Dihydronicotinamide-adenine dinucleotide, disodium salt

Endpoint summary

Description of key information

- Episkin test for skin irritation (OECD 439): 83% cell viability: Non irritant

- BCOP test (OECD 437): IVIS -0.6, NC (non irritant)

- Epiocular TM(OECD 492): 47% cell viability, C1/C2

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

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

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 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.

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

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 - 09 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

09 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; for the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

10 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: The EpiOcular™ human cell construct (MatTek Corporation)

Details on test animals or tissues and environmental conditions:

DETAILS ON TEST SYSTEM
RhCE tissue construct used: EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
-Lot No.: 27012
- Expiry date: 09 November 2017

- Storage: The EpiOcular™ (OCL-200-EIT) units were stored at refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 hours (± 15 min)

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes immersion incubation (Post-Soak)
18 hours ± 15 minutes (Post-treatment Incubation)

Number of animals or in vitro replicates:

Two

Details on study design:

DETAILS ON THE TEST PROCEDURE USED:

- Preparation of EpiOcular™ Tissues for Treatment
After the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes. The Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37±1°C in an incubator with 5±1% CO2, 90±10% humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).

- Application
Two replicates were used for the test item and control(s) respectively.
Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Test Item: Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface (e.g. the lid of a microtiter plate) and dosed by pouring the solid test article onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
Positive and Negative Control: A volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface if necessary.
Additional controls for MTT direct interacting chemicals: 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.
Additional controls for dyes and chemicals able to colour the tissue: In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation.
Additional controls for non-specific colour in killed tissues: In addition to the normal procedure, two killed treated tissues were used for avoiding a possible double correction for colour interference.

- Exposure
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±1°C in an incubator with 5±1% CO2, 90±10% humidified atmosphere).

- Rinsing
After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as following way: Three clean beakers (glass or plastic with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS were used per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care of not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container.
This process was performed two additional times in the first beaker. The tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

-Post-Soak and Post-incubation
After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, 90±10% humidified atmosphere.

- Formazan Extraction
Each insert was removed from the 24-well plate after 3 hours ± 10 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with parafilm (between the plate cover and upper edge of the wells). To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 3 hours at room temperature. At the end of the extraction period, the tissue was not pierced.

- Cell Viability Measurements
Following the formazan extraction, 200 µL sample(s) from each tube (2×200 µL) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using isopropanol solutions as the blank (8×200 µL).

OTHER INFORMATION ON MATERIALS AND METHODS
- Check-method for Possible Direct MTT Reduction by the Test Item
Approximately 50 mg test item was added to 1 mL MTT 1 mg/mL solution in a 6-well plate or tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere. The mixture was incubated for approximately three hours and then any colour change observed:
- Test item which do not interact with MTT: yellow
- Test item interacting with MTT: blue or purple
If the MTT solution’s colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed tissues).

- Additional controls for direct MTT interacting chemicals (MTT reducers): 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 (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batches of the living tissues (batch no. of killed EpiOcular™ tissues: 23785). The same treatment steps are followed for these tissues as for the living tissues.

- Check-method to Detect the Colouring Potential of Test Item: Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. Blue, dark purple and black test articles may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue MTT product.
Approximately 50 mg test item was added to 1mL of water in a tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere for one hour and then colour checked (unaided eye assessment).
Approximately 50 mg test item was added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in tubes. The tube was placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature and then colour checked (unaided eye assessment).

The test item has an intrinsic colour (yellowish). So Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
-Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.
Additional controls for non-specific colour in killed tissues:
The test item is a MTT-reducer and has an intrinsic colour (yellowish). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. However, the NSCliving % was 0.68 % (below 5 %), so the (NSCkilled) was not determined and used during the calculation of true MTT metabolic conversion.
In addition to the normal procedure, two killed treated tissues were used to avoid a possible double correction for colour interference. In this additional control, the test item was applied on two killed tissue replicates, which undergo the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step.

