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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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The substance was tested in two Ames Assays, one mammalian Mutation Assay and one mammalian Cell Micronucleus Assay according to Guidelines (OECD 471, OECD 490 and OECD 487 respectively),
Information on Gene Mutation in Mammalian Cells is provided by Predictions obtained with OECD QSAR Toolbox. Predictions are provided from two categories formed by application of
the profiles "organic functional groups: Azo" and "organic functional groups: Nitrile". This approach was followed as no category of substances was available having both (or all*) profiles of the substance.
*The substance has the profile "organic functional groups: Azo, Nitrile, Azonitrile, Isopropyl and Alkane, branched with tertiary carbon"
Link to relevant study records
- 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
- 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
- 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 - Target gene:
- Thymidine Kinase locus
- 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
- 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.
- 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.
- 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.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- May 2021 - Sep 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- 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: Sponsor, Batch Nr. 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: 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: stable under storage conditions
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no information provided in report
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: no information provided in report
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no information provided in report
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
no special treatment
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: no differences
INFORMATION ON NANOMATERIALS
no nano material
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
no biocide/pesticide
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: no information available - Target gene:
- histidine deficiency
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat Liver S9
- Test concentrations with justification for top dose:
- The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.
The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313, 156 and 78 µg/plate.
Due to toxicity observed in experiment 2, additional concentrations were tested
The following nominal test item concentrations were prepared for experiment 2b:
TA98 (+S9), TA102 (-S9), TA1535 (-S9):
625, 313, 156, 78, 39, 19.5, 9.8 µg/plate
TA1335 (+S9), TA1537 (+S9):
313, 156, 78, 39, 19.5, 9.8, 4.88 µg/plate
TA98 (-S9), TA1537 (-S9):
78, 39, 19.5, 9.8, 4.88, 2.44, 1.22 µg/plate
justification: according to Guideline - Vehicle / solvent:
- Ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene
- Remarks:
- The negative controls are consisting of solvent or vehicle alone, without the test item or the positive control substances and otherwise treated in the same way as the treatment samples. The control samples were used to provide the corresponding values fo
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) triplicate
- Number of independent experiments 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): no applicable
- Test substance added in agar (plate incorporation); preincubation;
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Incubation time: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition;
METHODS FOR MEASUREMENTS OF GENOTOXICIY
visual colony counting - Rationale for test conditions:
- according to Guideline
- Evaluation criteria:
- A result is considered as clearly positive if all following criteria are fulfilled:
- A concentration-related increase, in revertants
- a clear biological relevant increase in at least one concentration compared to the concurrent solvent control
- at least one concentration with an increase above the distribution of historical solvent control data (mean ± 3 SD).
A biologically relevant increase is described as follows:
- if in the bacteria strains TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
- if in the bacteria strains TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0). - Statistics:
- no statistical analysis applied, only mean and standard deviation are calculated and stated.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO
Sodium azide (Na-azide) in demin. water
Mitomycin C (MMC) in demin. water - With metabolic activation:
2-Amino anthracene (2-AA) in DMSO
Benzo-a-pyrene (BaP) in DMSO - Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO
Sodium azide (Na-azide) in demin. water
Mitomycin C (MMC) in demin. water - With metabolic activation:
2-Amino anthracene (2-AA) in DMSO
Benzo-a-pyrene (BaP) in DMSO - Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO
Sodium azide (Na-azide) in demin. water
Mitomycin C (MMC) in demin. water - With metabolic activation:
2-Amino anthracene (2-AA) in DMSO
Benzo-a-pyrene (BaP) in DMSO - Conclusions:
- Based on the results of this study it is concluded that 2,2'-Azobis(2,4-
dimethylvaleronitrile) is not mutagenic in the Salmonella typhimurium strains TA98,
TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activa-
tion under the experimental conditions in this study. - Executive summary:
Findings and Results:
The study procedures described in this report were based on the most recent Guideline
OECD 471 (2020) and EU Method B.13/14 (2008).
The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) was tested in the Bacterial reverse mu-
tation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and
TA1537).
The test was performed in three valid experiments in the presence and absence of metabolic
activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing
for absence of metabolic activation.
Exp. 2 had to be repeated (exp. 2b) due to an insufficient number of analyzable non-toxic
concentrations.Experiment 1:
In the first experiment, the test item (dissolved in ethanol) was tested up to concentrations
of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102,
TA1535 and TA1537 using the plate incorporation method.
The test item showed a few precipitates on the plates with TA98, TA100, TA1535 and
TA1537 at 5000 µg/plate (for details see chapter 8.1.2, page 22).
No signs of toxicity towards the bacteria strains were observed.
The results of this experiment showed that none of the tested concentrations induced a
relevant or dose-related increase in the number of revertants in all tested strains, in the
presence and the absence of metabolic activation.Experiment 2:
Based on the results of the first experiment, the test item was tested up to concentrations of
5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the pre-
incubation method.
The test item showed a few precipitates on the plates with TA98, TA100, TA102, TA1535
and TA1537 at 5000, 2500 and 1250 µg/plate.
Signs of toxicity towards the bacteria strains TA98 (+/-S9), TA100 (-S9), TA102 (-S9),
TA1535 (+/-S9), TA1537 (+/-S9) were observed (for details see chapter 8.2.2, page 24).
Only for the strains TA100 (+/-S9) and TA102 (+S9), sufficient (at least five) non-toxic con-
centrations could be evaluated in this experiment. In these strains, none of the tested con-
centrations induced a relevant or dose-related increase in the number of revertants.
For the other strains, a repetition was performed in exp. 2b.Experiment 2b:
Based on the results of exp. 2, the test item was tested up to individual concentrations per
strain (see chapter 7.2, page 18) in the presence and absence of S9 mix in all bacteria
strains using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations and no signs
of toxicity could be observed. Therefore, sufficient concentrations (at least five) could be
evaluated for mutagenicity.
