Registration Dossier

Administrative data

Description of key information

Based on all available data and using the weight of evidence approach, scopolamine is classified for skin sensitization Cat. 1 H317 (May cause an allergic reaction to skin), following a conservative approach.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-12-07 to 2017-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
- Preparation of the Test Item
The test item was freshly prepared immediately prior to use and dissolved in acetonitrile (vehicle).

Stock solutions with a concentration of 10 mM (lysine peptide run 2), 50 mM (lysine peptide run 3), and 100 mM (cysteine and lysine peptide run 1, experiment 1) were prepared.

- Controls
Reference controls, co-elution controls and a positive control were set up in parallel to the test item in order to confirm the validity of the test.

- Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run.

- Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run.

- Reference Control
Reference controls were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.

Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.

Reference control C was set up for the test item and the positive control. Reference control C for the test item was prepared using the respective solvent used to solubilize the test item. Reference control C for the positive control was prepared using acetonitrile. Reference control C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. Reference control was included in every assay run for both peptides and was injected together with the samples.

- HPLC system
HPLC/DAD: Agilent, 1200 Series, with Chemstation, Rev. B.04.01
Detection: 220 nm signal for quantitation; 258 nm signal used as indicator for co-elution
Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µ m, Agilent Art. Nr. 861753-902
Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm. Art. Nr: AJO-4286
Column Temperature: 30 °C
Sample Temperature: 25 °C
Run time: 20 minutes
Injection volume: 10µL

HPLC Mobile Phase (Cysteine Run, Experiment 1; Lysine Run 2 and 3)
HPLC Mobile Phase A: 0.1% ( v/v) trifluoroacetic acid in water
HPLC Mobile Phase B: 0.085% ( v/v) trifluoroacetic acid in acetonitrile

HPLC Mobile Phase (Lysine Run 1, Experiment 1)
HPLC Mobile Phase A: 0.085% ( v/v) trifluoroacetic acid in water
HPLC Mobile Phase B: 0.1% ( v/v) trifluoroacetic acid in acetonitrile


Positive control results:
Refer to "Any other information on result incl. tables"
Key result
Run / experiment:
other: Cysteine Peptide Depletion
Parameter:
other: Mean Peptide Depletion
Value:
0.54
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: Lysine Peptide Depletion Run 1
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
not determinable
Run / experiment:
other: Lysine Peptide Run 2
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
not measured/tested
Remarks:
Due to the influence of the test item on the elution behaviour of the lysine peptide the lysine peak areas from the test item samples of run 2 were not evaluated
Run / experiment:
other: Lysine Peptide Run 3
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
not measured/tested
Remarks:
Due to the influence of the test item on the elution behaviour of the lysine peptide the lysine peak areas from the test item samples of run 3 were not evaluated
Other effects / acceptance of results:
Acceptance Criteria
The run met the acceptance criteria if:
-the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
-the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
-the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
 
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM
Cellular proliferation data / Observations:
n/a

Solubility assessment

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using acetonitrile.

 

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

 

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for test item samples. A slight precipitation was observed for the samples of the positive control, no centrifugation was necessary to perform the HPLC analysis.

 

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A phase separation observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

 

For the 10 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis the lysine peptide samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. Turbidity and phase separation was observed for the samples of the positive control and the respective co-elution control. Samples were not centrifuged prior to the HPLC analysis.

 

For the 50 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis the lysine peptide samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A phase separation was observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

 

Since the positive control fulfilled all quality criteria the observed precipitation, turbidity and phase separations were considered as irrelevant.

 

Co-elution with the Peptide Peaks

Co-elution of the test item with the lysine peptide peak was observed.

 

Results of the cysteine peptide depletion

 

Table 3: Depletion of the Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Test item

4346.9439

4330.2656

4277.6216

0.4946

0.4927

0.4867

0.00*

0.20

1.42

0.54

0.77

142.1

Positive control

1209.3923

1131.5034

1167.8929

0.1372

0.1283

0.1325

72.13

73.92

73.08

73.04

0.90

1.23

* Value was set to 0 due to negative depletion.