ASSAY ACCEPTANCE CRITERIA
- The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is:
30 minute exposure: below 50% of control viability (liquids)
6 hours exposure: below 50% of control viability (solids)
- The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: Mean Tissue Viability (% of negative control)

Value:

47

Negative controls validity:

valid

Positive controls validity:

valid

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 0.107 %. As the NSMTT were below 60 % 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 and isopropanol.
However, as the test item has an intrinsic colour (yellowish), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.008. The Non Specific Colour % (NSC %) was calculated as 0.68 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD value 1.197
- Acceptance criteria met for positive control: 19 % viability at 6 h exposure
- Difference of viability between the two tissue replicates: 0.1% to 11.7%

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: Sterile deionized water

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	1.218	102	3.4
2	1.177	98
Mean	1.197	100

Positive Control: Methyl acetate

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	0.199	17	4.7
2	0.255	21
Mean	0.227	19

Test Item: Beta-NADH, disodium salt

Replicate	Optical Density (OD)	TODTT	Viability (%)	Relative Viability (%)	Delta %
1	0.499	0.498	42	42	11.7
2	0.640	0.638	53	53	11.7
Mean	0.569	0.568	48	47
Standard deviation (SD)			8.29	8.29

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

Aditional controls	Replicate	Optical Density (OD)
Negative control killed tissues: Sterile deionized water	1 2 Mean	0.054 0.050 0.052
Test item treated killed tissues: ß-NADH, disodium salt	1 2 Mean	0.052 0.054 0.053

OD values and NSC % of additional colour control for Beta-NADH, disodium salt

Replicate	Optical Densitiy (OD)	Non specific colour % (NSCliving%)	Delta %
1 2 mean	0.009 0.008 0.008	0.68	0.1

Remark: Delta %: The difference of viability between the two relating tissues

Interpretation of results:

other: Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1)

Remarks:

Test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Further testing is required.

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Beta-NADH, disodium salt (CAS Nr. 606-68-8) indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Therefore further testing with other test methods will be required to decide on its final classification.

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item Beta-NADH, disodium salt (CAS Nr. 606-68-8) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before treatment the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37± 1°C in an incubator with 5± 1% CO2, 90±10% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37± 1°C in an incubator with 5± 1% CO2 protected from light, 90±10% humidified atmosphere. The precipitated formazan was then extracted using 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 and isopropanol. However, the test item has an intrinsic colour (yellowish), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item is a MTT-reducer and has an intrinsic colour (yellowish). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (<=) to 60% compared to the negative control. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

The test item Beta-NADH, disodium salt showed significantly reduced cell viability in comparison to the negative control (mean relative viability: 47 %). All obtained test item viability results were below 60% when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Beta-NADH, disodium salt (CAS Nr. 606-68-8) indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Therefore further testing with other test methods will be required to decide on its final classification.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 - 24 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Corneas were prepared and used for testing as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples: Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity, pigmentation or neovascularisation) prior to use.Any eyes showing defects were rejected from further use.
- Indication of any antibiotics used: Not specified

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration Test Item: 20 %, w/v in physiological saline
NEGATIVE CONTROL: Physiological saline
- Amount(s) applied: 750 µL
- Concentration: Sodium Chloride 0.9%, w/v
POSITIVE CONTROL : Imidazole [CAS Number 288-32-4]
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20%, w/v in physiological saline

Duration of treatment / exposure:

4 h ± 10 min

Number of animals or in vitro replicates:

Three Corneas per test item four corneas for positive and negative controls

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and
neovascularization by removing them from the physiological saline and holding them in the
light. Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were
incubated for the minimum of 1 hour at 32 +/- 1°C.

After the incubation period, the medium was removed from both compartments and replaced ith fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT METHOD: closed chamber. The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with
the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were then taken using an Opacitometer
- Corneal permeability: Following the measurement of opacity,The medium of both compartments (anterior compartment first) was removed. The posterior
compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the
permeability calculation.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = Opacity change + (15 x OD490 Value)

DECISION CRITERIA:
In accordance with OECD Test Guideline No. 437, irritancy was assigned on the basis of in vitro irritancy scores (IVIS), calculated from the opacity and permeability value for each cornea, as follows:

IVIS<=3: not classified according to GHS/CLP
3IVIS>55: Classified as Eye Damaging Category 1 according to GHS/CLP

Irritation parameter:

in vitro irritation score

Run / experiment:

Single experiment

Value:

-0.6

Vehicle controls validity:

valid

Remarks:

IVIS: 2.4

Positive controls validity:

valid

Remarks:

IVIS: 137

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium
- Visible damage on test system: not observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean post treatment opacity of 2.1 (historical values range -5.4 – 5.2) / mean permeability corrected for blank of 0.014 (historical values range -0.010 - 0.205). The classification achieved was “No Category”.
- Acceptance criteria met for positive control: yes, mean IVIS 137 (historical range: 86.5 – 211.4). The classification achieved was “Category 1.
- The BCOP results were similar for all corneas and are presented in full in "Any other information on results incl. tables" section

Table 1

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score ^1, 2
Negative control	2.1	0.014	2.4
Positive control	95	2.802	137
ß-NADH, disodium salt	-0.5	-0.004	-0.6

¹ Calculated using the negative control mean opacity and mean permeability values for the positive control and

test item.

