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EC number: 209-513-6 | CAS number: 583-60-8
- 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:
- March 2019-March 2020
- Reliability:
- 1 (reliable without restriction)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
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)
- Version / remarks:
- Final
- Deviations:
- yes
- Remarks:
- Concerning: Experimental Performance Report: In the experiment with metabolic activation (for selection and plating efficiency) the cell culture medium was prepared without A. This deviation did not influence the quality or integrity of the present study.
- 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:
- 2-methylcyclohexanone
- EC Number:
- 209-513-6
- EC Name:
- 2-methylcyclohexanone
- Cas Number:
- 583-60-8
- Molecular formula:
- C7H12O
- IUPAC Name:
- 2-methylcyclohexan-1-one
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- water
- Reference substance name:
- Unknown impurities
- Molecular formula:
- not applicable
- IUPAC Name:
- Unknown impurities
- Test material form:
- liquid
- Details on test material:
- Batch No.: 01246M0002, purity confirmed by analytical certificate
Constituent 1
impurity 1
impurity 2
- Specific details on test material used for the study:
- Characterisation of the Test Item
The identity of the test item was inspected upon delivery at the test facility (e.g. test item name,
batch no. and additional data were compared with the label) based on the following specifications
provided by the sponsor. The following listed information applies to the sample as received.
Method
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- (clone TK+/- 3.7.2C)
- Details on mammalian cell type (if applicable):
- These cells are characterised by their high proliferation rate (10 - 12 h
doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than
50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the
Thymidine Kinase (TK) locus in order to detect mutation events at the TK-locus.
To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated
by culturing cells in RPMI 1640 supplemented with:
9.0 µg/mL hypoxanthine
15.0 µg/mL thymidine
22.5 µg/mL glycine
0.1 µg/mL methotrexate
The cells are resuspended in medium without methotrexate but with thymidine, hypoxanthine and
glycine for 1 - 3 days.
Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich. This allows the repeated use of the same cell batch in
experiments. Each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian Microsomal Fraction S9 Homogenate
An advantage of using in vitro cell cultures is the accurate control of concentration and exposure
time of cells to the test item under study. However, due to the limited capacity of cells growing in
vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is
necessary [8]. Many substances only develop mutagenic potential when they are metabolized by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). Since the L5178Y cells used in this assay do not have the cytochrome-based P450 metabolic oxidation system, an exogenous metabolic activation system, the S9 microsomal fraction was added. The cells were exposed to the test substance both, in the presence and the absence of metabolic activation.
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany.
Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone.
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at
≤ -75 °C.
The protein concentration in the S9 preparation (Lot: 4146) was 38.5 mg/mL.
S9 Mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to
result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
The final concentration of S9 mix in the cultures was 5%. - Test concentrations with justification for top dose:
- The test item was investigated at the following concentrations: without and with metabolic activation:
0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- methylmethanesulfonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RPMI 1640 medium supplemented with 5 % HS 100 U/100 µg/mL penicillin/streptomycin 1 mM sodium pyruvate 2 mM L-glutamine 25 mM HEPES 2.5 µg/mL amphotericin B
- True negative controls:
- no
- Positive controls:
- no
- Details on test system and experimental conditions:
- Experimental Design
Pre-Experiment for Toxicity
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Eight concentrations [0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM] were tested without and
with metabolic activation. The experimental conditions in these pre-experiments were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector. - Evaluation criteria:
- The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a concentration-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or
chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF or the
trend of the test is negative.
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:
- not specified
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
Any other information on results incl. tables
Table 1: Pre-Experiment for Toxicity, without metabolic activation.