The results of this experiment showed that none of the tested concentrations induced a
relevant or dose-related increase in the number of revertants in all tested strains, in the
presence and the absence of metabolic activation- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25. October 2017 - 16. February 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.49
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- no single target gene, assay aims for whole genom
- Species / strain / cell type:
- primary culture, other: peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- Blood samples were obtained from healthy donors who neither smoke nor receive medication.
- Cytokinesis block (if used):
- cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from
Trinova Biochem GmbH, Gießen, and stored at – 80 ± 5°C. - Test concentrations with justification for top dose:
- 200 / 100 / 50 / 25 / 12.5 mg/mL
The solubility of the test item was determined in a non-GLP pre-test using cell culture medium,
dimethyl sulfoxide (DMSO) and ethanol absolute. The test item was insoluble in medium,
DMSO and ethanol at the required concentrations (20 mg/mL in medium and
400 mg/mL in DMSO and ethanol) but completely soluble in DMSO at the next lower concentration
(200 mg/mL). - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- 7.2.1 Cell Cultivation
The blood cultures were set up in defined time intervals within 24 h after collection in sterile
culture vessels, each containing 1 part of heparinised blood and 9 parts of complete
culture medium RPMI 1640 for cell proliferation. The cultures were incubated for 72 h at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
7.2.2 Cell Treatment
After the initial cell cultivation, duplicate cultures were prepared for each test group. After
centrifugation (10 min, 500 * g), the cells were resuspended in serum free RPMI 1640 and
solvent control, positive control or the single test item concentrations were added.
In the case of metabolic activation, 50 μL S9 mix per mL medium were used. The cell cultures
were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h
(exposure period)
After the exposure time of 4 h, the cells were spun down by gentle centrifugation for 5 min
(500 * g). The supernatant was discarded, the cells were re-suspended in 5 mL Saline G
and centrifuged again. The washing procedure was repeated once as described.
After washing, the cells were resuspended in complete culture medium RPMI 1640, cytochalasin
B (final concentration 5 μg/ml) was added and the cells were incubated at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 19 h until preparation.
In experiment II without metabolic activation, 72 h after seeding, the blood cultures were
centrifuged (10 min, 500 * g). The cell pellet was re-suspended in complete culture medium
RPMI 1640, cytoB (final concentration 5 μg/mL) and solvent control, positive control or
the test item concentrations were added. The exposure duration was 23 h.
7.2.3 Harvesting Procedure
Each cell culture was harvested and processed separately. The cells were spun down by
gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were
re-suspended in 12 mL hypotonic KCl solution. The cell suspension was allowed to stand
for 10 min at room temperature (20 ± 5°C). After removal of the hypotonic solution by centrifugation
(10 min, 500 * g), the cell pellet was fixed with a mixture of methanol and glacial
acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the cell suspension was
spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and
the cell pellet was re-suspended in fixative again. The washing procedures were repeated
until the cell pellet was white.
7.2.4 Preparation of Slides
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.
7.2.5 Determination of the Cytokinesis-Block Proliferation Index
In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture)
was determined in order to assess the cytotoxicity of the test item. From these determinations,
the test item concentrations which were evaluated for scoring of micronuclei
were defined.
7.2.6 Determination of Binucleated Cells with Micronuclei
At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently
distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included
in the analysis. - Rationale for test conditions:
- according to Guideline
- Evaluation criteria:
- The test item is considered to have no genotoxic effects if:
Neither a statistically significant nor a concentration-related increase of the number of
micronucleate cells in the evaluated test concentrations is observed.
The obtained results lie within the range of the historical laboratory control data for solvent
controls, considering also e.g. 95.5 % control limits where appropriate.
The test item is considered to have genotoxic effects if all of the following conditions are
met:
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 using trend analysis.
Any of the results lies outside the range of the historical laboratory control data for solvent
controls, considering also e.g. 95.5 % control limits where appropriate. - Statistics:
- The number of binucleated 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 binucleated cells with micronuclei, the chi-squaretest was used - Key result
- Species / strain:
- primary culture, other: human peripheral lymphocytes
- 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
- Conclusions:
- The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) is considered as “not genotoxic” under
the conditions of the test. - Executive summary:
This study was performed to assess the genotoxic potential of 2,2'-Azobis(2,4-
dimethylvaleronitrile) to induce formation of micronuclei in human lymphocytes cultured in
vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix
from male rats, treated with Aroclor 1254).
The test item was dissolved in DMSO to prepare a stock solution with a concentration of
200 mg/mL, corresponding to the highest concentration (1000 μg/mL) in the test. In addition,
a geometric series of 4 dilutions was prepared from the stock solution.
Two valid experiments were performed.
Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by
addition of phytohaemagglutinin and exposed to solvent control, test item and positive control.
After the culture harvest time, the cells were harvested and slides were prepared. Then,
the proportion of cells containing micronuclei was determined.
Two independent experiments were performed. In each experiment, 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 (CBPI) was calculated for all cultures
treated with solvent control, positive control and test item. On the basis of the data of the
cytokinesis-block proliferation index, the appropriate concentrations were selected for micronuclei
scoring.
None of the test item concentrations induced a cytotoxic effect in experiment I and II.
In experiment I without metabolic activation, precipitates were observed at the two highest
test item concentrations (1000 μg/mL and 500 μg/mL) at the end of treatment. In the approach
with metabolic activation, precipitates were clearly visible at the concentrations
1000 μg/mL, 500 μg/mL and 250 μg/mL. According to the OECD 487, 250 μg/mL was
chosen as highest analysable concentration in the approach with metabolic activation and
500 μg/mL in the approach without metabolic activation. In experiment II without metabolic
activation, no precipitates were observed up to the maximum test item concentration.