 

Results of the lysine peptide depletion

 

Table 4: Depletion of the Lysine Peptide: Run 1

Sample

Peak Area at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Test item

Co-elution

Co-elution

Co-elution

-

-

-

-

-

-

-

-

-

Positive control

1802.2493

1749.5433

1855.6707

0.2125

0.2062

0.2188

57.49

58.73

56.23

57.48

1.25

2.18

 

Table 5: Depletion of the Lysine Peptide: Run 2 and 3

Sample

Peak Area at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive control: Run 2

2051.3940

2078.4993

2113.1423

0.2418

0.2450

0.2491

52.31

51.68

50.87

51.62

0.62

0.01

Positive control: Run 3

1817.4902

1655.4071

1768.4237

0.2133

0.1942

0.2075

57.09

60.92

58.25

58.75

1.96

0.03

Due to the influence of the test item on the elution behaviour of the lysine peptide the lysine peak areas from the test item samples of run 2 and 3 were not evaluated.

 

Categorisation of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

 

Since co-elution with the lysine peptide was observed, prediction model 2 should be considered.

 

Table 6: Categorisation of the Test Item

Prediction model

Prediction Model 1

(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2

(Cysteine Peptide / Ratio: 1:10)

Test substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test item

n/a

n/a

n/a

0.54

Minimal reactivity

Not a sensitiser

Positive control

65.26*

High reactivity

Sensitiser

73.04

Moderate reactivity

Sensitiser

* mean peptide depletion of cysteine peptide and lysine peptide

 

Interpretation of results:
other: Weight of Evidence approach
Conclusions:
In in chemico direct peptide reactivity assay (DPRA) under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as “non-sensitizer”. Mean cysteine peptide depletion for the test item was 0.54%.
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.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

 

In the present study (-) - Scopolamine was dissolved in acetonitrile. Based on a molecular weight of 303.35 g/mol different stock solutions were prepared. The test item solutions were tested by incubating the samples with peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

 

Experiment 1 (cysteine peptide run, lysine peptide run 1):

A 100 mM stock solution of the test item was prepared, the test item solutions were incubated with a peptide containing cysteine (cysteine peptide run) and with a peptide containing lysine (lysine peptide run 1).

After the 24 h ± 2 h incubation period but prior to the HPLC analysis the cysteine and lysine peptide samples were inspected for precipitation, turbidity or phase separation. For the cysteine peptide run no precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for the samples of the positive control, no centrifugation was necessary to perform the HPLC analysis. For the lysine peptide run no precipitation, turbidity or phase separation was observed for the test item samples. A phase separation was observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

 

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides.The mean depletion of both peptides was 65.26%.

In lysine peptide run 1 only a small peak was observed at the retention time of the lysine peptide (in comparison to the standards and reference controls), suggesting an almost complete depletion of the lysine peptide. However, the test item eluted closely to the retention time of the peptide peak and a peak not occurring in the test item co-elution control and near the expected elution of the lysine peptide was observed in the chromatogram (see Figure 5). The high concentration of the test item –indicating an overload of the system by the peak tailing– in comparison to the lysine peptide and the close elution of both compounds made an influence on the elution behaviour of the peptide peak seem possible. It was suspected that the lysine peptide had not been depleted but that the peak had been shifted (forward) due to the influence of the test substance. Since the OECD guideline 442c as well as the requirements of GLP do not allow the change of the chromatographic conditions without method validation, within the scope of this study and for better comparability, it was decided to only change the concentration of the test item. Two further lysine peptide runs with reduced test item concentrations (10 mM and 50 mM) were performed in order to observe a possible shifting of the lysine peptide peak. Since the positive control acceptance criteria are based on a combination of results from a lysine and a cysteine peptide run, these criteria were excluded from evaluation for lysine peptide runs 2 and 3.

