Thiamine hydrochloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July 2017 to 07 July 2017

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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guidelines.

Test material

Test material information

Constituent 1

Reference substance name:

Thiamine hydrochloride

EC Number:

200-641-8

EC Name:

Thiamine hydrochloride

Cas Number:

67-03-8

Molecular formula:

C12H17N4OS.ClH.Cl

IUPAC Name:

thiamine hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder
- Test item storage: At room temperature protected from light and humidity

Specific details on test material used for the study:

- No correction for the purity/composition of the test material was performed.
- Solubility of the test material in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test material completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN) and Milli-Q water (MQ).
- Test material stock solutions were prepared freshly for each reactivity assay.
- For both the cysteine and lysine reactivity assay 55.14 mg of the test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1635 μL MQ (tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test material was dissolved. The test material, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Any residual volumes were discarded.

In chemico test system

Details on the study design:

TEST SYSTEM
- Test System: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Storage: The peptides were stored in the freezer (≤-15 °C) for a maximum of 6 months.

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN. In addition, a RCcysCwater sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RCcysCwater sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL MQ.

- SPCC Calibration Curve: A SPCC calibration curve was prepared:
STDcys1 solution (0.534 mM): 1600 μL stock solution of 0.667 mM SPCC + 400 μL CAN
STDcys2 solution (0.267 mM): 1 mL STDcys1 + 1 mL STDcys7
STDcys3 solution (0.133 mM): 1 mL STDcys2 + 1 mL STDcys7
STDcys4 solution (0.067 mM): 1 mL STDcys3 + 1 mL STDcys7
STDcys5 solution (0.033 mM): 1 mL STDcys4 + 1 mL STDcys7
STDcys6 solution (0.017 mM): 1 mL STDcys5 + 1 mL STDcys7
STDcys7 solution (0 mM): 8 mL phosphate buffer (pH 7.5) + 2 mL CAN

- Co-elution Control, Test Material and Positive Control Samples: The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared:
Co-elution control (CC) (1 replicate: 1 CCcys-208708/A): 750 μL Phosphate buffer pH 7.5, 200 μL ACN and 50 μL 208708/A test solution (100 mM)
Cinnamic aldehyde (PC) (3 replicates: PCcys-1 to PCcys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN and 50 μL Cinnamic aldehyde solution (100 mM in ACN)
Test material 208708/A (3 replicates: 208708/A-cys-1 to 208708/A-cys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN and 50 μL 208708/A test solution (100 mM).

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCwater sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RClysCwater sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL MQ.

- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared:
STDlys1 solution (0.534 mM): 1600 μL stock solution of 0.667 mM SPCL + 400 μL ACN
STDlys2 solution (0.267 mM): 1 mL STDlys1 + 1 mL STDlys7
STDlys3 solution (0.133 mM): 1 mL STDlys2 + 1 mL STDlys7
STDlys4 solution (0.067 mM): 1 mL STDlys3 + 1 mL STDlys7
STDlys5 solution (0.033 mM): 1 mL STDlys4 + 1 mL STDlys7
STDlys6 solution (0.017 mM): 1 mL STDlys5 + 1 mL STDlys7
STDlys7 solution (0 mM): 8 mL ammonium acetate buffer (pH 10.2) + 2 mL CAN

- Co-elution Control, Test Material and Positive Control Samples: The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared:
Co-elution control (CC) (1 replicate: CClys-208708/A): 750 μL Ammonium acetate buffer pH 10.2 and 250 μL 208708/A test solution (100 mM)
Cinnamic aldehyde (PC) (3 replicates: PClys-1 to PClys-3): 750 μL Stock solution of 0.667 mM SPCL and 250 μL Cinnamic aldehyde solution (100 mM in ACN)
Test material 208708/A: (3 replicates: 208708/A-lys-1 to 208708/A-lys-3): 750 μL Stock solution of 0.667 mM SPCL and 250 μL 208708/A test solution (100 mM)

SAMPLE INCUBATIONS
- After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test material samples) were placed in the autosampler in the dark and incubated at 25±2.5 °C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 24 hours and 23.5 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

HPLC-PDA ANALYSIS
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:

System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)

System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

- Mobile phase: A: 0.1 % (v/v) TFA in Milli-Q water, B: 0.085 % (v/v) TFA in CAN
- Gradient: 0 min 10 % B, 10 min 25 % B, 11 min 90 % B, 13 min 90 % B, 13.5 min 10 % B and 20 min 10 % B.
- Flow: 0.35 mL/min
- Injection volume: 4 μL
- Sample tray temperature: Set at 25 °C
- Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 μm (Agilent Technologies, Santa Clara, CA, USA)
- Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
- Column temperature: Set at 30 °C
- Detection: Photodiode array detection, monitoring at 220 and 258 nm

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r^2>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8 and 100 % for SPCC and between 40.2 and 69.0 % for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9 % for the Percent Cysteine Peptide Depletion and <11.6 % for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0 %.

The following criteria had to be met for a test material’s results to be considered valid:
- The maximum SD for the test material replicates had to be <14.9 % for the Percent Cysteine Depletion and <11.6 % for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

ANALYSIS
- Data Evaluation: The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [ 1 – (Peptide Peak Area in Replicate Injection (at 220 nm) / Mean Peptide Peak Area in Reference Controls (at 220 nm))] x 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90 %
- Data Interpretation: The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test material. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser.