Therefore, the three highest test item concentrations were evaluated for micronuclei.
Neither a statistically significant nor a biologically relevant increase in the number of binucleated
cells containing micronuclei at the evaluated concentrations was observed.
All positive control compounds caused large, statistically significant increases in the proportion
of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.
In conclusion, under the experimental conditions reported, 2,2'-Azobis(2,4-
dimethylvaleronitrile) does not induce the formation of micronuclei in human lymphocytes
in vitro.
Referenceopen allclose all
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 |
1 Data of Experiment 1
1.1 Determination of Titre
Criterion: The determination of titre should give a number of at least 109 cells/mL, correlating to 100 colonies/plate after dilution.
Exact colony counts could not be determined due to the strong growth of the bacteria, but
by visual inspection it was obvious that the values exceed 100 colonies/ plate
Tabelle 14.1‑a Titre Values (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. |
Repl. 2 | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. |
Assessment | ok | ok | ok | ok | ok | ok | ok | ok | ok | ok |
s.g.= strong growth, too strong for counting of colonies
ok= the criterion was fulfilled
1.2 Sterility Control
Criterion: No colony per plate may grow.
Tabelle 14.2‑a Sterility (colonies per plate)
| Demin. water | DMSO | Ethanol |
Repl. 1 | 0 | 0 | 0 |
Repl. 2 | 0 | 0 | 0 |
Repl. 3 | 0 | 0 | 0 |
Repl. 4 | 0 | 0 | 0 |
Assessment | ok | ok | ok |
ok= the criterion was fulfilled
1.3 Spontaneous Revertants
1.3.1 Demin. Water
Tabelle 14.3‑a Spontaneous Revertants demin. water (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 16 | 17 | 71 | 74 | 140 | 148 | 9 | 9 | 4 | 5 |
Repl. 2 | 16 | 19 | 70 | 70 | 148 | 152 | 9 | 9 | 4 | 5 |
Repl. 3 | 18 | 20 | 62 | 69 | 160 | 156 | 11 | 10 | 5 | 6 |
Mean | 17 | 19 | 68 | 71 | 149 | 152 | 10 | 9 | 4 | 5 |
sd | 1.2 | 1.5 | 4.9 | 2.6 | 10.1 | 4.0 | 1.2 | 0.6 | 0.6 | 0.6 |
sd = standard deviation ±
1.3.2 DMSO
Tabelle 14.3‑b Spontaneous Revertants DMSO (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 15 | 19 | 65 | 74 | 140 | 144 | 8 | 9 | 5 | 5 |
Repl. 2 | 17 | 20 | 62 | 72 | 140 | 152 | 10 | 9 | 6 | 5 |
Repl. 3 | 17 | 21 | 58 | 60 | 152 | 160 | 11 | 10 | 6 | 7 |
Mean | 16 | 20 | 62 | 69 | 144 | 152 | 10 | 9 | 6 | 6 |
sd | 1.2 | 1.0 | 3.5 | 7.6 | 6.9 | 8.0 | 1.5 | 0.6 | 0.6 | 1.2 |
sd = standard deviation ±
1.3.3 Ethanol
Tabelle 14.3‑c Spontaneous Revertants Ethanol (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 14 | 17 | 70 | 74 | 132 | 144 | 8 | 9 | 5 | 4 |
Repl. 2 | 15 | 17 | 61 | 70 | 144 | 152 | 9 | 10 | 6 | 4 |
Repl. 3 | 15 | 20 | 58 | 66 | 156 | 160 | 11 | 12 | 6 | 6 |
Mean | 15 | 18 | 63 | 70 | 144 | 152 | 9 | 10 | 6 | 5 |
sd | 0.6 | 1.7 | 6.2 | 4.0 | 12.0 | 8.0 | 1.5 | 1.5 | 0.6 | 1.2 |
sd = standard deviation ±
1.4 Positive Controls
For used concentrations see chapter 6.2.4, page 14.
