Dihydronicotinamide-adenine dinucleotide, disodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 15 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Remarks:

EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiSkin Model has been validated for irritation testing in an international trial. The EpiSkin method is a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 17-EKIN-050
- Expiry date: 18 December 2017
- Date of initiation of testing: 13 December 2017

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE
- Test Item: The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
- Positive and negative control: A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
In addition to the normal procedure, 2 killed treated tissues and 2 killed negative control tissues were used for the MTT evaluation in one run.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Triplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1x solution, once. rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: none

POST-INCUBATION
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, >=95% humidified atmosphere.

MTT TEST
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION
A disk of epidermis was cut from the unit using a biopsy punch, the epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (0.04N HCl), mixed by using a vortex mixer and incubated for 4 hours at room temperature protected from light with gentle agitation (~150 rpm). At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIAVILITY MEASUREMENTS
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA:
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: 1x PBS (Phosphate Buffered Saline)

POSITIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5% aq. solution

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Three replicates were used for the test item and controls, respectively.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

83

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability value of three tissues

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item
Colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 7.774 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

- Colouring potential of test item
The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is colourless and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD value 1.155 and standard deviation value (SD) for the % viability: 8.49 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 6% mean viability range standard deviation value (SD) for the % viability: 1.77 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 3.59 % SD

Any other information on results incl. tables

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

Negative Control (1xPBS):

Replicate	Optical Density (OD)	Viability (%)
1	1.128	98
2	1.264	109
3	1.073	93
Mean	1.155	100
Standard Deviation (SD)		8.49

Positive Control (SDS 5% aq.):

Replicate	Optical Density (OD)	Viability (%)
1	0.098	9
2	0.064	6
3	0.062	5
Mean	0.075	6
Standard Deviation (SD)		1.77

Test item: beta-NADH, disodium salt OD values and viability percentages (including corrected values):

Replicate	Optical Density (OD)	TODTT	Viability (%)	Relative viability (%)
1	1.001	0.911	87	79
2	1.077	0.987	93	85
3	1.066	0.977	92	85
Mean	1.048	0.958	91	83
Standard Deviation (SD)			3.59	3.59

OD values of additional controls for MTT-interacting test item:

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1 2 3	0.102 0.055 0.060
	Mean	0.072
Test item treated killed tissues: beta-NADH, disodium salt	1 2 3	0.088 0.188 0.210
	Mean	0.162

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. The test item beta-NADH, disodium salt (CAS No.: 606-68-8) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).

Executive summary:

EpiSkin^TMSM test of beta-NADH, disodium salt (CAS No.: 606 -68 -8) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours in an incubator with 5±1 % CO2, >=95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1°C in 5±1% CO₂protected from light, >=95% humidified atmosphere. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissuesand lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (<=) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item beta-NADH, disodium salt did not show significantly reduced cell viability in comparison to the negative control (mean relative value: 83 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. Positive and negative control values were within the corresponding historical control data ranges.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. The test item beta-NADH, disodium salt (CAS No.: 606-68-8) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).