Negative DPRA prediction:
0 % ≤ Mean % depletion ≤ 6.38 % (No or minimal reactivity)

Positive DPRA prediction:
6.38 % < Mean % depletion ≤ 22.62 % (Low reactivity)
22.62 % < Mean % depletion ≤ 42.47 % (Moderate reactivity)
42.47 % < Mean % depletion ≤ 100 % (High reactivity)

Results and discussion

Positive control results:

Cysteine Reactivity Assay: The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.9 ± 2.2 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD <14.9 %).

Lysine Reactivity Assay: The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 58.5 ± 0.9 %. This was within the acceptance range of 40.2 to 69.0 % with a SD that was below the maximum (SD <11.6 %).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: The mean Percent SPCC Depletion

Value:

7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: ± 5.4 %

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: The mean Percent SPCL Depletion

Value:

5.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: ± 2.7 %

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT OF THE TEST MATERIAL
- At a concentration of 100 mM, the test material was not soluble in ACN, but was soluble in MQ. Therefore this solvent was used to dissolve the test material in this DPRA study.


CYSTEINE REACTIVITY ASSAY

Acceptability:
- The correlation coefficient (r^2) of the SPCC standard calibration curve was 0.991. Since the r^2 was >0.99, the SPCC standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.480±0.005 mM, the mean peptide concentration of Reference Controls C was 0.490±0.008 mM and the mean peptide concentration of Reference Controls Cwater was 0.479±0.023 mM. The means of Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test material did not impact the Percent SPCC Depletion.
- The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 3.1 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 18.69. The mean A220/A258 ratio ± 10 % range was 16.82-20.55. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCC Depletion of the positive control cinnamic aldehyde was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.9 ± 2.2 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD <14.9 %).

Results for the Test Material:
- No precipitate was observed in any of the test material samples.
- In the CC sample no peak was observed at the retention time of SPCC, this demonstrated that there was no co-elution of the test material with SPCC. For the 208708/A-cys samples, the mean SPCC A220/A258 area ratio was 18.98. Since this was within the 16.82-20.55 range, this again indicated that there was no coelution of the test material with SPCC.
-The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls Cwater. The mean Percent SPCC Depletion for the test material was 7.0 ± 5.4 %.


LYSINE REACTIVITY ASSAY

Acceptability:
- The correlation coefficient (r^2) of the SPCL standard calibration curve was 0.997. Since the r^2 was >0.99, the SPCL standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.506±0.008 mM, the mean peptide concentration of Reference Controls C was 0.502±0.012 mM and the mean peptide concentration of Reference Controls Cwater was 0.494±0.005 mM. The means of Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test material did not impact the Percent SPCL Depletion.
- The CV of the peptide areas for the nine Reference Controls B and C was 2.0 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 14.95. The mean A220/A258 ratio ± 10% range was 13.46-16.45. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCL Depletion of the positive control cinnamic aldehyde was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 58.5 ± 0.9 %. This was within the acceptance range of 40.2 to 69.0 % with a SD that was below the maximum (SD <11.6 %).

Results for the Test Material:
- No precipitate was observed in any of the samples.
- In the CC sample no peak was observed at the retention time of SPCL, this demonstrated that there was no co-elution of the test material with SPCL. For the 208708/A-lys samples, the mean SPCL A220/A258 area ratio was 15.13. Since this was within the 13.46-16.45 range, this again indicated that there was no coelution of the test material with SPCL.
- The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls Cwater. The mean Percent SPCL Depletion for Thiamine hydrochloride was 5.9 ± 2.7%.

DPRA PREDICTION AND REACTIVITY CLASSIFICATION
- In the cysteine reactivity assay the test material showed 7.0 % SPCC depletion while in the lysine reactivity assay the test material showed 5.9 % SPCL depletion. The mean of the SPCC and SPCL depletion was 6.5 % and as a result the test material was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, the test material was considered to be positive in the DPRA.

Any other information on results incl. tables

Table 1: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for The Test Material

Treatment	SPCC Depletion		SPCL Depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test Material	7.0 %	± 5.4 %	5.9 %	± 2.7 %	6.5 %	Positive: Low reactivity

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.991	>0.99	0.997
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.480 ± 0.005	0.50 ± 0.05	0.506 ± 0.008
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.490 ± 0.008	0.50 ± 0.05	0.502 ± 0.012
Mean peptide concentration RC-Cwater samples (mM)	0.50 ± 0.05	0.479 ± 0.023	0.50 ± 0.05	0.494 ± 0.005
CV (%) for RC samples B and C	<15.0	3.1	<15.0	2.0
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	72.9	40.2-69.0	58.5
SD of peptide depletion cinnamic aldehyde (%)	<14.9	2.2	<11.6	0.9
SD of peptide depletion for the test material	<14.9	5.4	<11.6	2.7

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

other: Positive in the DPRA (Low reactivity class)

Conclusions:

Under the conditions of this study the test material was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test material and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test material, were all within the acceptability criteria for the DPRA.

In the cysteine reactivity assay the test material showed 7.0 % SPCC depletion while in the lysine reactivity assay the test material showed 5.9 % SPCL depletion. The mean of the SPCC and SPCL depletion was 6.5 % and as a result the test material was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Under the conditions of this study the test material was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.