Tabelle 14.4‑a Diagnostic Mutagens (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Substance | NPD | BaP | Na-azide | 2-AA | MMC | 2-AA | Na-azide | 2-AA | NPD | 2-AA |
Repl. 1 | 688 | 156 | 720 | 1536 | 880 | 672 | 328 | 188 | 76 | 43 |
Repl. 2 | 704 | 168 | 704 | 1496 | 896 | 688 | 344 | 200 | 81 | 45 |
Repl. 3 | 752 | 184 | 664 | 1472 | 928 | 728 | 344 | 212 | 87 | 49 |
Mean | 715 | 169 | 696 | 1501 | 901 | 696 | 339 | 200 | 81 | 46 |
sd | 33.3 | 14.0 | 28.8 | 32.3 | 24.4 | 28.8 | 9.2 | 12.0 | 5.5 | 3.1 |
f(I) | 44.69 | 8.45 | 10.24 | 21.75 | 6.05 | 4.58 | 33.90 | 22.22 | 13.50 | 7.67 |
Rev. abs. | 699 | 149 | 628 | 1432 | 752 | 544 | 329 | 191 | 75 | 40 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
1.5 Mutagenicity Test Test Item 2,2'-Azobis(2,4-dimethylvaleronitrile)
Tabelle 14.5‑a Concentration 5000 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 14 | 12 | 45 | 55 | 136 | 144 | 9 | 9 | 3 | 3 |
Repl. 2 | 12 | 11 | 41 | 53 | 148 | 148 | 9 | 10 | 3 | 4 |
Repl. 3 | 11 | 9 | 37 | 42 | 160 | 156 | 9 | 10 | 4 | 5 |
Mean | 12 | 11 | 41 | 50 | 148 | 149 | 9 | 10 | 3 | 4 |
sd | 1.5 | 1.5 | 4.0 | 7.0 | 12.0 | 6.1 | 0.0 | 0.6 | 0.6 | 1.0 |
f(I) | 0.80 | 0.61 | 0.65 | 0.71 | 1.03 | 0.98 | 1.00 | 1.00 | 0.50 | 0.80 |
Rev. abs. | -3 | -7 | -22 | -20 | 4 | -3 | 0 | 0 | -3 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 14.5‑b Concentration 1500 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 10 | 17 | 60 | 54 | 140 | 144 | 8 | 8 | 3 | 3 |
Repl. 2 | 9 | 16 | 52 | 47 | 148 | 152 | 9 | 9 | 3 | 3 |
Repl. 3 | 9 | 15 | 48 | 48 | 152 | 160 | 10 | 9 | 4 | 5 |
Mean | 9 | 16 | 53 | 50 | 147 | 152 | 9 | 9 | 3 | 4 |
sd | 0.6 | 1.0 | 6.1 | 3.8 | 6.1 | 8.0 | 1.0 | 0.6 | 0.6 | 1.2 |
f(I) | 0.60 | 0.89 | 0.84 | 0.71 | 1.02 | 1.00 | 1.00 | 0.90 | 0.50 | 0.80 |
Rev. abs. | -6 | -2 | -10 | -20 | 3 | 0 | 0 | -1 | -3 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 14.5‑c Concentration 500 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 17 | 14 | 54 | 59 | 152 | 140 | 9 | 8 | 3 | 3 |
Repl. 2 | 11 | 14 | 45 | 54 | 164 | 152 | 10 | 8 | 3 | 5 |
Repl. 3 | 11 | 13 | 43 | 49 | 168 | 164 | 10 | 9 | 4 | 5 |
Mean | 13 | 14 | 47 | 54 | 161 | 152 | 10 | 8 | 3 | 4 |
sd | 3.5 | 0.6 | 5.9 | 5.0 | 8.3 | 12.0 | 0.6 | 0.6 | 0.6 | 1.2 |
f(I) | 0.87 | 0.78 | 0.75 | 0.77 | 1.12 | 1.00 | 1.11 | 0.80 | 0.50 | 0.80 |
Rev. abs. | -2 | -4 | -16 | -16 | 17 | 0 | 1 | -2 | -3 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 14.5‑d Concentration 150 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 19 | 15 | 56 | 60 | 136 | 144 | 9 | 9 | 3 | 3 |
Repl. 2 | 16 | 14 | 53 | 53 | 140 | 152 | 9 | 10 | 3 | 4 |
Repl. 3 | 12 | 9 | 46 | 51 | 156 | 160 | 11 | 12 | 4 | 5 |
Mean | 16 | 13 | 52 | 55 | 144 | 152 | 10 | 10 | 3 | 4 |
sd | 3.5 | 3.2 | 5.1 | 4.7 | 10.6 | 8.0 | 1.2 | 1.5 | 0.6 | 1.0 |
f(I) | 1.07 | 0.72 | 0.83 | 0.79 | 1.00 | 1.00 | 1.11 | 1.00 | 0.50 | 0.80 |
Rev. abs. | 1 | -5 | -11 | -15 | 0 | 0 | 1 | 0 | -3 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 14.5‑e Concentration 50 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 16 | 19 | 65 | 66 | 144 | 152 | 9 | 9 | 3 | 4 |
Repl. 2 | 15 | 18 | 56 | 63 | 164 | 168 | 10 | 11 | 4 | 4 |
Repl. 3 | 13 | 16 | 54 | 58 | 168 | 172 | 10 | 11 | 4 | 5 |
Mean | 15 | 18 | 58 | 62 | 159 | 164 | 10 | 10 | 4 | 4 |
sd | 1.5 | 1.5 | 5.9 | 4.0 | 12.9 | 10.6 | 0.6 | 1.2 | 0.6 | 0.6 |
f(I) | 1.00 | 1.00 | 0.92 | 0.89 | 1.10 | 1.08 | 1.11 | 1.00 | 0.67 | 0.80 |
Rev. abs. | 0 | 0 | -5 | -8 | 15 | 12 | 1 | 0 | -2 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
2 Data of Experiment 2
2.1 Determination of Titre
Criterion: The determination of titre should give a number of at least 109 cells/mL, correlating to 100 colonies/plate after dilution.
Exact colony counts could not be determined due to the strong growth of the bacteria, but
by visual inspection it was obvious that the values exceed 100 colonies/ plate
Tabelle 15.1‑a Titre Values (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. |
Repl. 2 | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. | s.g. |
Assessment | ok | ok | ok | ok | ok | ok | ok | ok | ok | ok |
s.g.= strong growth, too strong for counting of colonies
ok= the criterion was fulfilled
2.2 Sterility Control
Criterion: No colony per plate may grow.
