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EC number: 224-583-8 | CAS number: 4419-11-8
- 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 gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- September 2021 - January 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Test material
- Reference substance name:
- Unknown impurities
- Molecular formula:
- This reference substance refers to from one to several impurities with potentially differing molecular formulas.
- IUPAC Name:
- Unknown impurities
- Reference substance name:
- 2,2'-azobis[2,4-dimethylvaleronitrile]
- EC Number:
- 224-583-8
- EC Name:
- 2,2'-azobis[2,4-dimethylvaleronitrile]
- Cas Number:
- 4419-11-8
- Molecular formula:
- C14H24N4
- IUPAC Name:
- 2-[(E)-2-(1-cyano-1,3-dimethylbutyl)diazen-1-yl]-2,4-dimethylpentanenitrile
- Test material form:
- solid: crystalline
impurity 1
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: study sponsor, Batch No 201902029
- Purity, including information on contaminants, isomers, etc.: 98.42 %
RADIOLABELLING INFORMATION (if applicable)
not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in a closed vessel in the fridge (2 - 8 °C).
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: assumed stable for purposes of the test
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: assumed stable for purposes of the test
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The solubility of the test item was determined in a non-GLP pre-test in culture medium (RPMI 1640) at a concentration of 20 mg/mL and dimethyl sulfoxide (DMSO), ethanol as well as acetone at a concentration of 400 mg/mL. The test item was completely insoluble in RPMI 1640, DMSO and ethanol but completely dissolved in acetone.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): assumed unreactive for purposes of the test
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the 1st day of the pre-test, a stock solution containing 400 mg/mL (nominal) of the test item in acetone was prepared. This stock solution was afterwards used to prepare the ge-ometric series (factor 2) of the resulting test item concentrations. The concentrations of the stock solutions for experiment I and II were 50 mg/mL (experiment I +S9) and 25 mg/mL (experiment I -S9 and experiment II).
FORM AS APPLIED IN THE TEST (if different from that of starting material)
same as starting material
INFORMATION ON NANOMATERIALS
no Nanoform
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
no biocide
OTHER SPECIFICS
none stated
Method
- Target gene:
- Thymidine Kinase locus
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: purchased at ATCC (Wesel, Germany) as L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™)
- Suitability of cells: please refer to OECD Guideline
- Normal cell cycle time (negative control): 10-12 h in stock cultures
For cell lines:
- Absence of Mycoplasma contamination: yes, checked
- Number of passages if applicable: not stated
- Methods for maintenance in cell culture: stored in liquid nitrogen in the cell bank of LAUS GmbH
- Cell cycle length, doubling time or proliferation index : 10-12 h in stock cultures
- Modal number of chromosomes: 40 +- 2
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: yes
For lymphocytes:
not applicable
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 complete culture medium with 10 % HS, at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 (liver enzyme mixture used for the test with metabolic activation)
- Test concentrations with justification for top dose:
- Experiment I +S9: 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781 mg/mL
Experiment I -S9, Experiment II: 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391 mg/mL
According to the OECD guideline 490 the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is lowest), unless limited by the solubility or toxicity of the test item. RCE values below 20 % are considered toxic. In case of toxic effects, the high-est test item concentration of the main experiment should reduce the RSG value to 10 - 20 %. For poorly soluble test items that are not cytotoxic at concentrations below the low-est insoluble concentrations, the highest concentration analysed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test item.
In reference to the results of the pre-test (precipitation was taken into account), 7 concen-trations were chosen for experiment I and II - Vehicle / solvent:
- acetone
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Remarks:
- RPMI 1640 medium is used as solvent control for the positive control MMS in a final con-centration of 0.5 % (positive control) during treatment.
0.9 % NaCl is used as solvent control for the positive control CPAm in a final concentra-tion of 0.5 % during
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate,
- Number of independent experiments 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 4*10^3
- Test substance added in medium;
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: none
- Exposure duration/duration of treatment: 4h/24h
- Harvest time after the end of treatment (sampling/recovery times): 48 h
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h (= harvest time)
- Selection time (if incubation with a selective agent): 12 d
- Fixation time (start of exposure up to fixation or harvest of cells): no fixation, 48 h (=harvest time)
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. TFT, during selection time
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 4*10^3, counted manually
- Criteria for small (slow growing) and large (fast growing) colonies:
Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
Less than 25 % of the well’s diameter = small colony
More than 25 % of the well’s diameter = large colony
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative cloning efficiency (RCE) and relative total growth (RTG - Rationale for test conditions:
- according to Guideline
- Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
• the induced mutation frequency reproducibly exceeds a threshold of 126 colo-nies per 106 cells above the corresponding solvent control.