 

For the lysine run 1 the coefficient of determination for the calibration curve was > 0.99 (0.9999). The mean peptide depletion of the lysine peptide was between 40.2% and 69.0% (57.48%).The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.50 mM, RC Cacetonitrile: 0.50 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.28%, RC Cacetonitrile: 0.15%). The standard deviation of the peptide depletion for the replicates of the positive control was < 11.6% (PC: 1.25%; test item: n.a.%).

 

Lysine peptide run 2:

 A 10 mM stock solution of the test item was prepared, the test item solutions were incubated with a peptide containing lysine.

 

After the 24 h ± 2 h incubation period but prior to the HPLC analysis the lysine peptide samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. Turbidity and phase separation was observed for the samples of the positive control and the respective co-elution control. Samples were not centrifuged prior to the HPLC analysis.

 

Lysine peptide run 3:

A 50 mM stock solution of the test item was prepared, the test item solutions were incubated with a peptide containing lysine.

After the 24 h±2 h incubation period but prior to the HPLC analysis the lysine peptide samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A phase separation was observed for the samples of the positive control and the respective co-elution control. No centrifugation was necessary to perform the HPLC analysis.

 

The chromatograms of the different test item concentrations showed that the lysine peptide peak was shifted, depending on the test item concentration, as shown inFigure 5. It was concluded that the (chromatographic) conditions with 100 mM test item are not suitable for a (correct) separation of test item and peptide. The strong tailing proves an overload of the system and the peptide cannot be retained/eluted in the required way with this amount of test material. Therefore, the experiment is not considered suitable to properly display the depletion of the lysine peptide. Hence, the lysine reactivity was excluded from evaluation and only the cysteine was considered. Due to the influence of the test item on the elution behaviour of the lysine peptide, the peak areas from all three runs were not evaluated.

The sensitiing potential of the test item was predicted only from the peptide depletion of the cysteine peptide run (experiment 1) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was£6.38% (0.54%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 February 2017 to 23 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Details on the study design:
Method:
- Cell line:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number <30 (p 9 – main experiment 1, p 12 - main experiment 2) were used.

Cells were cultured in 75 cm^2 culture flasks (Greiner) in RPMI-1640, supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37°C and 5% CO2.

- CD54 and CD86 Expression:
THP-1 cells were pre-cultured for at least 48 h.

500 µL of cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 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.

Blocking solution: 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.

Staining: with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies (diluted in FACS buffer) in the dark for 30 min.

PI staining: was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).

Expression level and cell viability: the expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of lambda = 488 nm and an emission wavelength of lambda = 530 nm for FITC and lambda > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 and cell viability was calculated.

Each test item was tested in two independent runs using separate cultivated cells at passage 9 (first experiment) and 12 (second experiment).

- Concentrations:
Dose finding assay 1 and 2: 1000, 500, 250, 125, 62.50, 31.25, 15.63 and 7.81 µg/mL
Main expt. 1 and 2: 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

- Controls:
Negative control (NC): Culture medium
Positive control (PC): 2,4-dinitrochlorobenzene (DNCB, CAS no.: 97-00-7), 4.0 µg/mL in 0.2% DMSO in culture medium
Vehicle control (VC): 0.2% DMSO in culture medium
Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1.
No. of animals per dose:
n/a
Details on study design:
n/a
Challenge controls:
n/a
Concentration:
n/a
No. of animals per dose:
n/a
Details on study design:
n/a
Statistics:
n/a
Positive control results:
Refer to "Any other information on result incl. tables"
Run / experiment:
other: 1 and 2
Parameter:
other: RFI CD54 [%]
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1 and 2
Parameter:
other: RFI CD 86 [%]
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The controls confirmed the validity of the study.
The viability of the solvent control was >90% (96.7% - 96.8%).
The number tested test item concentrations with cell viability >50% was >=4 (8).
The RFI for CD86 and CD54 of cells treated with the solvent DMSO was <=150% (105% experiment 1; 116% experiment 2) and <=200% (137% experiment 1; 156% experiment 2).
The MFI ratio of the medium control and isotype IgG1control was >=105% for CD86 (171% experiment 1; 158% experiment 2) and CD54 (121% experiment 1; 111% experiment 2).
The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was >=105% for CD86 (173% experiment 1; 162% experiment 2) and CD54 (128% experiment 1; 115% experiment 2).
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (615% experiment 1; 508% experiment 2) and 200% for CD54 (519% experiment 1; 811% experiment 2) were clearly exceeded.