Tabelle 15.2‑a Sterility (colonies per plate)
| Demin. water | DMSO | Ethanol |
Repl. 1 | 0 | 0 | 0 |
Repl. 2 | 0 | 0 | 0 |
Repl. 3 | 0 | 0 | 0 |
Repl. 4 | 0 | 0 | 0 |
Assessment | ok | ok | ok |
ok= the criterion was fulfilled
2.3 Spontaneous Revertants
2.3.1 Demin. Water
Tabelle 15.3‑a Spontaneous Revertants demin. water (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 19 | 20 | 69 | 53 | 160 | 156 | 8 | 8 | 4 | 4 |
Repl. 2 | 20 | 22 | 80 | 61 | 164 | 160 | 9 | 9 | 6 | 6 |
Repl. 3 | 21 | 25 | 103 | 64 | 172 | 168 | 10 | 9 | 8 | 7 |
Mean | 20 | 22 | 84 | 59 | 165 | 161 | 9 | 9 | 6 | 6 |
sd | 1.0 | 2.5 | 17.3 | 5.7 | 6.1 | 6.1 | 1.0 | 0.6 | 2.0 | 1.5 |
sd = standard deviation ±
2.3.2 DMSO
Tabelle 15.3‑b Spontaneous Revertants DMSO (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 19 | 20 | 59 | 55 | 156 | 160 | 9 | 8 | 4 | 5 |
Repl. 2 | 19 | 22 | 60 | 65 | 164 | 164 | 10 | 9 | 6 | 5 |
Repl. 3 | 20 | 24 | 72 | 90 | 208 | 168 | 12 | 10 | 7 | 8 |
Mean | 19 | 22 | 64 | 70 | 176 | 164 | 10 | 9 | 6 | 6 |
sd | 0.6 | 2.0 | 7.2 | 18.0 | 28.0 | 4.0 | 1.5 | 1.0 | 1.5 | 1.7 |
sd = standard deviation ±
2.3.3 Ethanol
Tabelle 15.3‑c Spontaneous Revertants Ethanol (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 19 | 21 | 68 | 88 | 156 | 160 | 8 | 8 | 4 | 6 |
Repl. 2 | 20 | 21 | 72 | 96 | 164 | 168 | 9 | 9 | 5 | 7 |
Repl. 3 | 21 | 25 | 75 | 114 | 176 | 176 | 9 | 9 | 7 | 10 |
Mean | 20 | 22 | 72 | 99 | 165 | 168 | 9 | 9 | 5 | 8 |
sd | 1.0 | 2.3 | 3.5 | 13.3 | 10.1 | 8.0 | 0.6 | 0.6 | 1.5 | 2.1 |
sd = standard deviation ±
2.4 Positive Controls
For used concentrations see chapter 6.2.4, page 14.
Tabelle 15.4‑a Diagnostic Mutagens (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Substance | NPD | BaP | Na-azide | 2-AA | MMC | 2-AA | Na-azide | 2-AA | NPD | 2-AA |
Repl. 1 | 560 | 132 | 656 | 1112 | 968 | 616 | 240 | 104 | 112 | 184 |
Repl. 2 | 616 | 136 | 688 | 1152 | 976 | 632 | 280 | 108 | 136 | 184 |
Repl. 3 | 680 | 148 | 696 | 1208 | 1008 | 648 | 288 | 124 | 140 | 200 |
Mean | 619 | 139 | 680 | 1157 | 984 | 632 | 269 | 112 | 129 | 189 |
sd | 60.0 | 8.3 | 21.2 | 48.2 | 21.2 | 16.0 | 25.7 | 10.6 | 15.1 | 9.2 |
f(I) | 32.58 | 6.32 | 8.10 | 16.53 | 5.96 | 3.85 | 29.89 | 12.44 | 21.50 | 31.50 |
Rev. abs. | 600 | 117 | 596 | 1087 | 819 | 468 | 260 | 103 | 123 | 183 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
2.5 Mutagenicity Test Test Item 2,2'-Azobis(2,4-dimethylvaleronitrile)
Tabelle 15.5‑a Concentration 5000 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 2 | 4 | 7 | 60 | 14 | 140 | 0 | 3 | 0 | 0 |
Repl. 2 | 0 | 4 | 5 | 65 | 23 | 156 | 0 | 3 | 0 | 0 |
Repl. 3 | 1 | 5 | 10 | 63 | 30 | 168 | 0 | 3 | 0 | 0 |
Mean | 1 | 4 | 7 | 63 | 22 | 155 | 0 | 3 | 0 | 0 |
sd | 1.0 | 0.6 | 2.5 | 2.5 | 8.0 | 14.0 | 0.0 | 0.0 | 0.0 | 0.0 |
f(I) | 0.05 | 0.18 | 0.10 | 0.64 | 0.13 | 0.92 | 0.00 | 0.33 | 0.00 | 0.00 |
Rev. abs. | -19 | -18 | -65 | -36 | -143 | -13 | -9 | -6 | -5 | -8 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 15.5‑b Concentration 2500 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 2 | 3 | 52 | 80 | 26 | 144 | 0 | 4 | 0 | 0 |
Repl. 2 | 2 | 5 | 46 | 67 | 31 | 148 | 0 | 6 | 0 | 0 |
Repl. 3 | 1 | 6 | 67 | 73 | 35 | 156 | 0 | 8 | 0 | 0 |
Mean | 2 | 5 | 55 | 73 | 31 | 149 | 0 | 6 | 0 | 0 |
sd | 0.6 | 1.5 | 10.8 | 6.5 | 4.5 | 6.1 | 0.0 | 2.0 | 0.0 | 0.0 |
f(I) | 0.10 | 0.23 | 0.76 | 0.74 | 0.19 | 0.89 | 0.00 | 0.67 | 0.00 | 0.00 |
Rev. abs. | -18 | -17 | -17 | -26 | -134 | -19 | -9 | -3 | -5 | -8 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 15.5‑c Concentration 1250 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 5 | 3 | 80 | 70 | 35 | 140 | 0 | 7 | 0 | 0 |