Test Group | Concentration [mM] | Number of Cells 4 h after Treatment | Number of Cells 24 h after Treatment | Number of Cells 48 h after Treatment | SGa | RSGb [%] |
C1 | 0 | 384000 | 1010000 | 1300000 | 13.1 | 100.0 |
C2 | 405000 | 1040000 | 1320000 | 13.7 | ||
1 | 0.1 | 406000 | 1020000 | 1520000 | 15.5 | 115.5 |
2 | 0.2 | 445000 | 1150000 | 1270000 | 14.6 | 108.8 |
3 | 0.5 | 445000 | 1000000 | 1440000 | 14.4 | 107.2 |
4 | 1.0 | 386000 | 1030000 | 1410000 | 14.5 | 108.1 |
5 | 2.5 | 350000 | 906000 | 1510000 | 13.7 | 101.9 |
6 | 5.0 | 405000 | 980000 | 1390000 | 13.6 | 101.4 |
7 | 7.5 | 345000 | 897000 | 1500000 | 13.5 | 100.2 |
8 | 10 | 349000 | 933000 | 1490000 | 13.9 | 103.5 |
C: Negative control
a: Suspension Growth, SG = [((value 24 h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
Table 2. Pre-Experiment for Toxicity, with metabolic activation
Test Group | Concentration [mM] | Number of Cells 4 h after Treatment | Number of Cells 24 h after Treatment | Number of Cells 48 h after Treatment | SGa | RSGb [%] |
C1 | 0 | 337000 | 914000 | 1420000 | 13.0 | 100.0 |
C2 | 359000 | 978000 | 1230000 | 12.0 | ||
1 | 0.1 | 332000 | 867000 | 1370000 | 11.9 | 95.0 |
2 | 0.2 | 361000 | 961000 | 1400000 | 13.5 | 107.6 |
3 | 0.5 | 338000 | 908000 | 1210000 | 11.0 | 87.9 |
4 | 1.0 | 362000 | 959000 | 1260000 | 12.1 | 96.6 |
5 | 2.5 | 332000 | 959000 | 1420000 | 12.3 | 98.6 |
6 | 5.0 | 331000 | 937000 | 1490000 | 14.0 | 111.7 |
7# | 7.5 | 138000 | 358000 | 1410000 | 5.0 | 40.4 |
8# | 10 | 152000 | 382000 | 1480000 | 5.7 | 45.2 |
C: Negative control
a: Suspension Growth, SG = [((value 24 h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100] #
: for these concentrations only half of the cell number was used for treatment, but since this test was only used for toxicity determination, this was concluded as acceptable.
Table 3. Main Experiment - Toxicity Data, without metabolic activation
Test Group | Concentration [mM] | Number of Cells 4 h after Treatment | Number of Cells 24 h after Treatment | Number of Cells 48 h after Treatment | SGa | RSGb [%] | RCEc [%] | RTGd [%] |
C1 | 0 | 392000 | 1150000 | 1060000 | 12.2 | 100.0 | 100.0 | 100.0 |
C2 | 407000 | 1110000 | 1110000 | 12.3 | ||||
2 | 0.2 | 406000 | 851000 | 1100000 | 9.4 | 76.4 | 97.2 | 74.2 |
3 | 0.5 | 389000 | 787000 | 1200000 | 9.4 | 77.1 | 84.1 | 64.8 |
4 | 1.0 | 413000 | 802000 | 1090000 | 8.7 | 71.3 | 70.4 | 50.2 |