• the relative increase of the mutation frequency shows a dose relationship.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if:
• the induced mutation frequency does not exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
• the relative increase of the mutation frequency does not show a dose relationship.
A mutagenic response is considered to be reproducible if it occurs in both parallel cul-tures. - Statistics:
- A linear regression (least squares) of the test item concentrations was performed to as-sess a possible dose dependent increase of mutant frequencies.
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:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- two individual experiments performed leading to same results.
Any other information on results incl. tables
The results of the mutagenicity assay with metabolic activation are presented in the following tables.
Mutagenicity Experiment I with Metabolic Activation
Culture | Content | Cells seeded per well | No. of empty wells | Wells with small colonies | Wells with large colonies | |||
|
|
| plate 1 | plate 2 | plate 1 | plate 2 | plate 1 | plate 2 |
A | Solvent Control Test Item (Acetone 0.5%) | 71 | 60 | 23 | 31 | 9 | 10 | 71 |
B | Solvent Control Test Item (Acetone 0.5%) | 66 | 69 | 23 | 19 | 11 | 9 | 66 |
A | Solvent Control CPAm (NaCl 0.9%) | 66 | 64 | 23 | 24 | 13 | 13 | 66 |
B | Solvent Control CPAm (NaCl 0.9%) | 77 | 75 | 20 | 10 | 3 | 11 | 77 |
A | Positive Control CPAm | 49 | 48 | 56 | 58 | 19 | 14 | 49 |
B | Positive Control CPAm | 47 | 48 | 63 | 56 | 14 | 19 | 47 |
A | Test Item 0.25 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.25 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
A | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
A | Test Item 0.06 mg/mL | 66 | 55 | 34 | 34 | 6 | 15 | 66 |
B | Test Item 0.06 mg/mL | 70 | 59 | 29 | 40 | 6 | 9 | 70 |
A | Test Item 0.03 mg/mL | 64 | 66 | 35 | 20 | 8 | 11 | 64 |
B | Test Item 0.03 mg/mL | 60 | 58 | 37 | 31 | 16 | 16 | 60 |
A | Test Item 0.016 mg/mL | 70 | 70 | 24 | 24 | 8 | 9 | 70 |
B | Test Item 0.016 mg/mL | 63 | 55 | 34 | 43 | 7 | 9 | 63 |
A | Test Item 0.008 mg/mL | 67 | 65 | 29 | 31 | 7 | 6 | 67 |
B | Test Item 0.008 mg/mL | 57 | 68 | 38 | 26 | 11 | 6 | 57 |
A | Test Item 0.004 mg/mL | n/e | n/e | n/e | n/e | n/e | n/e | n/e |
B | Test Item 0.004 mg/mL | n/e | n/e | n/e | n/e | n/e | n/e | n/e |
Additional untreated control (medium control) | ||||||||
A | Solvent control (RPMI) | 73 | 62 | 24 | 21 | 7 | 17 | 73 |
B | Solvent control (RPMI) | 67 | 64 | 24 | 26 | 11 | 14 | 67 |
A | Acetone 0.5 % | 71 | 60 | 23 | 31 | 9 | 10 | 71 |
B | Acetone 0.5 % | 66 | 69 | 23 | 19 | 11 | 9 | 66 |
n/a = not analysed for mutagenicity because of cytotoxicity
n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations
Results Mutation frequency Experiment I with Metabolic Activation
Culture | Content | Small mutant colonies / 106cells | Large mutant colonies / 106cells | Mutant colonies / 106cells | Small mutant colonies / 106cells (Mean both cultures) |
| Mutant colonies / 106cells (Mean both cultures) |
|
A | Solvent Control Test Item (Acetone 0.5%) | 72.9 | 23.0 | 84.4 | 71 | 27 | 91 |
|