Repl. 2 | 3 | 7 | 75 | 71 | 41 | 144 | 3 | 4 | 0 | 0 |
Repl. 3 | 5 | 11 | 81 | 71 | 49 | 152 | 5 | 4 | 0 | 0 |
Mean | 4 | 7 | 79 | 71 | 42 | 145 | 3 | 5 | 0 | 0 |
sd | 1.2 | 4.0 | 3.2 | 0.6 | 7.0 | 6.1 | 2.5 | 1.7 | 0.0 | 0.0 |
f(I) | 0.20 | 0.32 | 1.10 | 0.72 | 0.25 | 0.86 | 0.33 | 0.56 | 0.00 | 0.00 |
Rev. abs. | -16 | -15 | 7 | -28 | -123 | -23 | -6 | -4 | -5 | -8 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 15.5‑d Concentration 625 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 5 | 10 | 85 | 85 | 160 | 152 | 4 | 3 | 0 | 0 |
Repl. 2 | 7 | 8 | 75 | 73 | 168 | 160 | 2 | 6 | 0 | 0 |
Repl. 3 | 13 | 7 | 78 | 50 | 180 | 164 | 1 | 4 | 0 | 0 |
Mean | 8 | 8 | 79 | 69 | 169 | 159 | 2 | 4 | 0 | 0 |
sd | 4.2 | 1.5 | 5.1 | 17.8 | 10.1 | 6.1 | 1.5 | 1.5 | 0.0 | 0.0 |
f(I) | 0.40 | 0.36 | 1.10 | 0.70 | 1.02 | 0.95 | 0.22 | 0.44 | 0.00 | 0.00 |
Rev. abs. | -12 | -14 | 7 | -30 | 4 | -9 | -7 | -5 | -5 | -8 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 15.5‑e Concentration 313 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 6 | 16 | 65 | 81 | 148 | 144 | 3 | 2 | 1 | 2 |
Repl. 2 | 11 | 13 | 75 | 81 | 152 | 148 | 3 | 3 | 4 | 5 |
Repl. 3 | 2 | 9 | 54 | 52 | 164 | 160 | 5 | 5 | 1 | 6 |
Mean | 6 | 13 | 65 | 71 | 155 | 151 | 4 | 3 | 2 | 4 |
sd | 4.5 | 3.5 | 10.5 | 16.7 | 8.3 | 8.3 | 1.2 | 1.5 | 1.7 | 2.1 |
f(I) | 0.30 | 0.59 | 0.90 | 0.72 | 0.94 | 0.90 | 0.44 | 0.33 | 0.40 | 0.50 |
Rev. abs. | -14 | -9 | -7 | -28 | -10 | -17 | -5 | -6 | -3 | -4 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 15.5‑f Concentration 156 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 9 | 12 | 55 | 80 | 140 | 144 | 9 | 5 | 4 | 6 |
Repl. 2 | 7 | 8 | 74 | 60 | 156 | 148 | 2 | 4 | 1 | 3 |
Repl. 3 | 6 | 15 | 74 | 72 | 160 | 164 | 4 | 4 | 2 | 6 |
Mean | 7 | 12 | 68 | 71 | 152 | 152 | 5 | 4 | 2 | 5 |
sd | 1.5 | 3.5 | 11.0 | 10.1 | 10.6 | 10.6 | 3.6 | 0.6 | 1.5 | 1.7 |
f(I) | 0.35 | 0.55 | 0.94 | 0.72 | 0.92 | 0.90 | 0.56 | 0.44 | 0.40 | 0.63 |
Rev. abs. | -13 | -10 | -4 | -28 | -13 | -16 | -4 | -5 | -3 | -3 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
Tabelle 15.5‑g Concentration 78 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 6 | 13 | 43 | 100 | 156 | 156 | 5 | 8 | 3 | 2 |
Repl. 2 | 6 | 16 | 56 | 97 | 156 | 160 | 2 | 6 | 1 | 6 |
Repl. 3 | 10 | 14 | 60 | 87 | 172 | 164 | 3 | 5 | 3 | 7 |
Mean | 7 | 14 | 53 | 95 | 161 | 160 | 3 | 6 | 2 | 5 |
sd | 2.3 | 1.5 | 8.9 | 6.8 | 9.2 | 4.0 | 1.5 | 1.5 | 1.2 | 2.6 |
f(I) | 0.35 | 0.64 | 0.74 | 0.96 | 0.98 | 0.95 | 0.33 | 0.67 | 0.40 | 0.63 |
Rev. abs. | -13 | -8 | -19 | -4 | -4 | -8 | -6 | -3 | -3 | -3 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
3 Data of Experiment 2b
3.1 Determination of Titre
Criterion: The determination of titre should give a number of at least 109 cells/mL, correlating to 100 colonies/plate after dilution.
Exact colony counts could not be determined due to the strong growth of the bacteria, but
by visual inspection it was obvious that the values exceed 100 colonies/ plate
Tabelle 16.1‑a Titre Values (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | s.g. | s.g. | -- | -- | s.g. | -- | s.g. | s.g. | s.g. | s.g. |
Repl. 2 | s.g. | s.g. | -- | -- | s.g. | -- | s.g. | s.g. | s.g. | s.g. |
Assessment | ok | ok | -- | -- | ok | -- | ok | ok | ok | ok |
s.g.= strong growth, too strong for counting of colonies
ok= the criterion was fulfilled
-- = not tested
3.2 Sterility Control
Criterion: No colony per plate may grow.
