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EC number: 219-976-6 | CAS number: 2589-57-3
- 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:
- December 2015 - June 2016
- 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)
- Qualifier:
- according to guideline
- Guideline:
- other: B.49 (In Vitro Mammalian Cell Micronucleus Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- Dimethyl 2,2'-azobis(2-methylpropionate)
- EC Number:
- 219-976-6
- EC Name:
- Dimethyl 2,2'-azobis(2-methylpropionate)
- Cas Number:
- 2589-57-3
- Molecular formula:
- C10H18N2O4
- IUPAC Name:
- dimethyl 2,2'-azobis(2-methylpropionate)
- Test material form:
- solid: flakes
Constituent 1
Method
- Target gene:
- whole genom is targeted
Species / strain
- Species / strain / cell type:
- lymphocytes: peripheral
- Details on mammalian cell type (if applicable):
- The blood sample was obtained from a healthy donor who neither smokes nor receives
medication. The following donor was chosen for the experimental part:
female, 34 years old
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9
- Test concentrations with justification for top dose:
- Nominal concentration of test item solution (mg/mL): 400, 200, 100, 50, 25, 12.5, 6.3, 3.2
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was determined in a non-GLP pre-test. The test item was sufficiently soluble in DMSO.
Therefore, DMSO was chosen as solvent.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable):
DURATION
- Preincubation period: 47.5 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 18 h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h (Exposure duration + expression time)
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): cytochalasin
B
STAIN (for cytogenetic assays): 10 % solution of Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension onto a clean microscope slide.
The cells were then stained with a 10 % solution of Giemsa. All slides were independently
coded before microscopic analysis.
NUMBER OF CELLS EVALUATED: at least 500 cells per culture, cytokinesis-block proliferation index
At least 1000 binucleate cells per culture were scored for micronuclei
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Only cells with sufficiently
distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included
in the analysis.
DETERMINATION OF CYTOTOXICITY
Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent
solvent control
OTHER EXAMINATIONS:
-none
- OTHER: - Rationale for test conditions:
- according to Guideline
- Evaluation criteria:
- The test item is considered to have genotoxic effects if:
At least one test concentration shows a statistically significant increase of micronucleate
cells compared to the concurrent solvent control.
In at least one experimental condition a dose-related increase of micronucleate cells
can be observed.
Any of the results lies outside the range of the historical laboratory control data for solvent
controls. - Statistics:
- The number of binucleate cells with micronuclei in each treatment group was compared
with the solvent control. Statistical significance was tested using Fisher’s exact test at the
five per cent level (p 0.05)
For positive controls with high values of binucleate cells with micronuclei, the chi-squaretest
was used
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: peripheral
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- In conclusion, under the experimental conditions reported, dimethyl-2,2'-
azobisisobutyrate induced the formation of micronuclei in human lymphocytes in
vitro.
The test item dimethyl-2,2'-azobisisobutyrate is considered as “genotoxic under the
conditions of the test”. - Executive summary:
One valid experiment was performed.
This study was performed to assess the genotoxic potential of dimethyl-2,2'-
azobisisobutyrate to induce formation of micronuclei in human lymphocytes cultured in
vitro in the absence and the presence of an exogenous metabolic activation system (liver
S9 mix from male rats, treated with Aroclor 1254).
The test item was dissolved in DMSO. A stock solution with a concentration of 400 mg/mL
(corresponding to a final test concentration of 2 mg/mL) and thereof a geometric series of
dilutions was prepared.
Human lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin
and exposed to solvent control, test item and positive control.
After exposure period and expression time in growth medium (= culture harvest time), the
cells were harvested and slides were prepared. Then, the proportion of binucleate cells
containing micronuclei was determined.
The following schedule was observed:
Procedure
Exp. I
Metabolic activation
Without S9 mix
With S9 mix
Exposure period
4 h
4 h
Expression time in growth
medium
18 h 18 h Culture harvest time
22 h
22 h
Concentrations selected for
scoring of micronuclei
2, 0.25 and 0.03 mg/mL
2, 1 and 0.5 mg/mL
One valid experiment with 2 experimental conditions - without and with metabolic activation
- was performed. All cell cultures were set up in duplicates. In order to assess the toxicity
of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation
index was calculated for all cultures treated with solvent control, positive control and test
item. On the basis of these data, the concentrations indicated in the table above were selected
for micronuclei scoring.
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