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 96.9% (CD86), 97.2% (CD54) and 97.3% (isotype IgG1 control) in the first experiment and to 95.5% (CD86), 95.6% (CD54) and 95.2% (isotype IgG1 control) in the second experiment.

Table 1: Results of the Cell Batch Activation Test

Sample

Conc. [µg/mL]

CD86

CD54

Activated

Cell Viability [%]

RFI

Cell Viability [%]

RFI

yes/no

DNCB

4 µg/mL

88.1

406

88.4

243

Yes

NiSO4

100 µg/mL

88.4

324

87.9

336

Yes

LA

1000 µg/mL

96.7

69

96.7

77

No

 

Table 2: Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Conc.

[µg/ml]

Cell

Viability [%]

Concentration applied

[µg/ml]

Cell

Viability [%]

Medium Control

0.00

97.70

0.00

98.00

Solvent Control

(0.2% DMSO (v/v))

0.00

97.00

0.00

97.70

Scopolamine

7.81

97.40

7.81

98.10

15.63

97.30

15.63

98.00

31.25

97.60

31.25

98.20

62.50

97.50

62.50

98.10

125.00

97.50

125.00

97.90

250.00

96.40

250.00

98.30

500.00

97.00

500.00

97.90

1000.00

97.10

1000.00

97.60

Calculated CV75

[µg/mL]

No CV75

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

No SD

 

Table 3: 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

-

98.0

97.9

97.7

1134

805

663

471

142

95

73

171

121

DMSO Control

0.20%

97.9

98.1

97.8

1179

877

683

496

194

100

100

173

128

DNCB

4.00

81.1

81.8

82.3

3968

1926

919

3049

1007

615

519

432

210

(-)-Scopolamine

1000

96.9

97.2

97.3

1190

922

794

396

128

80

66

150

116

833.33

97.0

97.0

97.5

1189

933

810

379

123

76

63

147

115

694.44

97.5

97.4

97.3

1322

932

823

499

109

101

56

161

113

578.70

97.3

96.8

97.2

1312

950

826

486

124

98

64

159

115

482.25

97.4

97.3

97.6

1133

870

774

359

96

72

49

146

112

401.88

97.5

97.6

97.2

1141

936

803

338

133

68

69

142

117

334.90

97.6

97.6

97.8

1208

917

812

396

105

80

54

149

113

279.08

97.5

97.4

97.4

1284

984

860

424

124

85

64

149

114

Table 4: 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

CD86

CD54

Medium Control

-

96.8

97.0

97.1

1270

892

806

464

86

86

64

158

111

DMSO Control

0.20%

96.7

96.8

96.8

1408

1002

868

540

134

100

100

162

115

DNCB

4.0

85.3

85.7

85.8

3582

1924

837

2745

1087

508

811

428

230

(-)-Scopolamine

1000.00

95.5

95.6

95.2

1211

1001

771

440

230

81

172

157

130

833.33

96.0

96.2

95.5

1319

1022

787

532

235

99

175

168

130

694.44

95.8

95.6

95.9

1523

1001

750

773

251

143

187

203

133

578.70

95.8

96.3

96.6

1318

942

740

578

202

107

151

178

127

482.25

96.5

96.5

96.7

1344

990

796

548

194

101

145

169

124

401.88

96.2

95.7

96.2

1536

1068

805

731

263

135

196

191

133

334.90

96.2

96.1

95.8

1476

1006

772

704

234

130

175

191

130

279.08

96.2

96.9

96.7

1444

946

713

731

233

135

174

203

133

 

