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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 October - 17 October 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- 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:
- June 2019
- 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:
- February 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 3-(aminocarbonyl)-1-ß-D-ribofuranosyl-pyridinium bromide
- Cas Number:
- 78687-39-5
- Molecular formula:
- C11H15BrN2O5
- IUPAC Name:
- 3-(aminocarbonyl)-1-ß-D-ribofuranosyl-pyridinium bromide
- Reference substance name:
- Nicotinamide
- EC Number:
- 202-713-4
- EC Name:
- Nicotinamide
- Cas Number:
- 98-92-0
- Molecular formula:
- C6H6N2O
- IUPAC Name:
- nicotinamide
- Reference substance name:
- Methanol
- EC Number:
- 200-659-6
- EC Name:
- Methanol
- Cas Number:
- 67-56-1
- Molecular formula:
- CH4O
- IUPAC Name:
- methanol
- Reference substance name:
- Ethyl acetate
- EC Number:
- 205-500-4
- EC Name:
- Ethyl acetate
- Cas Number:
- 141-78-6
- Molecular formula:
- C4H8O2
- IUPAC Name:
- ethyl acetate
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- Oxidane
- Reference substance name:
- inorganic salts by sulphated ash
- IUPAC Name:
- inorganic salts by sulphated ash
- Reference substance name:
- Bromic acid
- EC Number:
- 232-158-3
- EC Name:
- Bromic acid
- Cas Number:
- 7789-31-3
- Molecular formula:
- BrHO3
- IUPAC Name:
- bromic acid
- Reference substance name:
- Unknown impurities.
- Molecular formula:
- Not available as unknown impurities.
- IUPAC Name:
- Unknown impurities.
- Test material form:
- solid
- Details on test material:
- Batch / Lot No. 1000146/01
Constituent 1
impurity 1
impurity 2
impurity 3
impurity 4
impurity 5
impurity 6
impurity 7
- Specific details on test material used for the study:
- Batch-No.: 1000146/01
Storage: Freezer (-20°C)
Test animals / tissue source
- Species:
- other: The EpiOcular™ human cell construct (MatTek Corporation)
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount applied: Approx. 50 mg
POSITIVE AND NEGATIVE CONTROL
- Amount applied:
Positive Control: 50µL
Negative Control: 50µL - 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:
- 2
- Details on study design:
- 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.: 30630
- Expiry date: 17 October 2019
- Storage: 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 OF 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.
DETAILS ON 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±2°C in an incubator with 5±1% CO2, =95% 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:
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.
- Exposure:
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2°C in an incubator with 5±1% CO2, =95% humidified atmosphere).
- Rinsing:
After the incubation time the plastic films were removed and the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as follows: 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 (± 15 min) at 37±2°C in an incubator with 5±1 % CO2 protected from light, =95% humidified atmosphere.
- Formazan extraction:
Inserts were removed from the 24-well plate after 3 hours ± 15 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was 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 (120 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.
- Cell viability measurements:
Following the formazan extraction, 200 µL sample(s) from each tube (preferably 2×200 µL if possible) was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at the wavelength of 570 nm ((±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2908 – 2.6589) using isopropanol solutions as the blank (8×200 µL).
Results and discussion
In vitro
Results
- Irritation parameter:
- other: Mean Tissue Viability (%)
- Value:
- 77
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- OTHER EFFETCS
- Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item. The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item:
The test item showed no ability to become coloured in contact with water and isopropanol. 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 2.538
- Acceptance criteria met for positive control: 5% viability at 6h exposure
- Difference of viability between the two tissue replicates: 2.1% - 11.3%
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: Sterile deionized water
Replicate | Optical Density (OD) | Viability (%) |
1 | 2.467 | 97 |
2 | 2.609 | 103 |
mean | 2.538 | 100 |
Positive Control: Methyl acetate
Replicate | Optical Density (OD) | Viability (%) |
1 | 0.088 | 3 |
2 | 0.142 | 6 |
mean | 0.115 | 5 |
Test item
Replicate | Optical Density (OD) | Viability (%) |
1 | 1.812 | 71 |
2 | 2.100 | 83 |
mean | 1.956 | 77 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, Nicotinamide-beta-D-riboside bromide (CAS 78687-39-5) is, thus, considered as non-irritant to eye (UN GHS No Category).
- Executive summary:
The purpose of this study was to determine the acute ocular irritation potential of the test item Nicotinamide-beta-D-riboside bromide on on three-dimensional RhCE tissue in the EpiOcular™ modelin 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 test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2°C in an incubator with 5 ± 1% CO2, =95% 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 (± 15 min) with MTT solution at 37 ± 2°C in an incubator with 5 ± 1% CO2protected from light, =95% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.
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 Nicotinamide-beta-D-riboside bromide (CAS 78687-39-5)d id not show significantly reduced cell viability in comparison to the negative control (mean tissue viability: 77 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye.
Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.
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