12.94

pass

Induction PC at 64 µM

2 .00 < x < 8.00

3.05

pass

4.90

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.6

3.5

96

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96
Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

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 DMSO. Based on a molecular weight of 751.98 g/mol a stock solution of 200 mM was prepared. A stable suspension was formed.

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, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No 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 non-sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-04-05 to 2019-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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”, adopted 25 June 2018
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
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 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 (283% experiment 1; 260% experiment 2) and
200% for CD54 (346% experiment 1; 347% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
123
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 208.33 µg/mL
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
103
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 250 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
121
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 173.61 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 173.61 µg/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 mets the acceptance criteria.

Results of the Cell Batch Activation Test

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

86.1

520

>150

85.3

650

>200

yes

pass

NiSO4

100 µg/mL

81.0

418

>150

79.3

661

>200

yes

pass

LA

1000 µg/mL

96.5

84

≤150

96.6

107

≤200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

94.30

Solvent Control

THF

--

94.70

Test item

C8

3.91

94.40

C7

7.81

95.40

C6

15.63

94.90

C5

31.25

94.50

C4

62.50

93.80

C3

125.00

94.30

C2

250.00

94.40

C1

500.00

95.00

Calculated CV75 [µg/mL]

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

No CV75

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

-

94.5

94.3

93.7

3114

1152

730

2384

422

92

96

427

158

Solvent Control 2 (THF)

0.20%

93.8

94.1

93.6

3295.0

1204.0

762.0

2533

442

100

100

432

158

Solvent Control 1 (DMSO)

0.20%

94.5

93.7

93.4

3357

1198

759

2598

439

100

100

442

158

DNCB

4.00

80.5

80.0

80.0

8099

2257

739

7360

1518

283

346

1096

305

Test item

250

93.5

92.7

92.9

3477

1245

789

2688

456

106

103

441

158

208.33

93.0

93.4

93.1

3844

1177

728

3116

449

123

102

528

162

173.61

93.5

92.9

93.1

3590

1135

741

2849

394

112

89

484

153

144.68

93.7

92.5

93.3

3216

1181

728

2488

453

98

102

442

162

120.56

93.8

93.4

93.3

2037

996

711

1326

285

52

64

286

140

100.47

93.8

93.6

93.5

2681

1017

985

1696

32

67

7

272

103

83.72

93.2

92.4

92.5

2252

1040

742

1510

298

60

67

304

140

69.77

93.3

92.7

93.2

2240

1035

774

1466

261

58

59

289

134

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

-

94.7

95.3

95.4

3642

1371

775

2867

596

86

83

470

177

Solvent Control 2 (THF)

0.20%

94.3

94.3

94.7

4010

1744

782

3228

962

100

100

513

223

Solvent Control 1 (DMSO)

0.20%

95.0

94.8

95.3

4085

1478

756

3329

722

100

100

540

196

DNCB

4.0

77.2

76.9

77.8

9504

3366

860

8644

2506

260

347

1105

391

 

Test item

250.00

93.80

93.50

93.70

4397

1593

822

3575

771

111

80

535

194

208.33

93.30

93.80

93.20

4450

1512

807

3643

705

113

73

551

187

173.61

93.60

93.40

94.00

4723

1670

823

3900

847

121

88

574

203

144.68

93.60

93.60

93.70

4435

1626

872

3563

754

110

78

509

186

120.56

93.40

93.10

93.50

4121

1617

847

3274

770

101

80

487

191

100.47

93.10

93.80

93.40

4401

1638

852

3549

786

110

82

517

192

83.72

93.80

93.60

93.50

4392

1637

814

3578

823

111

86

540

201

69.77

92.20

92.80

93.50

4302

1631

868

3434

763

106

79

496

188

Acceptance Criteria

Acceptance Criterion

range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability medium and solvent controls [%]

>90

93.4

-

94.5

pass

94.3

-

95.4

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

283

pass

260

pass

RFI of positive control of CD54

≥200

346

pass

347

pass

RFI of DMSO solvent control of CD86

<150

109

pass

116

pass

RFI of DMSO solvent control of CD54

<200

104

pass

121

pass

RFI of THF solvent control of CD86

<150

106

pass

113

pass

RFI of THF solvent control of CD54

<200

105

pass

161

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

427

pass

470

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

442

pass

540

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

432

pass

513

pass

MFI ratio CD54/IgG1 for medium control [%]

>105

158

pass

177

pass

MFI ratio CD54/IgG1 for DMSO control [%]

>105

158

pass

196

pass

MFI ratio CD54/IgG1 for THF control [%]

>105

158

pass

223

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
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 marker in at least two independent experiment runs. Because of the Log Kow of >6.57 the test item is considered as inconclusive.

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 250 mg/mL to 1.96 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:

500.00, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 µg/mL

In both experiments precipitation 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 93.5% (CD86), 92.7% (CD54) and 92.9% (isotype IgG1 control) in the first experiment and 93.8% (CD86), 93.5% (CD54) and 93.7% (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. The test item would be considered as non-sensitiser but because of the Log Kow of >6.57 it is considered as inconclusive.

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: ECHA Guidance QSAR
GLP compliance:
no
Run / experiment:
other: 1
Parameter:
other:
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:
GHS criteria not met
Conclusions:
The QSAR analysis using a knowlegde based QSAR system shows no indication for skin sensitisation.
Endpoint:
skin sensitisation, other
Remarks:
Weight of Evidence
Type of information:
other: Weight of Evidence
Adequacy of study:
weight of evidence
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
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 assays addressing key events of the biological mechanism associated with sensitisation.
GLP compliance:
no
Type of study:
other: Weigth of Evidence
Parameter:
SI
Value:
0
Remarks on result:
other: no indication for skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The absence of structural alerts, the exposure assessment and the negative in vitro data genereated in GLP complinat studies provide sufficient evidence to conclude that for the test material there is no safety concern for skin sensitisation.
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.