5 | 2.5 | 353000 | 750000 | 1120000 | 8.4 | 68.5 | 84.1 | 57.7 |
6 | 5.0 | 351000 | 692000 | 1110000 | 7.7 | 62.7 | 85.4 | 53.5 |
7 | 7.5 | 343000 | 610000 | 1160000 | 7.1 | 57.7 | 92.5 | 53.4 |
8 | 10 | 326000 | 601000 | 1140000 | 6.9 | 55.9 | 82.8 | 46.3 |
EMS | 300 µg/mL | 387000 | 803000 | 1050000 | 8.4 | 68.8 | 56.0 | 38.5 |
MMS | 10 µg/mL | 418000 | 896000 | 907000 | 8.1 | 66.3 | 49.3 | 32.7 |
C: Negative control
a: Suspension Growth, SG = [((value 24 h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
c: Relative Cloning Efficiency, RCE = [(CE test group / CE of corresponding controls) x 100], Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
d: Relative Total Growth, RTG = (RSG x RCE) / 100
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
Table 4. Main Experiment - Mutagenicity Data, without metabolic activation
Test Group | Concentration [mM] | Cloning Efficiency (CE) | Mutagenicity Data | ||||||||
Plate 1e | Plate 2e | CEf [%] | Number of cultures / 96 wells | MFg [mutants / 106 cells] | IMFh [mutants / 106 cells] | ||||||
Plate 1e | Plate 2e | Plate 3e | Plate 4e | Mean | |||||||
C1 | 0 | 79 | 76 | 102.9 | 14 | 15 | 12 | 16 | 14.3 | 78.1 | / |
C2 | 79 | 76 | 116.0 | 12 | 9 | 12 | 9 | 10.5 | 50.0 | / | |
2 | 0.2 | 79 | 78 | 106.4 | 11 | 11 | 6 | 15 | 10.8 | 56.1 | -7.9 |
3 | 0.5 | 73 | 75 | 92.1 | 11 | 6 | 14 | 11 | 10.5 | 63.2 | -0.9 |
4 | 1.0 | 67 | 69 | 77.0 | 14 | 7 | 13 | 10 | 11.0 | 79.3 | 15.3 |
5 | 2.5 | 74 | 74 | 92.1 | 14 | 10 | 7 | 10 | 10.3 | 61.5 | -2.5 |
6 | 5.0 | 71 | 78 | 93.5 | 13 | 9 | 14 | 12 | 12.0 | 71.5 | 7.5 |
7 | 7.5 | 81 | 73 | 101.2 | 12 | 7 | 11 | 20 | 12.5 | 69.7 | 5.6 |
8 | 10 | 69 | 78 | 90.7 | 10 | 11 | 8 | 9 | 9.5 | 57.5 | -6.6 |
EMS | 300 µg/mL | 56 | 64 | 61.3 | 63 | 60 | 65 | 59 | 61.8 | 842.6 | 778.6 |
MMS | 10 µg/mL | 59 | 52 | 53.9 | 34 | 40 | 38 | 37 | 37.3 | 455.8 | 391.7 |
C: Negative control
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
g: Mutant frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
Table 5. Main Experiment - Colony Sizing, without metabolic activation
Test Group* | Concentration [mM] | Wells with at least 1 colony | Large colonies | Small colonies | % small colonies |
C1 | 0 | 57 | 38 | 19 | 33.3 |
C2 | 42 | 27 | 15 | 35.7 | |
EMS | 300 µg/mL | 247 | 208 | 39 | 15.8 |
MMS | 10 µg/mL | 149 | 59 | 90 | 60.4 |
C: Negative control
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.
