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EC number: 944-336-4 | 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Jan - 26 Feb 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 guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Ministère de l'Économie et des Finances, vry-sur-Seine Cedex, France
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- 7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate
- EC Number:
- 276-957-5
- EC Name:
- 7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate
- Cas Number:
- 72869-86-4
- Molecular formula:
- C23H38N2O8
- IUPAC Name:
- Reaction mass of 7,7,9-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diylbismethacrylate and 7,9,9-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diylbismethacrylate
Constituent 1
Method
- Target gene:
- Not applicable
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Japan Health Science Foundation
- Suitability of cells: recommended test system in international guidelines
- Number of passages if applicable: 24 and 39
- Methods for maintenance in cell culture: Cell cultures were maintained in suspension cell culture flasks at 37 °C and 5% CO2.
MEDIA USED
- Type and identity of media including CO2 concentration: The culture medium used was the R10 medium: Roswell Park Memorial Institute medium (RPMI 1640) + 10% horse serum, heat inactivated (v/v),
50 IU/mL penicillin, 50 µg/mL streptomycin + 1 mM sodium pyruvate + 0.05% pluronic acid.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no
- Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment I
4 h treatment (with and without metabolic activation): 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 and 500 µg/mL
Experiment I Repetition
4 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL
Experiment II
24 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL
Test item concentrations were initially selected based on the solubility in the solvent ethanol. In cases where the test item is cytotoxic, the highest concentration should aim to reach a maximum cytotoxicity (cytostasis) of 55 ± 5%. In the first experiment (4 h) without metabolic activation, only two of the eight tested concentrations met the acceptability criteria, therefore the test was not valid. Cytotoxicity (cytostasis) of more than 71% was observed at concentrations of ≥ 15.6 µg/mL, but only when tested without metabolic activation. Therefore the first experiment (without metabolic activation) was repeated at lower concentrations. - Vehicle / solvent:
- - Vehicle used: ethanol (1.0% (v/v))
Controls
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine (-S9), 0.1 µg/mL in ethanol (4 h exposure) and 0.01 µg/mL in ethanol (24 h exposure)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E6 cells/tube in 8 mL cultivation medium
DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20-24 h; 24 h treatment: 44-48 h
STAIN: Giemsa
NUMBER OF REPLICATIONS: duplicates in all experiments
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, the cell cultures were homogenised, centrifuged and underwent hypotonic treatment. Afterwards, the cells were fixed with methanol-acetic acid (3:1 ratio) and spread on glas slides for microscopic analysis.
NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: cytostasis
- Any supplementary information relevant to cytotoxicity: please refer to “Any other informations on materials and methods” - Evaluation criteria:
- A test substance was considered positive (clastogenic or aneugenic) in the micronucleolus test if:
- At least one of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared to the concurrent negative control
- A dose effect was observed in at least one experimental condition when evaluated with an appropriate trend test
- The results were outside the data distribution range of historic negative controls
A test substance was considered negative (not clastogenic or aneugenic) in the micronucleus test if:
- None of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared with the concurrent negative control
- There was no concentration-related increase in the frequency of micronucleated cells when evaluated with an appropriate trend test
- All results were inside the distribution of the historical negative control data - Statistics:
- Chi square test (p < 0.05)
Results and discussion
Test results
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- HISTORICAL CONTROL DATA: please refer to Table 2 under “Any other informations on results incl. tables”
Any other information on results incl. tables
Table 1: Experimental results of the micronucleus test
Mononuclear cells number | Micronucleated cells number | P-value | Interpretation | |
Experiment I: 4 h + S9 mix | ||||
Vehicle control | 994 | 6 | ||
995 | 5 | |||
CP 1.5 µg/mL | 979 | 21 | 1.84E-08 | significant |
964 | 36 | |||
250 µg/mL | 996 | 4 | 4.90E-01 | non significant |
996 | 4 | |||
125 µg/mL | 995 | 5 | 8.27E-01 | non significant |
995 | 5 | |||
62.5 µg/mL | 997 | 3 | 4.90E-01 | non significant |
995 | 5 | |||
31.25 µg/mL | 993 | 7 | 8.27E-01 | non significant |
997 | 3 | |||
Negative control | 999 | 1 | ||
995 | 5 | |||
Experiment I (Repetition): 4 h - S9 mix | ||||
Vehicle control | 995 | 5 | ||
997 | 3 | |||
MMC 0.2 µg/mL | 814 | 186 | 1.50E-94 | significant |
781 | 219 | |||
Col 0.1 µg/mL | 879 | 121 | 1.61E-43 | non significant |
917 | 83 | |||
250 µg/mL | 996 | 4 | 1.00E+00 | non significant |
996 | 4 | |||
125 µg/mL | 998 | 2 | 7.96E-01 | non significant |
995 | 5 | |||
62.5 µg/mL | 996 | 4 | 1.00E+00 | non significant |
996 | 4 | |||
31.25 µg/mL | 998 | 2 | 7.96E-01 | non significant |
995 | 5 | |||
Negative control | 997 | 3 | ||
995 | 5 | |||
Experiment II: 24 h - S9 mix | ||||
Vehicle control | 994 | 6 | ||
996 | 4 | |||
MMC 0.02 µg/mL | 916 | 84 | 3.34E-28 | significant |
940 | 60 | |||
Col 0.01 µg/mL | 961 | 39 | 1.23E-11 | non significant |
969 | 31 | |||
250 µg/mL | 997 | 3 | 4.66E-01 | non significant |
996 | 4 | |||
125 µg/mL | 998 | 2 | 4.66E-01 | non significant |
995 | 5 | |||
62.5 µg/mL | 997 | 3 | 3.16E-01 | non significant |
997 | 3 | |||
31.25 µg/mL | 998 | 2 | 6.37E-01 | non significant |
994 | 6 | |||
Negative control | 995 | 5 | ||
997 | 3 | |||
CP: Cyclophosphamide; MMC: Mitomycin C; Col: Colchicine | ||||
Table 2: Historical control data
Without S9 | With S9 | |||||||
4 h treatment (n=30) | 24 h treatment (n=29) | 4 h treatment (n=30) | ||||||
Vehicle* | Mitomycin | Colchicine | Vehicle* | Mitomycin | Colchicine | Vehicle* | Cyclophosphamide | |
Concentration (µg/mL) | 0.2 | 0.1 | 0.02 | 0.01 | 1.5 | |||
Minimum | 5 | 261 | 91 | 5 | 93 | 43 | 5 | 58 |
Maximum | 13 | 489 | 326 | 14 | 228 | 231 | 15 | 233 |
Mean | 8 | 368 | 180 | 8 | 135 | 76 | 9 | 128 |
Standard deviation | 2 | 70 | 55 | 2 | 28 | 42 | 3 | 40 |
Vehicle*: culture medium or ethanol; data were generated in 2015-2018 |
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in mouse lymphoma L5178 cells with and without metabolic activation.
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