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EC number: 953-863-9 | 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2014-09-10 to 2014-10-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study was performed in accordance with standard test protocols (OECD 429 resp. EU B.42) 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:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- Nicotine
- EC Number:
- 200-193-3
- EC Name:
- Nicotine
- Cas Number:
- 54-11-5
- Molecular formula:
- C10H14N2
- IUPAC Name:
- 3-(1-methyl-2-pyrrolidinyl)pyridine
- Test material form:
- liquid
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: CBA/CaOlaHsd , Harlan Laboratories B.V., AD Horst / The Netherlands
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 19.3-22.8 g
- Housing: group
- Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination
ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature 22 ± 2°C
- Humidity (%): relative humidity approx. 45-65% (The relative humidity in the animal room was between approximately 45 - 100 % instead of 45 – 65% for few hours. This deviation does not affect the validity of the study)
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.
IN-LIFE DATES: daily
Study design: in vivo (LLNA)
- Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- 0.5%; 1% and 2% (which are approximately 12.2; 24.4 and 48.8 mg/kg body weight, calculated with the mean body weight of the animals before start of treatment and a density of nicotine of 1 g/mL)
- No. of animals per dose:
- 4
- Details on study design:
- TREATMENT PREPARATION AND ADMINISTRATION:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (4+1, v/v) as vehicle.
- Irritation: The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments (see "Any other information on materials and methods incl. tables").
MAIN STUDY
- Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.5, 1, and 2% (w/v) in acetone/olive oil (4+1, v/v).
- The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( diameter 8 mm) of each ear once daily for three consecutive days.
- A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.4 µCi of ³H-methyl thymidine (equivalent to 81.5 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.
- Approximately five hours after treatment with ³HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
- The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).
- Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size).
- After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
- The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed.
- The level of ³HTdR incorporation was then measured in a beta-scintillation counter.
- Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The beta-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute.
- The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of ³HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).
- Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with ³HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The mean values and standard deviations were calculated.
Results and discussion
- Positive control results:
- The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed in April 2014 using CBA/CaOlaHsd mice. The S.I. results were stated as follows (see also table in "Any other information on materials and methods incl. tables"):
0%: 1.00
5%: 2.51
10%: 1.76
25%: 6.79
Calculation of EC3 value:
EC3: (10% - 25%) [(3 - 6.79) / (1.76 - 6.79)] + 25 = 13.7% with the co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- 1.06
- Test group / Remarks:
- 0.5 % nicotine
- Key result
- Parameter:
- SI
- Value:
- 0.91
- Test group / Remarks:
- 1 % nicotine
- Key result
- Parameter:
- SI
- Value:
- 0.92
- Test group / Remarks:
- 2% nicotine
- Parameter:
- other: disintegrations per minute (DPM)
- Remarks on result:
- other: See "Any other information on results incl. tables"
Any other information on results incl. tables
Viability / Mortality
No deaths occurred during the study period.
Clinical Signs
From day 1 to 3, the animals treated with 2% test item concentration showed reduced spontaneous activity and on days 1 (here only animal no. 15), 4, and 5 ruffled fur. On day 3, the animals treated with a test item concentration of 2% showed an erythema of the ear skin (Score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation and toxicity.
Body Weights
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.
Calculation and Results of Individual Data
Test item concentration % |
Group |
Measurement DPM |
Calculation |
Result |
||
DPM-BGa) |
number of lymph nodes |
DPM per lymph nodeb) |
S.I. |
|||
--- |
BG I |
14 |
--- |
--- |
--- |
--- |
--- |
BG II |
14 |
--- |
--- |
--- |
--- |
0 |
1 |
5899 |
5885.0 |
8 |
735.6 |
1.00 |
0.5 |
2 |
6251 |
6237.0 |
8 |
779.6 |
1.06 |
1 |
3 |
5382 |
5368.0 |
8 |
671.0 |
0.91 |
2 |
4 |
5451 |
5437.0 |
8 |
679.6 |
0.92 |
1 = Control Group
2-4= Test Group
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid. The test item nicotine was not a skin sensitiser and is therefore not classified according to Regulation (EC) No 1272/2008.
- Executive summary:
In order to study a possible skin sensitising potential of nicotine, three groups each of four female mice were treated once daily with the test item at concentrations of 0.5, 1, and 2% (w/v) in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments. A control group of four mice was treated with the vehicle (acetone/olive oil (4+1, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.
All treated animals survived the scheduled study period. From day 1 to 3, the animals treated with 2% test item concentration showed reduced spontaneous activity and on days 1 (here only animal no. 15), 4, and 5 ruffled fur. On day 3, the animals treated with a test item concentration of 2% showed an erythema of the ear skin (Score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation and toxicity.
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.06, 0.91, and 0.92 were determined with the test item at concentrations of 0.5, 1, and 2% (w/v) in acetone/olive oil (4+1, v/v). A dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The dossier substance "Distillate from tobacco leaves of Nicotiana tabacum (Solanaceae) is therefore expected to be non-sensitizing to skin.
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