Table 5: Acceptance Criteria

Acceptance Criteria

range

Expt. 1

pass/fail

Expt.2

pass/fail

cell viability solvent controls

[%]

>90

97.7 - 98.1

pass

96.7 - 97.1

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

615

pass

508

pass

RFI of positive control of

CD54

>200

519

pass

811

pass

RFI of solvent control of CD86

<150

105

pass

116

pass

RFI of solvent control of CD54

<200

137

pass

156

pass

MFI ratio IgG1/CD86 for

medium control [%]

>105

171

pass

158

pass

MFI ratio IgG1/CD86 for

DMSO control [%]

>105

173

pass

162

pass

MFI ratio IgG1/CD54 for

medium control [%]

>105

121

pass

111

pass

MFI ratio IgG1/CD54 for

DMSO control [%]

>105

128

pass

115

pass

 

Interpretation of results:
other: Weight of Evidence 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 hCLAT experimental runs when tested up to the maximum recommended concentration in accordance with current guideline requirements (1000 mcg/mL, in DMSO). Therefore the test item is considered not to no a skin sensitiser in this test system.
Executive summary:

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 sensitiser and non-sensitisers.

 

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

 

In the present study (-)-Scopolamine was dissolved in DMSO. For the dose finding assay stock solutions with a concentration of 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. No CV75 could be derived in the dose finding assay.

 

Based on this, 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

 

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 96.9% (CD86), 97.2% (CD54) and 97.3% (isotype IgG1 control) in the first experiment and to 95.5% (CD86), 95.6% (CD54) and 95.2% (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 (615% experiment 1; 508% experiment 2) and 200% for CD54 (519% experiment 1; 811% experiment 2) were clearly exceeded.

 

The controls confirmed the validity of the study. The viability of the solvent control was >90% (96.7% - 96.8%). The number tested test item concentrations with cell viability >50% was =4 (8). The RFI for CD86 and CD54 of cells treated with the solvent DMSO was =150% (105% experiment 1; 116% experiment 2) and =200% (137% experiment 1; 156% experiment 2). The mean (geometric) fluorescence intensity (MFI) ratio of the medium control and isotype IgG1 control was =105% for CD86 (171% experiment 1; 158% experiment 2) and CD54 (121% experiment 1; 111% experiment 2). The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was =105% for CD86 (173% experiment 1; 162% experiment 2) and CD54 (128% experiment 1; 115% experiment 2).

 

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 hCLAT experimental runs when tested up to the maximum recommended concentration in accordance with current guideline requirements (1000 mcg/mL, in DMSO). Therefore the test item is considered not to no a skin sensitiser in this test system.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-12-12 to 2017-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
n/a
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Principles of method if other than guideline:
n/a
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
n/a
Details on the study design:
TEST SYSTEM
- Cell line: transgenic cell line KeratinoSens™ (Givaudan, Switzerland) derived from human keratinocytes (HaCaT)

TEST-SUBSTANCE PREPARATION
- Concentrations: 2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
- Stock: 4x concentration of the highest concentration (diluted 1:4 when incubated with the cells)
- Vehicle: DMSO (1% (v/v)

CONTROLS
- Positive control (PC): Cinnamic in 1% DMSO: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Vehicle control (VC): DMSO: 1% (v/v) in test item exposure medium
- Blank control: Culture medium without cells

MEDIUM
- Maintenance Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885- 025) with 1.0 g/L D-glucose and Na-Pyruvate + 10% fetal bovine calf serum + 1% geneticin (500 µg/mL)
- Assay Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025) with 1.0 g/L D-glucose and Na-Pyruvate + 10% fetal bovine calf serum
- Test Item Exposure Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885- 025) with 1.0 g/L D-glucose and Na-Pyruvate + 1% fetal bovine calf serum