² In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 2: Individual Results

Treatment	Final Opacity	Final OD490	In vitro Irritancy Score
Physiological saline (Negative control)	3.5 1.8 1.1	0.008 0.027 0.008	3.7 2.2 1.3
Imidazole (Positive control)	116 61 108	2.837 2.379 3.189	158 97 156
ß-NADH, disodium salt	-1.5 0.4 -0.5	-0.013 -0.012 -0.014	-1.7 0.2 -0.3

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since beta-NADH, disodium salt (CAS No. 606-68-8) induced an IVIS = 3 in the BCOP test (OECD 437 test method), no classification is required for eye irritation or serious eye damage according to the UN GHS classification and the CLP classification system.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of beta-NADH, disodium salt (CAS No. 606-68-8) as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP) test (OECD 437 test method)..

This report describes the potential of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes.

The study procedures described in this report were based on the most recent OECD guideline 437.

The test item was a light yellow crystalline powder. The test item was applied as a 20% (w/v) solution (750 µl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 137 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.6 after 4 hours of treatment.

In conclusion, since the test item beta-NADH, disodium salt (CAS No. 606-68-8) induced an

IVIS = 3 in the BCOP test (OECD 437 test method), no classification is required for eye irritation or serious eye damage according to the UN GHS classifications and the CLP classification system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A valid EpiSkinTM SM test of beta-NADH, disodium salt was performed under GLP 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. EPISKIN discs were exposed in triplicate to beta-NADH, disodium salt for 15 min at room temperature. The EPISKIN discs treated with the test item did not show significantly reduced cell viability (83% viability compared to the negative controls). Therefore beta-NADH, disodium salt is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified). The study is rated as key study and as Klimisch 1 "reliable without restriction".

Eye irritation:

A valid study was performed under GLP to determine the acute ocular irritation potential of the test item beta-NADH, disodium salt on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to OECD guideline Nr. 492, 28 July 2015. EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±1°C) in an incubator with 5±1 % CO2, 90±10% humidified atmosphere). The test item beta-NADH, disodium salt showed significantly reduced cell viability, </= 60%, in comparison to the negative control (mean relative viability: 47 %). The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item beta-NADH, disodium salt indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. Therefore an additional study was performed.

The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

A valid assessment of ocular irritation of beta-NADH, disodium salt In Vitro Using the Bovine Corneal Opacity and Permeability Assay was performed under GLP according the the OECD guideline Nr, 437, 26 July 2013. Beta-NADH, disodium salt ( 20 %, w/v; ca 750 µL in physiological saline), was applied to three corneas. Following exposure for 4 h ± 10 min, it was rinsed off. The in vitro irritancy score (IVIS) calculated for this substance was -0.6. If the IVIS score is </= 3, the substance does not require classification for eye damage according to the UN GHS classifications and the CLP classification system.

In conclusion, the BCOP assay categorised beta-NADH, disodium salt as ‘No category’ according to the UN GHS classifications and the GLP classification system.

The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

Justification for classification or non-classification

According to the results obtained with the EpiSkinTM SM test for irritation potential (OECD 439) beta-NADH, DiNa (CAS 606-68-8) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category /CLP not classified).

The results of the BCOP (OECD 437) and Epiocular (OECD 492) tests are contradictory.

However, BCOP can be used as key test for identifying substances that do do not need to be classified for Eye damage (NC). BCOP has a false negative rate of 0% when used for identifying substances that do not require classification (as stated on the OECD 437 guideline). Epiocular, on the other hand, has a false positive rate of 37% (63% specificity) (according the OECD 492 guideline). This result could therefore be considered a false positive in this case.

Moreover, no cytotoxic effects were observed on the Skin irritation/corrosion tests.

Taking all this information into account, it can be concluded that beta-NADH, DiNa (CAS 606-68-8), does not have to be classified for Eye Irritation/corrosion (UN GHS No Category /CLP not classified).