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EC number: 260-694-8 | CAS number: 57357-85-4
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-03-24 to 2016-11-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2014-09-26
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- [1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine
- EC Number:
- 260-694-8
- EC Name:
- [1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine
- Cas Number:
- 57357-85-4
- Molecular formula:
- C11H21N
- IUPAC Name:
- 1-[(1S,2S,3S,5R)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl]methanamine
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: 16/0055-1
- Expiration date of the lot/batch: 2016-10-26
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixing before preparation of the test substance preparations.
OTHER SPECIFICS: Liquid, colorless, clear
Method
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: The V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
- Doubling time: 12 - 14 h
- Methods for maintenance in cell culture if applicable: 1 mL portions of V79 stocks were maintained at -196 °C in liquid nitrogen using 7 % (v/v) dimethyl sulfoxide (DMSO) in culture medium as a cryoprotectant.
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL), and 1 % (v/v) amphotericine B (250 μg/mL). Cells were grown with 5 % (v/v) CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Dose selection according to OECD 487 guideline.
With S9 mix: 6.25, 12.5, 25.0, 50.0, and 100.0 µg/mL
Without S9 mix: 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 µg/mL - Vehicle / solvent:
- - Solvent used: DMSO 1 % (v/v)
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without S9 mix
- Positive controls:
- yes
- Positive control substance:
- other: ethyl methanesulfonate
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without S9 mix
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 3 - 5 x10E6 cells/culture
DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time: 24 - 44 h
- Fixation time: at the end of exposure time.
SPINDLE INHIBITOR: Cytochalasin B
STAIN: Prepared slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (PI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 5x10^4 cells per slide were centrifuged at 600 rpm for 7 min onto labeled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 1x10^4 cells per flask no slides were prepared. After drying, the slides were fixed in 90 % (v/v) methanol for 10 minutes.
NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
- The diameter of the micronucleus is less than 1/3 of the main nucleus.
- The micronucleus and main nucleus retain the same color.
- The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
- Only binucleated cells clearly surrounded by a membrane were scored.
DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD)
OTHER EXAMINATIONS:
- Determination of the cytokinesis-block proliferation index (CBPI). - Evaluation criteria:
- Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data. Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.
Assessment criteria:
A test substance is considered to be clearly positive if the following criteria are met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95 % control limit).
A test substance is considered to be clearly negative if the following criterion is met:
- Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
- The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95 % control limit). - Statistics:
- The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- according to OECD guideline 487
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Table 1: Summary table - experimental parts without S9 mix
Exp. |
Exposure/preparation interval [h] |
Test groups [µg/mL] |
Prec.* |
Genotoxicity** Micronucleated cells [%] |
Cytotoxicity |
|
Proliferation index cytostasis (CBPI) [%] |
Relative population doubling (RPD) [%] |
|||||
1 |
4/24 |
Vehicle control(1) |
n.d. |
0.2 |
0.0 |
100.0 |
3.13 |
- |
n.d. |
n.d. |
92.2 |
||
6.25 |
- |
0.4 |
5.4 |
106.8 |
||
12.50 |
- |
0.5 |
12.0 |
89.2 |
||
25.00 |
- |
0.3 |
10.1 |
75.0 |
||
50.00 |
- |
n.d. |
n.d. |
1.2 |
||
100.00 |
- |
n.d. |
n.d. |
-164.6 |
||
Positive control(2) |
n.d. |
1.3 (s) |
11.3 |
100.0 |
||
Positive control(3) |
n.d. |
2.3 (s) |
10.2 |
105.9 |
||
|
||||||
2 |
24/24 |
Vehicle control(1) |
n.d. |
0.2 |
0.0 |
100.0 |
Negative control |
- |
0.6 |
-18.1 |
131.2 |
||
0.78 |
- |
n.d. |
n.d. |
111.4 |
||
1.56 |
- |
n.d. |
n.d. |
95.8 |
||
3.13 |
- |
n.d. |
n.d. |
119.1 |
||
6.25 |
- |
0.5 |
5.5 |
106.8 |
||
12.50 |
- |
0.3 |
12.0 |
75.4 |
||
25.00 |
- |
0.2 |
21.8 |
64.4 |
||
Positive control(2) |
n.d. |
1.6 (s) |
14.1 |
154.8 |
||
Positive control(3) |
n.d. |
2.8 (s) |
16.9 |
150.9 |
||
|
||||||
3 |
24/24 |
Vehicle control(1) |
n.d. |
0.3 |
0.0 |
100.0 |
Negative control |
- |
0.4 |
-14.5 |
137.5 |
||
3.13 |
- |
n.d. |
n.d. |
71.7 |
||
6.25 |
- |
0.3 |
5.2 |
66.2 |
||
12.50 |
- |
0.2 |
9.4 |
66.4 |
||
25.00 |
- |
0.1 |
18.5 |
46.2 |
||
50.00 |
- |
n.d. |
n.d. |
-162.5 |
||
100.00 |
- |
n.p. |
n.p. |
-221.6 |
||
Positive control(2) |
n.d. |
2.9 (s) |
13.1 |
112.5 |
* Precipitation in culture medium at the end of exposure period (macroscopic)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(s) Frequency statistically significant higher than corresponding control values
n.d. Not determined
n.p. No cytospin slides prepared due to strong cytotoxicity
n.s. Not scorable due to strong cytotoxicity
(1) DMSO 1 % (v/v) (2) EMS 500 μg/mL (3) EMS 600 μg/mL
Table 2: Summary table - experimental parts with S9 mix
Exp. |
Exposure/preparation interval [h] |
Test groups [µg/mL] |
Prec.* |
Genotoxicity** Micronucleated cells [%] |
Cytotoxicity |
|
Proliferation index cytostasis (CBPI) [%] |
Relative population doubling (RPD) [%] |
|||||
1 |
4/24 |
Vehicle control(1) |
n.d. |
0.3 |
0.0 |
100.0 |
3.13 |
- |
n.d. |
n.d. |
75.7 |
||
6.25 |
- |
n.d. |
n.d. |
80.0 |
||
12.50 |
- |
0.5 |
0.4 |
83.2 |
||
25.00 |
- |
0.3 |
13.0 |
75.8 |
||
50.00 |
- |
0.4 |
13.1 |
42.6 |
||
100.00 |
- |
n.d. |
n.d. |
-94.6 |
||
Positive control(2) |
n.d. |
1.8 (s) |
22.5 |
61.5 |
||
|
||||||
2 |
4/44 |
Vehicle control (1) |
n.d. |
0.5 |
0.0 |
100.0 |
6.25 |
- |
n.d. |
n.d. |
103.2 |
||
12.50 |
- |
0.6 |
7.8 |
100.8 |
||
25.00 |
- |
0.5 |
8.0 |
86.3 |
||
50.00 |
- |
0.4 |
13.6 |
53.6 |
||
100.00 |
- |
n.s. |
n.s. |
94.1 |
||
Positive control(2) |
n.d. |
3.6 (s) |
-14.7 |
70.2 |
* Precipitation in culture medium at the end of exposure period (macroscopic)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(s) Frequency statistically significant higher than corresponding control values
n.d. Not determined
n.p. No cytospin slides prepared due to strong cytotoxicity
n.s. Not scorable due to strong cytotoxicity
(1) DMSO 1 % (v/v) (2) CPP 0.5 μg/mL
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