Dihydronicotinamide-adenine dinucleotide, disodium salt

Endpoint summary

Description of key information

-       QSAR DEREK: Alert for sensitization (equivocal) but after analysis it is concluded that substance should not be classified for Skin Sensitization

-       DPRA (OECD 442C): % peptide depletion: 9.6%, low reactivity, Sensitizer

-       Keratinosens^TM(OECD 442D): Negative, Non sensitizer

No cytotoxicity observed. Imax: 1.30 and 1.4 fold.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

28 September 2017

Reliability:

2 (reliable with restrictions)

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. SOFTWARE: DEREX NEXUS
2. MODEL (incl. version number): DEREK NEXUS 5.0.2.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: N1(C2=C(N=C1)C(=NC=N2)N)[C@@H]3O[C@@H]([C@@H]([C@H]3O) O)COP(OP(OC[C@H]4O[C@H]([C@H]([C@@H]4O)O)N5C=C(CC=C5)C( N)=O)(=O)[O-])(=O)[O-].[Na+].[Na+]
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached
5. APPLICABILITY DOMAIN (OECD principle 3)

Domains:
- Descriptor domain:The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule in Derek, a result of ‘no alerts fired’ is presented to the user. This can be interpreted as a negative prediction or that the query compound is outside the domain of the model. Which of these is more appropriate may depend on the endpoint of interest. For the endpoint of skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘no alerts fired’ may be extrapolated to a negative prediction

- Structural fragment domain: DEREK NEXUS is applicable to organic structures including their salts, e.g. sodium, potassium. For skin sensitisation, which features 80 alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ’no alerts fired’ may be extrapolated to a negative prediction. The beta-NADH, disodium salt (CAS No. 606-68-8) contains an alpha, beta-unsaturated amide which is substituted in the alpha position with a carbon and in the beta position with a nitrogen and, therefore, falls in the applicability domain of the alpha, beta unsaturated amides.

- Mechanism domain: The chemical may act as a hapten acting as an electrophilic Michael acceptor.

- Metabolic domain: no evident metabolism that might lead to skin sensitization is predicted for this structure

The uncertainty of the prediction (OECD principle 4):
DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 77%.This specific alert for alpha, beta-unsaturated amides was considered to be equivocal for this substance, which indicates that an equal weight of evidence against the proposition as for it is available.

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5).
The alert describes the skin sensitisation of alpha, beta-unsaturated amides and precursors which interact with skin proteins via a Michael addition mechanism.

6. ADEQUACY OF THE RESULT
-Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Guideline:

other: REACH Guidance on QSARs R.6

Version / remarks:

Prediction on the potential for skin sensitization with the in silico model DEREK NEXUS, version 5.0.2.

Principles of method if other than guideline:

- Software tool(s) used including version: in silico model DEREK NEXUS version 5.0.2
- Knowledge Base: Derek KB 2015 2.0
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

Specific details on test material used for the study:

SMILES:N1(C2=C(N=C1)C(=NC=N2)N)[C@@H]3O[C@@H]([C@@H]([C@H]3O) O)COP(OP(OC[C@H]4O[C@H]([C@H]([C@@H]4O)O)N5C=C(CC=C5)C( N)=O)(=O)[O-])(=O)[O-].[Na+].[Na+]

Key result

Parameter:

other: alerts for skin sensitization

Remarks on result:

other: DEREK NEXUS version 5.0.2 yielded an alert for beta-NADH, disodium salt for skin sensitization based on the presence of an alpha,beta-unsaturated amide. beta-NADH, disodium salt (CAS No. 606-68-8) is predicted to be sensitizing to the skin (equivocal).

The alert was triggered from the following structural analogue: Acrylamide, CAS number 79-06-1, weak sensitizer in the guinea pig maximisation test.

The alert describes the skin sensitisation of alpha,beta-unsaturated amides and precursors which interact with skin proteins via a Michael addition mechanism.

Considerations on structural analogues:

Acrylamide has no substituents on the double bond, while the current substance has a substituent on the alpha and beta position, which may lead to steric hindrance and therefore acrylamide may be seen as a worst case.

