Hyoscine

Endpoint summary

Description of key information

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

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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 C_acetonitrile: 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 C_acetonitrile: 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.

Reference 2

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 of test system:

THP-1 cell line [442E]

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.

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.

Reference 3

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 of test system:

Keratinoses transgenic cell line [442D]

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: 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 µM

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: Cell viability (%)

Value:

135.9 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Parameter:

other: EC1.5 (µM)

Value:

1 070.54 µM

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:

First experiment: Imax 2.6 at 2000 µM
Second experiment: Imax 3.09 at 2000µM
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 (I_max) 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 EC_1.5 was > 1000 µM (1209.60 µM).

 

In the second experiment, a max luciferase activity (I_max) 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 EC_1.5 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.

Reference 4

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:

A review of the available human reports concludes that the substance can causes allergic contact dermatitis (type IV delayed hypersensitivity) in humans using well established clincal criteria.

 

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. 1A H317 (May cause an allergic reaction to skin).

 

 

 

Justification for classification or non-classification

Based on a weight of evidence approach, scopolamine is classified for skin sensitization Cat. 1A H317 (May cause an allergic reaction to skin).