B | Solvent Control Test Item (Acetone 0.5%) | 68.8 | 30.7 | 98.2 |
| |||
A | Solvent Control CPAm (NaCl 0.9%) | 82.5 | 42.8 | 114.7 | 62 | 31 | 86 |
|
B | Solvent Control CPAm (NaCl 0.9%) | 41.7 | 18.6 | 57.3 |
| |||
A | Positive Control CPAm | 499.6 | 104.6 | 378.7 | 598 | 120 | 443 |
|
B | Positive Control CPAm | 696.2 | 135.8 | 506.6 |
| |||
A | Test Item 0.25 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a |
|
B | Test Item 0.25 mg/mL | n/a | n/a | n/a |
| |||
A | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a |
|
B | Test Item 0.13 mg/mL | n/a | n/a | n/a |
| |||
A | Test Item 0.06 mg/mL | 109.3 | 29.0 | 115.4 | 108 | 24 | 106 |
|
B | Test Item 0.06 mg/mL | 107.2 | 19.6 | 95.7 |
| |||
A | Test Item 0.03 mg/mL | 77.9 | 24.1 | 90.0 | 110 | 42 | 124 |
|
B | Test Item 0.03 mg/mL | 141.8 | 59.1 | 157.8 |
| |||
A | Test Item 0.016 mg/mL | 74.7 | 24.1 | 82.0 | 110 | 24 | 110 |
|
B | Test Item 0.016 mg/mL | 145.7 | 24.7 | 138.4 |
| |||
A | Test Item 0.008 mg/mL | 99.0 | 18.5 | 99.0 | 109 | 23 | 112 |
|
B | Test Item 0.008 mg/mL | 119.0 | 27.2 | 125.9 |
| |||
A | Test Item 0.004 mg/mL | n/e | n/e | n/e | n/e | n/e | n/e |
|
B | Test Item 0.004 mg/mL | n/e | n/e | n/e |
| |||
Additional untreated control (medium control) | ||||||||
A | Solvent control (RPMI) | 71.8 | 35.9 | 94.7 | 79 | 38 | 102 | |
B | Solvent control (RPMI) | 85.8 | 39.7 | 108.7 | ||||
A | Acetone 0.5 % | 72.9 | 23.0 | 84.4 | 71 | 27 | 91 | |
B | Acetone 0.5 % | 68.8 | 30.7 | 98.2 |
n/a = not analysed for mutagenicity because of cytotoxicity
n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations
Mutagenicity Experiment I without Metabolic Activation
Culture | Content | Cells seeded per well | No. of empty wells | Wells with small colonies | Wells with large colonies | |||
|
|
| plate 1 | plate 2 | plate 1 | plate 2 | plate 1 | plate 2 |
A | Solvent Control Test Item (Acetone 0.5%) | 67 | 72 | 25 | 26 | 11 | 7 | 67 |
B | Solvent Control Test Item (Acetone 0.5%) | 59 | 68 | 37 | 19 | 7 | 13 | 59 |
A | Solvent Control MMS (RPMI 1640) | 71 | 69 | 19 | 20 | 9 | 12 | 71 |
B | Solvent Control MMS (RPMI 1640) | 66 | 62 | 17 | 24 | 16 | 12 | 66 |
A | Positive Control MMS | 45 | 44 | 64 | 55 | 15 | 11 | 45 |
B | Positive Control MMS | 49 | 50 | 64 | 57 | 12 | 12 | 49 |
A | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
A | Test Item 0.06 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.06 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
A | Test Item 0.03 mg/mL | 61 | 65 | 38 | 33 | 8 | 7 | 61 |
B | Test Item 0.03 mg/mL | 50 | 65 | 54 | 37 | 11 | 9 | 50 |
A | Test Item 0.016 mg/mL | 63 | 57 | 30 | 35 | 11 | 10 | 63 |
B | Test Item 0.016 mg/mL | 55 | 59 | 37 | 41 | 9 | 9 | 55 |
A | Test Item 0.008 mg/mL | 65 | 65 | 23 | 26 | 13 | 11 | 65 |
B | Test Item 0.008 mg/mL | 73 | 62 | 17 | 31 | 9 | 11 | 73 |
A | Test Item 0.004 mg/mL | 67 | 64 | 20 | 21 | 12 | 14 | 67 |
B | Test Item 0.004 mg/mL | 65 | 68 | 33 | 15 | 11 | 17 | 65 |
A | Test Item 0.002 g/mL | n/e | n/e | n/e | n/e | n/e | n/e | n/e |
B | Test Item 0.002 g/mL | n/e | n/e | n/e | n/e | n/e | n/e | n/e |
Additional untreated control (medium control) | ||||||||
A | Solvent control (RPMI) | 59 | 67 | 32 | 23 | 15 | 10 | 59 |