Tabelle 16.2‑a Sterility (colonies per plate)
| Demin. water | DMSO | Ethanol |
Repl. 1 | 0 | 0 | 0 |
Repl. 2 | 0 | 0 | 0 |
Repl. 3 | 0 | 0 | 0 |
Repl. 4 | 0 | 0 | 0 |
Assessment | ok | ok | ok |
ok= the criterion was fulfilled
3.3 Spontaneous Revertants
3.3.1 Demin. Water
Tabelle 16.3‑a Spontaneous Revertants demin. water (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 15 | 15 | -- | -- | 164 | -- | 5 | 6 | 4 | 4 |
Repl. 2 | 17 | 17 | -- | -- | 168 | -- | 7 | 6 | 4 | 5 |
Repl. 3 | 17 | 20 | -- | -- | 172 | -- | 8 | 7 | 4 | 5 |
Mean | 16 | 17 | -- | -- | 168 | -- | 7 | 6 | 4 | 5 |
sd | 1.2 | 2.5 | -- | -- | 4.0 | -- | 1.5 | 0.6 | 0.0 | 0.6 |
sd = standard deviation ±
-- = not tested
3.3.2 DMSO
Tabelle 16.3‑b Spontaneous Revertants DMSO (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 15 | 15 | -- | -- | 168 | -- | 7 | 5 | 3 | 3 |
Repl. 2 | 18 | 18 | -- | -- | 176 | -- | 7 | 6 | 4 | 4 |
Repl. 3 | 19 | 18 | -- | -- | 176 | -- | 7 | 11 | 5 | 4 |
Mean | 17 | 17 | -- | -- | 173 | -- | 7 | 7 | 4 | 4 |
sd | 2.1 | 1.7 | -- | -- | 4.6 | -- | 0.0 | 3.2 | 1.0 | 0.6 |
sd = standard deviation ±
-- = not tested
3.3.3 Ethanol
Tabelle 16.3‑c Spontaneous Revertants Ethanol (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 16 | 17 | -- | -- | 152 | -- | 3 | 4 | 3 | 4 |
Repl. 2 | 17 | 18 | -- | -- | 160 | -- | 6 | 6 | 3 | 5 |
Repl. 3 | 18 | 19 | -- | -- | 164 | -- | 7 | 6 | 4 | 5 |
Mean | 17 | 18 | -- | -- | 159 | -- | 5 | 5 | 3 | 5 |
sd | 1.0 | 1.0 | -- | -- | 6.1 | -- | 2.1 | 1.2 | 0.6 | 0.6 |
sd = standard deviation ±
-- = not tested
3.4 Positive Controls
For used concentrations see chapter 6.2.4, page 14.
Tabelle 16.4‑a Diagnostic Mutagens (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Substance | NPD | BaP | Na-azide | 2-AA | MMC | 2-AA | Na-azide | 2-AA | NPD | 2-AA |
Repl. 1 | 720 | 76 | -- | -- | 816 | -- | 296 | 248 | 72 | 33 |
Repl. 2 | 776 | 88 | -- | -- | 848 | -- | 328 | 256 | 80 | 36 |
Repl. 3 | 816 | 100 | -- | -- | 888 | -- | 344 | 264 | 92 | 37 |
Mean | 771 | 88 | -- | -- | 851 | -- | 323 | 256 | 81 | 35 |
sd | 48.2 | 12.0 | -- | -- | 36.1 | -- | 24.4 | 8.0 | 10.1 | 2.1 |
f(I) | 45.35 | 5.18 | -- | -- | 5.07 | -- | 46.14 | 36.57 | 20.25 | 8.75 |
Rev. abs. | 754 | 71 | -- | -- | 683 | -- | 316 | 249 | 77 | 31 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
3.5 Mutagenicity Test Test Item 2,2'-Azobis(2,4-dimethylvaleronitrile)
Tabelle 16.5‑a Concentration 625 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | -- | 11 | -- | -- | 84 | -- | 5 | -- | -- | -- |
Repl. 2 | -- | 11 | -- | -- | 92 | -- | 6 | -- | -- | -- |
Repl. 3 | -- | 12 | -- | -- | 96 | -- | 8 | -- | -- | -- |
Mean | -- | 11 | -- | -- | 91 | -- | 6 | -- | -- | -- |
sd | -- | 0.6 | -- | -- | 6.1 | -- | 1.5 | -- | -- | -- |
f(I) | -- | 0.61 | -- | -- | 0.57 | -- | 1.20 | -- | -- | -- |
Rev. abs. | -- | -7 | -- | -- | -68 | -- | 1 | -- | -- | -- |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑b Concentration 313 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | -- | 15 | -- | -- | 156 | -- | 3 | 4 | -- | 4 |
Repl. 2 | -- | 16 | -- | -- | 160 | -- | 6 | 5 | -- | 5 |
Repl. 3 | -- | 17 | -- | -- | 176 | -- | 7 | 6 | -- | 5 |
Mean | -- | 16 | -- | -- | 164 | -- | 5 | 5 | -- | 5 |
sd | -- | 1.0 | -- | -- | 10.6 | -- | 2.1 | 1.0 | -- | 0.6 |
f(I) | -- | 0.89 | -- | -- | 1.03 | -- | 1.00 | 1.00 | -- | 1.00 |
Rev. abs. | -- | -2 | -- | -- | 5 | -- | 0 | 0 | -- | 0 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑c Concentration 156 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | -- | 16 | -- | -- | 156 | -- | 4 | 5 | -- | 3 |
Repl. 2 | -- | 17 | -- | -- | 160 | -- | 4 | 8 | -- | 4 |
Repl. 3 | -- | 17 | -- | -- | 164 | -- | 4 | 11 | -- | 5 |
Mean | -- | 17 | -- | -- | 160 | -- | 4 | 8 | -- | 4 |
sd | -- | 0.6 | -- | -- | 4.0 | -- | 0.0 | 3.0 | -- | 1.0 |
f(I) | -- | 0.94 | -- | -- | 1.01 | -- | 0.80 | 1.60 | -- | 0.80 |
Rev. abs. | -- | -1 | -- | -- | 1 | -- | -1 | 3 | -- | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑d Concentration 78 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 11 | 18 | -- | -- | 152 | -- | 3 | 5 | 3 | 4 |
Repl. 2 | 11 | 18 | -- | -- | 160 | -- | 5 | 9 | 4 | 5 |