Outcome: Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

Conclusion: The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Interpretation of results:

other: alert Sensitizer (equivocal) but the analysis concludes substance should not be classified for Skin Sensitization

Remarks:

QSAR will be used for classification in combination with other studies (Weight of Evidence)

Conclusions:

DEREK NEXUS version 5.0.2 yielded an alert for beta-NADH, disodium salt for skin sensitization based on the presence of an alpha,beta-unsaturated amide. beta-NADH, disodium salt (CAS No. 606-68-8) is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS could not perform an EC3 prediction due to lack of sufficient data on closest structurally-related substances.
Outcome: Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of beta-NADH, disodium salt (CAS No. 606-68-8) with the in-silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts: The DEREK NEXUS system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by DEREK NEXUS are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. “No alerts fired” may be extrapolated to a negative prediction.

DEREK NEXUS version 5.0.2 yielded an alert for beta-NADH, disodium salt for skin sensitization based on the presence of an alpha,beta-unsaturated amide. beta-NADH, disodium salt (CAS No. 606-68-8) is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS could not perform an EC3 prediction due to lack of sufficient data on closest structurally-related substances.

The alert describes the skin sensitisation of alpha,beta-unsaturated amides and precursors which interact with skin proteins via a Michael addition mechanism.

This specific alert for alpha, beta-unsaturated amides was considered to be equivocal for this substance, which indicates that an equal weight of evidence against the proposition as for it is available.

Considerations on structural analogues: Acrylamide has no substituents on the double bond, while the current substance has a substituent on the alpha and beta position, which may lead to steric hindrance and therefore acrylamide may be seen as a worst case.

Outcome: Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

The result is adequate to be used in a weight-of-evidence approach together within chemico/in vitro studies to complete the endpoint skin sensitization.

Reference 2

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

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)

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

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%.

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%).

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

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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 October - 3 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Test concentrations: final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.987 µM (final concentration vehicle DMSO of 1%)
- All concentrations of the test item were tested in triplicate.
- Positive control: Ethylene dimethacrylate glycol, final concentration 7.8 to 250 µM (final concentration DMSO of 1%)
- Negative control: vehicle: 1% DMSO in exposure medium

- Test System A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).

- Cell culture:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Manteinance Medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

- Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 28 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 46.6°C).

EXPERIMENTAL DESIGN
- Plating of cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was p+15 in experiment 1 and p+3 in experiment 2.
- Treatment of cells
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 experiments were performed.

- Luciferase activity measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Positive control results:

• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.08 and the EC1.5 99 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.00 and the EC1.5 96 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

1.3

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.08

Key result

Run / experiment:

other: 2

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

1.24

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.00

Key result

Run / experiment:

other: 1

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 99 µM.

Remarks on result:

other: no EC1.5 could be calculated

Key result

Run / experiment:

other: 2

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 96 µM

Remarks on result:

other: no EC1.5 could be calculated

Other effects / acceptance of results:

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (99 µM and 96 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was 2.08-fold and 2.00-fold in experiment 1 and 2, respectively.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (4.4% and 9.3% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Table1          
Overview Luminescence Induction and Cell Viability of ß-NADH, disodium salt in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	0.89	1.01	1.04	1.04	1.10	1.17	1.11	1.16	1.28	1.17	1.30	1.17
Exp 1 viability (%)	108.4	96.4	101.7	103.7	99.1	99.1	96.8	98.1	92.3	97.0	91.7	94.7
Exp 2 luminescence	0.83	0.63	0.75	0.80	1.04	1.08	1.12	1.15	1.16	0.80	1.11	1.24
Exp 2 viability (%)	107.0	156.3	126.7	122.1	101.9	96.7	95.6	94.9	94.5	108.2	89.7	98.7

 

Table2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.04	1.07	1.19	1.36	1.60^***	2.08^***
Exp 1 viability (%)	101.4	95.7	107.9	108.0	114.2	117.3
Exp 2 luminescence	0.88	0.94	1.03	1.21	1.75^***	2.00^***
Exp 2 viability (%)	108.5	104.1	113.9	127.2	116.4	133.0

^***p<0.001 Student’s t test

 

Table3          
Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.30	NA	NA
Test item Experiment 2	NA	1.24	NA	NA
Pos Control Experiment 1	99	2.08	NA	NA
Pos Control Experiment 2	96	2.00	NA	NA

NA = Not applicable

Interpretation of results:

other: Test item did not induce activation of the ARE-dependant pathway in keratinocytes

Remarks:

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

Conclusions:

In conclusion, ß-NADH, disodium salt (CAS No. 606-68-8) is classified as negative (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of ß-NADH, disodium salt (CAS No. 606-68-8) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 18243100 of ß-NADH, disodium salt was a light yellow crystalline powder.  The test item was suspended in dimethyl sulfoxide (DMSO) at 200 mM.  From this stock 11 spike solutions in DMSO were prepared.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series).  The highest test concentration was the highest dose required in the current guideline.  No precipitate was observed at any dose level tested.  Two independent experiments were performed.