EXPERIMENTAL PROCEDURE
Luciferase assay:
in Test Item Exposure Medium
- Replicates: 3
- Experiments: 2 independent run
- Exposure period: 48 hours
- Exposure temp.: 37°C (and 5% CO2)
- Washing cells: DPBS
- Incubation: 2 hours at room temperature in the absence of light
- Plate reader for luminescence measurement: Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms.
MTT assay:
in 200 µL Test Item Exposure Medium, 27 µL MTT solution (5 mg/mL MTT in DPBS), incubation 4h at 37°C and 5% CO2. Afterwards medium was removed and replaced by 200 µL 10% SDS solution (sodium dodecyl sulfate in dist. water), incubation overnight at 37°C and 5% CO2. After incubation, plate was shaken for 10 min and the OD was measured at ¿ = 600 nm.

All plates were sealed during incubation using a sealing tape.

ANALYSIS
- Calculation of Cell Viability
- Calculation of the Maximal Induction of the Luciferase Activity (Imax)
- Calculation of the EC1.5
- Calculation of IC50 and IC30

ACCEPTANCE CRITERIA
- 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.

EVALUATION CRITERIA
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
Positive control results:
Refer to "Any other information on result incl. tables"
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
2.6
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
at 2000 µM
Run / experiment:
other: 1
Parameter:
other: Cell viability (%)
Value:
101.8
Vehicle controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: EC1.5 (µM)
Value:
1 209.6
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Only at the highest test item concentration a significant luciferase induction >1.5 was
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
3.09
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
at 2000 µM
Run / experiment:
other: 2
Parameter:
other: Cell viability (%)
Value:
135.9
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: EC1.5 (µM)
Value:
1 070.54
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Only at the highest test item concentration a significant luciferase induction >1.5 was
Other effects / acceptance of results:
The controls confirmed the validity of the study. The luciferase activity induced by the positive controlat a concentration of 64 µMwas between 2 and 8 (2.16 (experiment 1); 6.60 (experiment 2)). The calculated EC1.5was between 7 and 30 µM (22.70 (experiment 1); 7.58 (experiment 2)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (9.4% (experiment 1); 12.3% (experiment 2)).

Table 1: Results of the Cytotoxicity Measurement

 

Conc. [µM]

Cell Viability [%]

Expt. 1

Expt.2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

116.2

107.8

112.0

5.9

8.00

126.3

106.6

116.4

14.0

16.00

131.8

118.6

125.2

9.4

32.00

137.1

123.2

130.2

9.9

64.00

142.1

129.4

135.8

8.9

Test Item

0.98

96.2

103.2

99.7

4.9

1.95

95.7

110.3

103.0

10.3

3.91

90.4

117.8

104.1

19.4

7.81

101.2

116.1

108.7

10.5

15.63

100.2

116.8

108.5

11.7

31.25

93.4

118.8

106.1

18.0

62.50

101.6

122.5

112.0

14.8

125.00

99.4

122.8

111.1

16.5

250.00

104.5

121.4

113.0

12.0

500.00

101.2

119.4

110.3

12.9

1000.00

106.3

132.8

119.6

18.7

2000.00

101.8

135.9

118.8

24.1

Table 2: Induction of Luciferase Activity Experiment 1

Expt.1

Conc. [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

1.24

1.09

1.07

1.13

0.10

8.00

1.20

1.25

1.31

1.25

0.06

16.00

1.42

1.33

1.32

1.36

0.06

32.00

1.81

1.60

1.69

1.70

0.10

*

64.00

2.48

1.93

2.15

2.19

0.28

*

Test Item

0.98

0.75

0.85

1.10

0.90

0.18

1.95

0.73

0.77

0.78

0.76

0.03

3.91

0.79

0.80

0.89

0.83

0.05

7.81

0.59

0.74

0.79

0.71

0.11

15.63

0.71

0.76

0.94

0.80

0.12

31.25

0.81

0.83

0.92

0.85

0.06

62.50

1.03

0.79

0.97

0.93

0.12

125.00

0.83

0.80

1.07

0.90

0.14

250.00

0.85

0.95

1.27

1.02

0.22

500.00

0.86

0.97

1.09

0.97

0.11

1000.00

1.09

1.09

1.45

1.21

0.21

2000.00

2.09

2.35

3.35

2.60

0.67

*

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

 