B | Solvent control (RPMI) | 67 | 67 | 21 | 24 | 13 | 8 | 67 |
A | Acetone 0.5 % | 67 | 72 | 25 | 26 | 11 | 7 | 67 |
B | Acetone 0.5 % | 59 | 68 | 37 | 19 | 7 | 13 | 59 |
n/a = not analysed for mutagenicity because of cytotoxicity
n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations
Results Mutation frequency Experiment I without Metabolic Activation
Culture | Content | Small mutant colonies / 106cells | Large mutant colonies / 106cells | Mutant colonies / 106cells | Small mutant colonies / 106cells (Mean both cultures) | Large mutant colonies / 106cells (Mean both cultures) | Mutant colonies / 106cells (Mean both cultures) |
A | Solvent Control Test Item (Acetone 0.5%) | 80.3 | 25.6 | 84.0 | 82 | 26 | 93 |
B | Solvent Control Test Item (Acetone 0.5%) | 84.6 | 27.0 | 101.4 | |||
A | Solvent Control MMS (RPMI 1640) | 55.7 | 28.4 | 77.5 | 59 | 35 | 91 |
B | Solvent Control MMS (RPMI 1640) | 62.5 | 41.0 | 105.4 | |||
A | Positive Control MMS | 601.8 | 90.6 | 478.5 | 573 | 82 | 421 |
B | Positive Control MMS | 544.8 | 73.1 | 362.8 | |||
A | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.13 mg/mL | n/a | n/a | n/a | |||
A | Test Item 0.06 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.06 mg/mL | n/a | n/a | n/a | |||
A | Test Item 0.03 mg/mL | 126.7 | 22.3 | 115.6 | 148 | 26 | 126 |
B | Test Item 0.03 mg/mL | 169.9 | 29.1 | 135.6 | |||
A | Test Item 0.016 mg/mL | 83.1 | 23.3 | 94.5 | 112 | 25 | 118 |
B | Test Item 0.016 mg/mL | 140.7 | 26.6 | 140.7 | |||
A | Test Item 0.008 mg/mL | 79.5 | 36.0 | 105.2 | 76
| 32 | 97 |
B | Test Item 0.008 mg/mL | 71.9 | 27.5 | 88.0 | |||
A | Test Item 0.004 mg/mL | 61.3 | 37.1 | 97.5 | 72 | 41 | 102 |
B | Test Item 0.004 mg/mL | 82.9 | 45.4 | 105.8 | |||
A | Test Item 0.002 g/mL | n/e | n/e | n/e | n/e | n/e | n/e |
B | Test Item 0.002 g/mL | n/e | n/e | n/e | |||
Additional untreated control (medium control) | |||||||
A | Solvent control (RPMI) | 89.3 | 36.9 | 111.5 | 80 | 34 | 103 |
B | Solvent control (RPMI) | 70.7 | 30.7 | 95.2 | |||
A | Acetone 0.5 % | 80.3 | 25.6 | 84.0 | 82 | 26 | 93 |
B | Acetone 0.5 % | 84.6 | 27.0 | 101.4 |
n/a = not analysed for mutagenicity because of cytotoxicity
n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations
Mutagenicity Experiment II without Metabolic Activation
Culture | Content | Cells seeded per well | No. of empty wells | Wells with small colonies | Wells with large colonies | |||
|
|
| plate 1 | plate 2 | plate 1 | plate 2 | plate 1 | plate 2 |
A | Solvent Control Test Item (Acetone 0.5%) | 3997 | 61 | 65 | 11 | 11 | 28 | 30 |
B | Solvent Control Test Item (Acetone 0.5%) | 4001 | 69 | 68 | 8 | 8 | 25 | 27 |
A | Solvent Control MMS (RPMI 1640) | 3999 | 61 | 65 | 9 | 8 | 30 | 27 |
B | Solvent Control MMS (RPMI 1640) | 4001 | 71 | 81 | 7 | 5 | 22 | 12 |
A | Positive Control MMS | 3995 | 36 | 34 | 54 | 64 | 44 | 50 |
B | Positive Control MMS | 4000 | 45 | 41 | 40 | 45 | 36 | 40 |
A | Test Item 0.13 mg/mL | 4001 | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.13 mg/mL | 3995 | n/a | n/a | n/a | n/a | n/a | n/a |
A | Test Item 0.06 mg/mL | 4003 | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.06 mg/mL | 3996 | n/a | n/a | n/a | n/a | n/a | n/a |