Repl. 3 | 12 | 18 | -- | -- | 168 | -- | 6 | 9 | 4 | 5 |
Mean | 11 | 18 | -- | -- | 160 | -- | 5 | 8 | 4 | 5 |
sd | 0.6 | 0.0 | -- | -- | 8.0 | -- | 1.5 | 2.3 | 0.6 | 0.6 |
f(I) | 0.65 | 1.00 | -- | -- | 1.01 | -- | 1.00 | 1.60 | 1.33 | 1.00 |
Rev. abs. | -6 | 0 | -- | -- | 1 | -- | 0 | 3 | 1 | 0 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑e Concentration 39 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 16 | 17 | -- | -- | 152 | -- | 3 | 4 | 3 | 3 |
Repl. 2 | 17 | 19 | -- | -- | 152 | -- | 6 | 4 | 4 | 3 |
Repl. 3 | 17 | 20 | -- | -- | 156 | -- | 7 | 6 | 5 | 5 |
Mean | 17 | 19 | -- | -- | 153 | -- | 5 | 5 | 4 | 4 |
sd | 0.6 | 1.5 | -- | -- | 2.3 | -- | 2.1 | 1.2 | 1.0 | 1.2 |
f(I) | 1.00 | 1.06 | -- | -- | 0.96 | -- | 1.00 | 1.00 | 1.33 | 0.80 |
Rev. abs. | 0 | 1 | -- | -- | -6 | -- | 0 | 0 | 1 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑f Concentration 19.5 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 17 | 17 | -- | -- | 160 | -- | 4 | 5 | 3 | 3 |
Repl. 2 | 18 | 18 | -- | -- | 164 | -- | 4 | 6 | 3 | 5 |
Repl. 3 | 18 | 19 | -- | -- | 176 | -- | 5 | 9 | 5 | 5 |
Mean | 18 | 18 | -- | -- | 167 | -- | 4 | 7 | 4 | 4 |
sd | 0.6 | 1.0 | -- | -- | 8.3 | -- | 0.6 | 2.1 | 1.2 | 1.2 |
f(I) | 1.06 | 1.00 | -- | -- | 1.05 | -- | 0.80 | 1.40 | 1.33 | 0.80 |
Rev. abs. | 1 | 0 | -- | -- | 8 | -- | -1 | 2 | 1 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑g Concentration 9.8 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 17 | 19 | -- | -- | 152 | -- | 4 | 6 | 3 | 3 |
Repl. 2 | 18 | 19 | -- | -- | 156 | -- | 5 | 7 | 3 | 4 |
Repl. 3 | 18 | 19 | -- | -- | 168 | -- | 8 | 10 | 5 | 4 |
Mean | 18 | 19 | -- | -- | 159 | -- | 6 | 8 | 4 | 4 |
sd | 0.6 | 0.0 | -- | -- | 8.3 | -- | 2.1 | 2.1 | 1.2 | 0.6 |
f(I) | 1.06 | 1.06 | -- | -- | 1.00 | -- | 1.20 | 1.60 | 1.33 | 0.80 |
Rev. abs. | 1 | 1 | -- | -- | 0 | -- | 1 | 3 | 1 | -1 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑h Concentration 4.88 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 16 | -- | -- | -- | -- | -- | -- | 4 | 3 | 5 |
Repl. 2 | 16 | -- | -- | -- | -- | -- | -- | 4 | 4 | 5 |
Repl. 3 | 18 | -- | -- | -- | -- | -- | -- | 4 | 5 | 5 |
Mean | 17 | -- | -- | -- | -- | -- | -- | 4 | 4 | 5 |
sd | 1.2 | -- | -- | -- | -- | -- | -- | 0.0 | 1.0 | 0.0 |
f(I) | 1.00 | -- | -- | -- | -- | -- | -- | 0.80 | 1.33 | 1.00 |
Rev. abs. | 0 | -- | -- | -- | -- | -- | -- | -1 | 1 | 0 |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑i Concentration 2.44 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 17 | -- | -- | -- | -- | -- | -- | -- | 3 | -- |
Repl. 2 | 17 | -- | -- | -- | -- | -- | -- | -- | 4 | -- |
Repl. 3 | 19 | -- | -- | -- | -- | -- | -- | -- | 4 | -- |
Mean | 18 | -- | -- | -- | -- | -- | -- | -- | 4 | -- |
sd | 1.2 | -- | -- | -- | -- | -- | -- | -- | 0.6 | -- |
f(I) | 1.06 | -- | -- | -- | -- | -- | -- | -- | 1.33 | -- |
Rev. abs. | 1 | -- | -- | -- | -- | -- | -- | -- | 1 | -- |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Tabelle 16.5‑j Concentration 1.22 µg/plate (colonies per plate)
Strain | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
Induction | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Repl. 1 | 15 | -- | -- | -- | -- | -- | -- | -- | 3 | -- |
Repl. 2 | 16 | -- | -- | -- | -- | -- | -- | -- | 4 | -- |
Repl. 3 | 19 | -- | -- | -- | -- | -- | -- | -- | 5 | -- |
Mean | 17 | -- | -- | -- | -- | -- | -- | -- | 4 | -- |
sd | 2.1 | -- | -- | -- | -- | -- | -- | -- | 1.0 | -- |
f(I) | 1.00 | -- | -- | -- | -- | -- | -- | -- | 1.33 | -- |
Rev. abs. | 0 | -- | -- | -- | -- | -- | -- | -- | 1 | -- |
sd = standard deviation ±
f(I) = increase factor, calculation see chapter 7.4, page 21
Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21
-- = not tested
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
as no genotoxic effects are present for the substance, no in vivo studies are needed.
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
as no genotoxic effects are present, no mode of action can be identified.
Additional information
Justification for classification or non-classification
The available information is conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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