Table 3: Induction of Luciferase Activity Experiment 2

Expt. 2

Conc. [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

1.09

1.11

1.26

1.15

0.10

 

8.00

1.28

1.39

1.95

1.54

0.36

 

16.00

1.52

1.57

2.68

1.92

0.65

 

32.00

2.29

2.16

3.78

2.74

0.90

*

64.00

3.45

4.76

11.59

6.60

4.37

*

Test Item

0.98

1.08

1.19

1.01

1.09

0.09

 

1.95

0.75

0.91

0.59

0.75

0.16

 

3.91

0.82

1.07

1.18

1.02

0.19

 

7.81

0.79

0.85

0.62

0.75

0.12

 

15.63

0.93

0.90

1.78

1.20

0.50

 

31.25

0.74

1.00

0.89

0.88

0.13

 

62.50

1.09

0.88

0.94

0.97

0.11

 

125.00

1.26

1.03

1.28

1.19

0.14

 

250.00

0.93

1.10

1.21

1.08

0.15

 

500.00

1.22

1.08

1.26

1.18

0.09

 

1000.00

1.05

1.44

1.65

1.38

0.30

 

2000.00

2.12

2.44

4.70

3.09

1.40

*

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

Interpretation of results:
other: Weight of Evidence 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 ARE-Nrf2 Luciferase experimental runs when tested up to the maximum recommended concentration in accordance with current guideline requirements (2000 uM, in DMSO). Therefore the test item is considered not to no a skin sensitiser in this test system.
Executive summary:

The in vitro ARE (antioxidative responsive element)-Nrf2 (nuclear factor (erythroid-derived 2)-like 2)Luciferase test method (KeratinoSens™) 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.

 

In the present study (-)-Scopolamine was dissolved in DMSO.

 

Based on a molecular weight of 303.35 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 :0.98 - 2000 µM (the maximum recommended concentration in accordance with current guideline requirements).

 

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 2.60 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 101.8%. Only at the highest test item concentration a significant luciferase induction >1.5 was found. However, the calculated EC1.5 was > 1000 µM (1209.60 µM).

 

In the second experiment, a max luciferase activity (Imax) induction of 3.09 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 135.9%. Only at the highest test item concentration a significant luciferase induction >1.5 was found. However, the calculated EC1.5 was > 1000 µM (1070.54 µM).

 

No clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

 

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 ARE-Nrf2 Luciferase experimental runs when tested up to the maximum recommended concentration in accordance with current guideline requirements (2000 uM, in DMSO). Therefore the test item is considered not to no a skin sensitiser in this test system.

Endpoint:
skin sensitisation, other
Remarks:
QSAR analysis
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. OECD Toolbox

2. OECD Toolbox (v. 3.4.0.17)

3. Input into model via CAS number / SMILES

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Workflow:
Profiling the structure of scopolamine from the chemical databases in the Toolbox produced positive results for:
- Aquatic toxicity classification by ECOSAR (primary grouping)
The following categories were therefore formed using this information

Mechanistic:
- DPRA cysteine peptide depletion
- DPRA lysine peptide depletion
- Protein binding by OASIS v1.4
- Protein binding by OECD

Sub-categorization
- Chemical elements (subcategorization)
- Lipinski Rule Oasis

Organic function group similarity and protein binding categories were then combined to give a category that was both structurally and mechanistically similar so as to increase the robustness of the estimations, resulting in the group of 28 analogues.


