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EC number: 605-146-4 | CAS number: 158451-78-6
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- Sep 26, 2014
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No 640/2012; B.49
- Version / remarks:
- May 30, 2008
- Principles of method if other than guideline:
- The data concerning the composition and/or purity of the test substance were updated by the sponsor after performing the 2nd Experiment. Thus, scoring of slides of the 2nd Experiment was omitted. Then, two additional experiments were performed with dose selection following the recommendations of the current OECD Guideline 487.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)
- EC Number:
- 605-146-4
- Cas Number:
- 158451-78-6
- Molecular formula:
- C16H38N2O2
- IUPAC Name:
- 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)
Constituent 1
- Specific details on test material used for the study:
- - Name of test substance as cited in study report: 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated
- Test substance No.: 14/0384-1
- Batch No.: 0011784570
- Purity/composition: The identity of the test substance was confirmed. Main peak: 86.4 area-% (CE-fingerprint); Content of water: 82.7g/100g;
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions was guaranteed until Apr 2016.
- Date of production/supply: April 2014
- Physical state, appearance: Liquid, yellowish, clear
- Storage conditions: Room temperature, avoid temperatures > 40°C and < 0°C
Method
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- The V79 cell line (permanent cell line)
- Doubling time: 12-14 hours
- Plating efficiency: ≥ 90%
- Stable karyotype (modal number of 22 chromosomes)
MEDIA USED
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)
− 1% (v/v) amphotericine B (250 μg/mL)/no]
- Cytokinesis block (if used):
- Actin polymerisation inhibitor cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital and ß-napthoflavone induced S9 liver fraction
- Test concentrations with justification for top dose:
- According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested:
- 1st experiment: (failed validation criteria concerning top concentration);
4 hours exposure, 24 hours harvest time, without S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
- 2nd experiment: (failed validation criteria concerning top concentration)
24 hours exposure, 24 hours harvest time, without S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
- 3rd experiment:
4 hours exposure, 24 hours harvest time, without S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4 hours exposure, 24 hours harvset time, with S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
- 4th experiment:
24 hours exposure, 24 hours harvest time, without S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL - Vehicle / solvent:
- - Vehicle/solvent used: Minimal Essential Medium
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 3x10^5 per culture
DURATION
- Preincubation period: 2-4 days
- Exposure duration: 4 hours without S9 mix (Exp. 1 and 3), 24 hours without S9 mix (Exp. 2 and 4), 4 hours with S9 mix (Exp. 1 and 3), 4 hours with S9 mix (Exp. 2 and 4)
- Recovery time: 20 hrs for experiment 1 and 3, and 40 hours for experiment 2 and 4
- Harvest time: 24 hours without S9 mix (Exp. 1, 2, 3, 4) and with S9 mix (Exp. 1 and 3), and 48 hours with S9 mix (Exp. 2 and 4)
SPINDLE INHIBITOR: actin polymerisation inhibitor cytochalasin B
STAIN: a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (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.
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by enzymatic dissociation. Then, the cell number per flask of each cell suspension was determined using a cell counter. Subsequently, 5x10^4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 10x10^4 cells per flask no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.
Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide in Fluoroshield™ at a concentration of 0.25 μg/mL each.
NUMBER OF CELLS EVALUATED: at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
− 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 were scored.
MICRONUCLUES ANALYSIS
The cytospin slides were scored by fluorescence microscopy. As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei. The analysis of micronuclei was carried out following these criteria:
− 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 were scored.
Slides were coded before microscopic analysis. Cultures with only few isolated cells were not analysed for micronuclei.
DETERMINATION OF CYTOTOXICITY
- Relative population doubling
- Proliferation index - 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 the 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 the 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 the laboratory’s historical negative control data (95% control limit). - Statistics:
- One-sided Fisher's exact test
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- except at 4600 μg/mL in the 2nd Experiment in the absence of S9 mix (relative population doubling of 37.7%)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- CELL MORPHOLOGY: In this study, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for micronuclei.
TREATMENT CONDITIONS: Osmolarity and pH values were not influenced by test substance treatment. However, in the 3rd and 4th Experiment the pH of the stock solutions were adjusted to a physiological value using small amounts of HCl. No precipitation of the test substance in culture medium was observed.
Applicant's summary and conclusion
- Conclusions:
- In this study, no relevant increase in the number of micronucleated cells was found after exposure to the test substance either with or without addition of a metabolizing system.
- Executive summary:
In this GLP-compliant study performed according to OECD guideline 487 and Commission Regulation (EC) No 640/2012, the test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Four independent experiments were carried out, all with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated for cytogenetic damage.
1st Experiment (failed validation criteria concerning top concentration)
4 hours exposure, 24 hours harvest time, without S9 mix
0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
2nd Experiment (failed validation criteria concerning top concentration)
24 hours exposure, 24 hours harvest time, without S9 mix
0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
3rd Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4th Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
In general, a sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The data concerning the composition and/or purity of the test substance were updated by the sponsor after performing the 2nd Experiment. Thus, scoring of slides of the 2nd Experiment was omitted. Then, two additional experiments were performed with dose selection following the recommendations of the current OECD Guideline 487. The negative controls gave frequencies of micronucleated cells within the range of the 95% control limit of the historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected experimental part. In this study, no relevant cytotoxicity indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest applied test substance concentration under all experimental conditions. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
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