Dihydronicotinamide-adenine dinucleotide, disodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 October - 7 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Test material

Test material information

Constituent 1

Reference substance name:

Dihydronicotinamide-adenine dinucleotide, disodium salt

EC Number:

210-123-3

EC Name:

Dihydronicotinamide-adenine dinucleotide, disodium salt

Cas Number:

606-68-8

Molecular formula:

C21H27N7O14P2.2Na

IUPAC Name:

disodium [(2R,3S,4R,5R)-5-(3-carbamoyl-1,4-dihydropyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl ({[(2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphonato}oxy)phosphonate

impurity 1

Reference substance name:

Dihydronicotinamide-adenine dinucleotide

EC Number:

200-393-0

EC Name:

Dihydronicotinamide-adenine dinucleotide

Cas Number:

58-68-4

Molecular formula:

C21H29N7O14P2

IUPAC Name:

[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoyl-4H-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid: crystalline

Remarks:

crystaline powder

Details on test material:

Batch No.: 18243100
Storage conditions: Store at -15 to -25°C,
Handling: Keep frozen on dry ice.

In vitro test system

Details on the study design:

EXPERIMENTAL PROCEDURES

PEPTIDES:
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

BUFFERS USED:
- Phosphate buffer: ca 100 mM, pH 7.5.
- Ammonium acetate buffer: ca 100 mM, pH 10.2.

PREPARATION PEPTIDE STOCK SOLUTIONS:
- CYSTEINE: 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
- LYSINE: 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

REFERENCE CONTROL SOLUTIONS
- CYSTEINE: 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 RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MQ.
- LYSINE: 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 RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MQ.

SAMPLE INCUBATION;
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. 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)
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
-The standard calibration curve had to have an r2>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 item’s results to be considered valid:
-The maximum SD for the test item 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)))]×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%
ANALYSIS-DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Cysteine 1:10 / Lysine 1:50 Prediction Model

0% = Mean % depletion = 6.38% No or minimal reactivity Negative
6.38% < Mean % depletion = 22.62% Low reactivity Positive
22.62% < Mean % depletion = 42.47% Moderate reactivity Positive
42.47% < Mean % depletion = 100% High reactivity Positive

Results and discussion

Positive control results:

The mean depletion value for the positive control was 69.4% showing a high reactivity (Sensitizer)
The mean Percent SPCCysteine Depletion for the positive control cinnamic aldehyde was 69.4% ± 0.7%.
The mean Percent SPCLysine Depletion for the positive control cinnamic aldehyde was 54.8% ± 1.7%.

In vitro / in chemico

Results

Key result

Run / experiment:

other: DPRA cysteine and lysine prediction model

Parameter:

other: %peptide depletion (mean value)

Value:

9.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

other:

Remarks:

Low reactivity class

Other effects / acceptance of results:

No co-elution of the test item with either peptide was observed.

SYSTEM SUITABILITY FOR THE CYSTEINE ASSAY
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.996. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.500±0.003 mM, the mean peptide concentration of Reference Controls C was 0.502±0.005 mM and the mean peptide concentration of Reference Controls CMQ was 0.506±0.007 mM. The means of Reference Control samples A, and C and CMQ 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 item 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 2.1%. 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 18.97. The mean A220/A258 ratio ± 10% range was 17.07-20.86. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
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 69.4% ± 0.7%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

SYSTEM SUITABILITY FOR THE LYSINE ASSAY
The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9993. Since the r2 was >0.99, the SPCL standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.461±0.008 mM, the mean peptide concentration of Reference Controls C was 0.472±0.011 mM and the mean peptide concentration of Reference Controls CMQ was 0.456±0.016 mM. The means of Reference Control samples A, and C and CMQ 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 item did not impact the Percent SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 1.4%. 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 16.43. The mean A220/A258 ratio ± 10% range was 14.78-18.07. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
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 54.8% ± 1.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Any other information on results incl. tables

Test item	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
ß-NADH, disodium salt	15.7%	±2.7%	3.5%	±3.3%	9.6%	Positive: Low reactivity

Using the cysteine and lysine prediction model (see Table below) the test material was categorised as low reactive and a sensitiser.

Mean depletion values (Cys Lys)	Mean Depletion values (cys only)	Reactivity classification	DPRA Prediction
<6.38 %	<13.89%	Minimal	Non Sensitizer
6.38 -22.62%	13.89 -23.09%	Low	Sensitizer
22.62 -42.47%	23.09%-98.24%	Moderate	Sensitizer
>42.47	>98.24%	High	Sensitizer

Applicant's summary and conclusion

Interpretation of results:

other: Low reactivity: sensitizer

Remarks:

Study will be used for classificatin in combination with other studies (Weight of Evidence)

Conclusions:

In conclusion, this DPRA test is valid. beta-NADH, disodium salt (CAS Nr. 606-68-8) 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 objective of this study was to determine the reactivity of beta-NADH, disodium salt towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item 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 item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guideline 442C (04 February 2015).

Milli-Q MQ (MQ) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of theDirect 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	0.996	>0.99	0.9993
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.500 ± 0.003	0.50 ± 0.05	0.461 ± 0.008
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.502 ± 0.005	0.50 ± 0.05	0.472 ± 0.011
Mean peptide concentration RC-CMQsamples (mM)	0.50 ± 0.05	0.506 ± 0.007	0.50 ± 0.05	0.456 ± 0.016
CV (%) for RC samples B and C	<15.0	2.1	<15.0	1.4
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	69.4	40.2-69.0	54.8
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.7	<11.6	1.7
SD of peptide depletion for  the test item (%)	<14.9	2.7	<11.6	3.3

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

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and C_MQ, 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 item, were all within the acceptability criteria for the DPRA.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 15.7% SPCC depletion while in the lysine reactivity assay the test item showed 3.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 9.6% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forß-NADH, disodium salt

Test item	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
ß-NADH, disodium salt	15.7%	±2.7%	3.5%	±3.3%	9.6%	Positive: Low reactivity

SD = Standard Deviation.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. beta-NADH, disodium salt (CAS Nr. 606-68-8) was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.