A | Test Item 0.03 mg/mL | 4003 | 71 | 70 | 11 | 6 | 22 | 22 |
B | Test Item 0.03 mg/mL | 4000 | 70 | 78 | 6 | 8 | 21 | 12 |
A | Test Item 0.016 mg/mL | 3998 | 60 | 63 | 7 | 10 | 32 | 28 |
B | Test Item 0.016 mg/mL | 3998 | 65 | 64 | 7 | 10 | 29 | 27 |
A | Test Item 0.008 mg/mL | 4003 | 77 | 76 | 4 | 8 | 17 | 15 |
B | Test Item 0.008 mg/mL | 4000 | 69 | 65 | 6 | 10 | 24 | 26 |
A | Test Item 0.004 mg/mL | 4002 | 75 | 62 | 9 | 9 | 19 | 33 |
B | Test Item 0.004 mg/mL | 3999 | 67 | 71 | 9 | 9 | 26 | 21 |
A | Test Item 0.002 g/mL | 4000 | n/e | n/e | n/e | n/e | n/e | n/e |
B | Test Item 0.002 g/mL | 4003 | n/e | n/e | n/e | n/e | n/e | n/e |
Additional untreated control (medium control) | ||||||||
A | Solvent control (RPMI) | 3999 | 61 | 65 | 9 | 8 | 30 | 27 |
B | Solvent control (RPMI) | 4001 | 71 | 81 | 7 | 5 | 22 | 12 |
A | Acetone 0.5 % | 3997 | 61 | 65 | 11 | 11 | 28 | 30 |
B | Acetone 0.5 % | 4001 | 69 | 68 | 8 | 8 | 25 | 27 |
n/a = not analysed for mutagenicity because of cytotoxicity
n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations
Results Mutation frequency Experiment II without Metabolic Activation
Culture | Content | Small mutant colonies / 106cells | Large mutant colonies / 106cells | Mutant colonies / 106cells | Small mutant colonies / 106cells (Mean both cultures) | Large mutant colonies / 106cells (Mean both cultures) | Mutant colonies / 106cells (Mean both cultures) |
A | Solvent Control Test Item (Acetone 0.5%) | 29.9 | 88.3 | 103.4 | 27 | 87 | 97 |
B | Solvent Control Test Item (Acetone 0.5%) | 23.5 | 85.1 | 91.0 | |||
A | Solvent Control MMS (RPMI 1640) | 27.2 | 103.4 | 123.6 | 23 | 81 | 97 |
B | Solvent Control MMS (RPMI 1640) | 19.3 | 58.2 | 69.7 | |||
A | Positive Control MMS | 844.0 | 595.3 | 893.1 | 734 | 566 | 875 |
B | Positive Control MMS | 623.2 | 537.1 | 856.0 | |||
A | Test Item 0.13 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.13 mg/mL | n/a | n/a | n/a | |||
A | Test Item 0.06 mg/mL | n/a | n/a | n/a | n/a | n/a | n/a |
B | Test Item 0.06 mg/mL | n/a | n/a | n/a | |||
A | Test Item 0.03 mg/mL | 26.7 | 75.1 | 89.1 | 25 | 67 | 85 |
B | Test Item 0.03 mg/mL | 23.4 | 58.3 | 80.4 | |||
A | Test Item 0.016 mg/mL | 25.9 | 104.6 | 124.3 | 23 | 89 | 104 |
B | Test Item 0.016 mg/mL | 19.6 | 72.9 | 84.0 | |||
A | Test Item 0.008 mg/mL | 15.8 | 44.7 | 55.7 | 21 | 67 | 82 |
B | Test Item 0.008 mg/mL | 26.0 | 90.2 | 107.5 | |||
A | Test Item 0.004 mg/mL | 28.4 | 91.1 | 97.4 | 30 | 90 | 100 |
B | Test Item 0.004 mg/mL | 30.9 | 88.1 | 103.6 | |||
A | Test Item 0.002 g/mL | n/e | n/e | n/e | n/e | n/e | n/e |
B | Test Item 0.002 g/mL | n/e | n/e | n/e | |||
Additional untreated control (medium control) | |||||||
A | Solvent control (RPMI) | 27.2 | 103.4 | 123.6 | 23 | 81 | 97 |
B | Solvent control (RPMI) | 19.3 | 58.2 | 69.7 | |||
A | Acetone 0.5 % | 29.9 | 88.3 | 103.4 | 27 | 87 | 97 |
B | Acetone 0.5 % | 23.5 | 85.1 | 91.0 |
n/a = not analysed for mutagenicity because of cytotoxicity
n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations
Statistical results
A linear regression (least squares) of the test item concentrations was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) is considered as critical.