5. APPLICABILITY DOMAIN
The target chemical, scopolamine met the applicability domain used to provide a read-across estimation

The applicability domain is defined by following scheme
a) Referential boundary:
The target chemical should be classified as SN2 AND SN2 >> Ring opening SN2 reaction AND SN2 >> Ring opening SN2 reaction >> Epoxides, Aziridines and Sulfuranes by Protein binding by OASIS v1.4

b) Referential boundary:
The target chemical should be classified as Low reactive AND Low reactive >> Epoxides by DPRA Lysine peptide depletion
c) Referential boundary:
The target chemical should be classified as Group 14 - Carbon C AND Group 15 - Nitrogen N AND Group 16 - Oxygen O by Chemical elements

d) Referential boundary:
The target chemical should be classified as Group 14 - Metalloids Si,Ge OR Group 17 - Halogens Cl OR Group 17 - Halogens F,Cl,Br,I,At by Chemical elements
e) Referential boundary:
The target chemical should be classified as Bioavailable by Lipinski Rule Oasis ONLY

f) Parametric boundary:
The target chemical should have a value of log Kow which is >= -0.151

g) Parametric boundary:
The target chemical should have a value of log Kow which is <= 0.861

6. ADEQUACY OF THE RESULT
The predicition is based on 12 neighbours' values, 11 of them equal to prediction. Prediction confidence is measured by the p value = 0.00317
Qualifier:
no guideline available
Principles of method if other than guideline:
QSAR analysis
GLP compliance:
no
Type of study:
other: in silico QSAR analysis
Specific details on test material used for the study:
Refer to report 0275962-TOX2 attached
Details on the study design:
Refer to report 0275962-TOX2 attached
Details on the study design:
Refer to report 0275962-TOX2 attached
Details on test animals and environmental conditions:
n/a
No. of animals per dose:
n/a
Details on study design:
n/a
Challenge controls:
n/a
Concentration:
n/a
No. of animals per dose:
n/a
Details on study design:
n/a
Statistics:
n/a
Positive control results:
n/a
Key result
Run / experiment:
other: n/a
Parameter:
other: QSAR
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
not measured/tested
Remarks:
the in silico estimates from the OECD Toolbox indicates that scopolamine is predicted to be a skin sensitizer based on protein binding domain
Other effects / acceptance of results:
n/a
Cellular proliferation data / Observations:
n/a

n/a

Interpretation of results:
other: Weight of Evidence approach
Conclusions:
In conclusion, the in silico estimates from the OECD Toolbox indicates that scopolamine is predicted to be a skin sensitizer based on protein binding, deemed to be an applicable domain.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The available human reports containing data on humans are inconclusive and without suitable experimental detail to make a decision. Thus, the available human data are insufficient to determine the CLP dermal sensitization classification for scopolamine. 

For determination regarding the dermal sensitization potential of scopolamine,in chemico and in vitro tests were conducted (OECD 422C, OEC 422D and OECD 422E). Each of these three in vitro tests are considered to be reliable and showed no evidence of skin sensitization. According to CLP the in vitro / in chemico tests are not regarded as stand alone tests and the result from such a test should be used together with other data in an overall weight of evidence assessment.

The in silico estimates from the OECD Toolbox indicates that scopolamine is predicted to be a skin sensitizer based on protein binding, deemed to be an applicable domain.

There are no animal data available to make decisions about classification. Scopolamine is acutely toxic by the dermal route (H310) and dermal application for sensitization testing using in vivo models is not scientifically justified. It is unlikely that dermal exposures sufficient to elicit an immune response could be achieved before acute toxic responses, including lethality.

Based on a weight of evidence approach using all available data (human data, in chemico / in vitro data, in sillico / QSAR), scopolamine is classified for skin sensitization Cat. 1 H317 (May cause an allergic reaction to skin) following a conservative approach.

 

 

Justification for classification or non-classification

Based on a weight of evidence approach, scopolamine is classified for skin sensitization Cat. 1 H317 (May cause an allergic reaction to skin). The available data are insufficient to allow sub-categorisation to Category 1A or 1B.