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EC number: 200-607-2 | CAS number: 65-31-6
- 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-10-10 to 2016-10-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was performed in accordance with standard test protocols (OECD 439 resp. EU B.46) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- nicotine bitartrate dihydrate
- IUPAC Name:
- nicotine bitartrate dihydrate
- Reference substance name:
- 6019-06-3
- Cas Number:
- 6019-06-3
- IUPAC Name:
- 6019-06-3
- Reference substance name:
- Nicotine dihydrogen ditartrate
- EC Number:
- 200-607-2
- EC Name:
- Nicotine dihydrogen ditartrate
- Cas Number:
- 65-31-6
- Molecular formula:
- C10-H14-N2.2C4-H6-O6
- IUPAC Name:
- nicotine dihydrogen ditartrate
- Test material form:
- solid: particulate/powder
- Remarks:
- powder
- Details on test material:
- white or almost white powder
Constituent 1
Constituent 2
Constituent 3
- Specific details on test material used for the study:
- - Homogeneity: homogeneous
- Appearance: white powder
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C)
In vitro test system
- Test system:
- human skin model
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: human-derived epidermal keratinocytes, cultured to form a multi-layered, highly differentiated model of the human epidermis
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have
been cultured to form a multi-layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers representing main lipid classes analogous
to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell
culture inserts.
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 11. Oct. 2016
Batch no.: 23362 - Amount/concentration applied:
- Amounts of Test Item
Tissue 1: 25.7 mg
Tissue 2: 25.2 mg
Tissue 3: 25.1 mg - Duration of treatment / exposure:
- Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were
transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps
and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into
a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours and 27 minutes at 37 ± 1°C and
5.0 ± 0.5% CO2. - Duration of post-treatment incubation (if applicable):
- After post-incubation, 0.9 mL assay medium were filled in the lower row of the 6-well-plate.
The tissues were removed from the incubator and shaken for 5 minutes (120 rpm). Then
the inserts were transferred into the lower row of the 6-well-plate and set into the incubator
for 19 hours and 20 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2. - Number of replicates:
- 3 Tissues
Test system
- Details on study design:
- MTT Assay
After a total incubation time of 42 hours and 47 minutes, a 24-well-plate was prepared with
300 μL freshly prepared MTT-medium in each well. The tissues were blotted on the bottom
and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator
for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
After this time, the MTT-medium was aspirated and replaced by DPBS buffer. This was
then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate.
Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the
insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts were pierced with an injection needle, taking care that all colour
was extracted. The inserts were then discarded and the content of each well was thoroughly
mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate
which was read in a plate spectrophotometer at 570 nm.
The photometric absorbance of the negative controls is considered as 100%. For each
replicate of test item and positive control, tissue viability is calculated as % photometric
absorbance compared with the mean of the negative controls
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1
- Value:
- 99.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
Any other information on results incl. tables
Absorbance Values negative control, test item and positive control (OD 570nm)
Designation | Measurement | Negative Control | Nicotine bitartrate dihydrate | Positive Control |
Tissue 1 | 1 | 2.021 | 1.934 | 0.097 |
2 | 2.033 | 1.898 | 0.097 | |
Tissue 2 | 1 | 2.050 | 2.005 | 0.098 |
2 | 2.026 | 2.010 | 0.097 | |
Tissue 3 | 1 | 1.994 | 2.094 | 0.096 |
2 | 1.954 | 2.078 | 0.096 |
Mean absorbance values
Designation | Negative Control | Nicotine bitartrate dihydrate | Positive Control |
Mean - blank (tissue 1) | 1.987 | 1.876 | 0.057 |
Mean - blank (tissue 2) | 1.998 | 1.968 | 0.058 |
Mean - blank (tissue 3) | 1.934 | 2.046 | 0.056 |
Mean of the three tissues | 1.973 | 1.963 | 0.057 |
% Viability
Designation | Nicotine bitartrate dihydrate | Positive Control |
% viability (tissue 1) | 95.1% | 2.9% |
% viability (tissue 2) | 99.7% | 2.9% |
% viability (tissue 3) | 103.7% | 2.8% |
% viability (mean) | 99.5% | 2.9% |
+/- SD of mean viability (%) | 4.3% | 0.1% |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item is considered as non-irritant to skin.
After the treatment, the relative absorbance values were reduced to 99.5%. This value is
above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion
of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of
the test system.
Variation within replicates was within the accepted range for negative control, positive control
and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid. - Executive summary:
Three tissues of the human skin model EpiDermTM were treated with Nicotine bitartrate
dihydrate for 60 minutes.
The test item was applied directly to each tissue and spread to match the tissue size
(0.63 cm2; as indicated by the supplier).
DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.
After treatment with the negative control, the absorbance values were within the required
acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed
clear irritating effects. Relative absorbance was reduced to 2.9 % (required: < 20%).
Variation within the tissue replicates was acceptable (required: ≤ 18%).
After the treatment with the test item, the relative absorbance values were reduced to
99.5 %. This value is above the threshold for skin irritation potential (50%).
Therefore, Nicotine bitartrate dihydrate is considered as non-irritant to skin in the
Reconstructed Human Epidermis (RHE) Test Method.
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