The following results were obtained:
Table Regression Parameters of Test Item
Treatment | Correlation coefficient |r| | F krit |
Exp. I with metabolic activation | 0.321 | 0.679 |
Exp. I without metabolic activation | 0.919 | 0.081 |
Exp. II without metabolic activation | 0.300 | 0.700 |
The positive controls were tested at one concentration only. Therefore, no dose-dependency could be evaluated, although the positive controls showed considerable increases in mutants. In the following table, the statistical significance values are presented. The chi-square test was used.
Statistically significant increase in mutants is considered as given if p is below 0.01.
Table Statistical Significance
Substance | Concentration | p-Values | |
without S9 | with S9 | ||
Positive Control MMS | 19.5 (Experiment I) 12.5 (Experiment II) | < 0.001 | -- |
Positive Control CPAm | 5.5 | -- | < 0.001 |
Applicant's summary and conclusion
- Conclusions:
- under the experimental conditions reported the test item did not induce gene mutations at the thymidine kinase locus (Tk1) in heterozygous mouse lymphoma L5178Y Tk+/- cells.
Therefore, the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) is considered to be “not mu-tagenic under the conditions of the mouse lymphoma assay”. - Executive summary:
The study was performed to investigate the potential of the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) to induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk +/- cells.
The assay was performed in a pre-test and two independent and valid experiments, using two parallel cultures each (replicates).
In experiment I, 7 concentrations of the test item were tested with and without metabolic activation and a treatment period of 4 h. Afterwards, 4 concentrations were evaluated for mutant frequency. In experiment II, 7 concentrations of the test item were tested without metabolic activation and a treatment period of 24 h. Afterwards, 4 concentrations were evaluated for mutant frequency.
MMS (19.5 µg/mL in experiment I and 12.5 µg/mL in experiment II) and CPAm (5.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and exceeded the number of mutant colonies of more than 300 in comparison to the corresponding solvent control.
In both experiments of this study (short exposure with and without S9 mix, extended exposure without S9 mix), the range of the spontaneous mutant frequency of the solvent controls was in the range of the historical data.
Since all further acceptability criteria of the assay were also met the study is valid.
The following nominal concentrations of the test item were investigated in experiment I:
+S9: 0.25 mg/mL, 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL
-S9: 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL, 0.002 mg/mL
The following nominal concentrations of the test item were investigated in experiment II: 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL, 0.002 mg/mL.
None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration.
In experiment I with metabolic activation, precipitation of the test item, visible to the unaided eye, was noted at the concentrations 0.25 mg/mL and 0.13 mg/mL and without metabolic activation at 0.13 mg/mL and 0.06 mg/mL.
In experiment II, precipitation of the test item, visible to the unaided eye, was noted at the concentrations 0.13 mg/mL and 0.06 mg/mL.
No significant reduction of growth was observed with and without S9 after 4 h treatment with the test item (all concentrations) in all experimental parts.
According to the Guideline OECD490, the highest concentrations analysed, showed precipitates, therefor the following 4 test item concentrations could be evaluated for mutagenicity:
+S9: 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL
-S9: 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL
In experiment II (- S9) a strong reduction of viability was observed in the three highest concentrations, but the RTG values for all concentrations were higher than 20 %. Since the two highest concentrations showed precipitates, the following test item concentrations could be evaluated for mutagenicity: 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL
In all evaluated concentrations of the test item no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.
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