Hyoscine

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Hyoscine

EC Number:

200-090-3

EC Name:

Hyoscine

Cas Number:

51-34-3

Molecular formula:

C17H21NO4

IUPAC Name:

(1R,2R,4S,5S,7S)-9-methyl-3-oxa-9-azatricyclo[3.3.1.0^{2,4}]nonan-7-yl (2S)-3-hydroxy-2-phenylpropanoate

Test material form:

solid: particulate/powder

Details on test material:

in vitro sens

In chemico test system

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

Results and discussion

Positive control results:

Refer to "Any other information on result incl. tables"

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

In vivo (LLNA)

Cellular proliferation data / Observations:

n/a

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.