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.  

- The EC1.5 of the positive control was between 5 and 125 µM (99 µM and 96 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was 2.08-fold and 2.00-fold in experiment 1 and 2, respectively.

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (4.4% and 9.3% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

ß-NADH, disodium salt showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 1.30-fold and 1.24-fold in experiment 1 and 2 respectively.  

ß-NADH, disodium salt is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations >=1000 µM.

In conclusion, ß-NADH, disodium salt (CAS No. 606-68-8) is classified as negative (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 5.0.2 yielded an alert for beta-NADH, disodium salt for skin sensitization based on the presence of an alpha,beta-unsaturated amide the substance beta-NADH, disodium salt (CAS No. 606-68-8) is predicted to be sensitizing to the skin (equivocal). DEREK NEXUS could not perform an EC3 prediction due to lack of sufficient data on closest structurally-related substances. However, after analysis it is concluded that substance should not be classified for Skin Sensitization.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in water at 100 mM. There was no evidence of co-elution of beta-NADH, disodium salt with either cysteine or lysine peptide. Peptide depletion was calculated as 15.7% +/- 2.7% and 3.5% +/- 3.3% in cysteine and lysine assays, respectively, resulting in a mean peptide depletion of 9.6%. This value places beta-NADH, disodium salt in the Low Reactivity Classification resulting in a DPRA prediction of "sensitiser" when using the Cysteine 1:10/Lysine 1:50 prediction model.

A valid Keratinosens^TMassay was performed according to OECD 442D and GLP principles. The test item was suspended in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The test item showed no precipitation at any of the test concentrations. Two independent experiments were performed. The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.30-fold and 1.24-fold in experiment 1 and 2, respectively beta-NADH, disodium salt is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations of =1000 µM.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

DEREK predicts beta-NADH, disodium salt to be a weak skin sensitizer with equivocal probability, which means that there is an equal weight of evidence for and against the proposition. The DEREK prediction is based on the fact that acrylamide is a weak skin sensitizer, but the two analogues included in the database with the amide substituted with a tertiary carbon on the N are both not sensitizers. The structure of all three analogues is quite dissimilar to that of beta-NADH, disodium salt. After analysis it is concluded that substance should not be classified for Skin Sensitization.

The DPRA showed a very low reactivity mean of cysteine and lysine depletion of 9.6% which is just above the threshold of 6.38%. Taking into consideration the rather high standard deviation for both cysteine and lysine depletion, the reliability of this result may be doubted.

The most logical mechanism for reaction with proteins is Michael addition via the alpha,beta-unsaturated amide. More substituents on the alkene and substituents like nitrogen will result in deactivation of the alpha,beta-unsaturated amide towards nucleophilic attack by increasing the electron density at the electrophilic centre. This deactivation of the amide will remove the Michael acceptor moiety and as a result significant rates of Michael addition reactions with proteins in the skin will not be reached.

Based on this mechanism which is considered to be relevant for beta-NADH, disodium salt, the DEREK prediction based on the presence of the alpha,beta-unsaturated amide, is considered to be not relevant and the DPRA test a false positive.

The KeratinoSens^TMassay was negative, which means that even if the substance might bind to a protein, the following step, induction ofgene expression associated with specific cell signaling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways, will not occur.

Moreover, beta-NADH, disodium salt is an endogenous substance involved in many processes in the eukaryotic cell, for example as part of the energy system, glycolysis and citric acid cycle of any cell, including those in the skin. When lesions of the skin occur, the skin is being exposed to cell contents, including beta-NADH, disodium salt and the substance is, consequently, very unlikely to be a skin sensitizer.

In conclusion, as the equivocal DEREK is considered not relevant (substance should not be classified for Skin Sensitization), the result of the DPRA a false positive and the KeratinoSensTM is negative, and taking also into consideration that NADH is an endogenic substance and therefore very unlikely to be a Sensitzer, it is concluded that beta-NADH, disodium salt (CAS No. 606-68-8) is Non sensitizing.