Table 6. Main Experiment - Toxicity Data, with metabolic activation
Test Group | Concentration [mM] | Number of Cells 4 h after Treatment | Number of Cells 24 h after Treatment | Number of Cells 48 h after Treatment | SGa | RSGb [%] | RCEc [%] | RTGd [%] |
C1 | 0 | 301000 | 826000 | 1440000 | 11.9 | 100.0 | 100.0 | 100.0 |
C2 | 288000 | 826000 | 1420000 | 11.7 | ||||
2 | 0.2 | 325000 | 896000 | 1380000 | 12.4 | 104.7 | 103.8 | 108.7 |
3 | 0.5 | 288000 | 860000 | 1420000 | 12.2 | 103.4 | 103.8 | 107.4 |
4 | 1.0 | 305000 | 914000 | 1450000 | 13.3 | 112.2 | 112.3 | 126.0 |
5 | 2.5 | 310000 | 849000 | 1330000 | 11.3 | 95.6 | 117.8 | 112.7 |
6 | 5.0 | 290000 | 830000 | 1400000 | 11.6 | 98.4 | 121.8 | 119.8 |
7 | 7.5 | 269000 | 815000 | 1420000 | 11.6 | 98.0 | 117.8 | 115.5 |
8 | 10 | 286000 | 794000 | 1430000 | 11.4 | 96.1 | 123.9 | 119.1 |
B[a]P | 2.5 µg/mL | 266000 | 563000 | 1420000 | 8.0 | 67.7 | 86.9 | 58.8 |
a: Suspension Growth, SG = [((value 24 h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h* x 20))];
* : for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
c: Relative Cloning Efficiency, RCE = [(CE test group / CE of corresponding controls) x 100]
Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
d: Relative Total Growth, RTG = (RSG x RCE) / 100
B[a]P: Benzo[a]pyrene
Table 7. Main Experiment - Mutagenicity Data, with metabolic activation
Test Group | Concentration [mM] | Cloning Efficiency (CE) | Mutagenicity Data | ||||||||
Plate 1e | Plate 2e | CEf [%] | Number of cultures / 96 wells | MFg [mutants / 106 cells] | IMFh [mutants / 106 cells] | ||||||
Plate 1e | Plate 2e | Plate 3e | Plate 4e | Mean | |||||||
C1 | 0 | 73 | 70 | 85.4 | 11 | 8 | 13 | 10 | 10.5 | 68.0 | / |
C2 | 73 | 73 | 89.3 | 11 | 8 | 14 | 9 | 10.5 | 65.1 | / | |
2 | 0.2 | 68 | 78 | 89.3 | 16 | 7 | 18 | 7 | 12.0 | 75.8 | 9.3 |
3 | 0.5 | 76 | 71 | 90.7 | 13 | 10 | 7 | 8 | 9.5 | 57.7 | -8.9 |
4 | 1.0 | 82 | 70 | 98.0 | 11 | 11 | 10 | 13 | 11.3 | 63.6 | -2.9 |
5 | 2.5 | 79 | 76 | 102.9 | 16 | 8 | 13 | 12 | 12.3 | 66.6 | 0.1 |
6 | 5.0 | 79 | 78 | 106.4 | 14 | 13 | 11 | 11 | 12.3 | 64.2 | -2.3 |
7 | 7.5 | 81 | 74 | 102.9 | 9 | 9 | 12 | 14 | 11.0 | 59.3 | -7.2 |
8 | 10 | 78 | 80 | 108.2 | 13 | 14 | 17 | 16 | 15.0 | 78.6 | 12.1 |
B[a]P | 2.5 µg/mL | 61 | 74 | 75.9 | 69 | 72 | 66 | 73 | 70.0 | 864.1 | 797.5 |
C: Negative control
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
g: Mutant frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
B[a]P: Benzo[a]pyrene
Table 8. Main Experiment - Colony Sizing, with metabolic activation
Test Group* | Concentration [mM] | Wells with at least 1 colony | Large colonies | Small colonies | % small colonies |
C1 | 0 | 42 | 33 | 9 | 21.4 |
C2 | 42 | 31 | 11 | 26.2 | |
B[a]P | 2.5 µg/mL | 280 | 168 | 112 | 40.0 |
C: Negative control
B[a]P: Benzo[a]pyrene
*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.
Applicant's summary and conclusion
- Conclusions:
- In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 2-MCH is considered to be non-mutagenic in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
- Executive summary:
Summary Results
The test item 2-MCH was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations:
without and with metabolic activation:
0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
No precipitation of the test item was noted in the experiment.
No growth inhibition was observed with metabolic activation. Growth inhibition was observed in the experiment without metabolic activation: The relative total
growth (RTG) was 46.3% for the highest concentration evaluated.
No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of thenegative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.
No concentration-related increase was observed.
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of
small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.Summary: Main Experiment, without and with metabolic activation
Test Group
Conc. [mM]
RCEa [%]
RTGb [%]
MFc [mutants/ 106 cells]
IMFd [mutants/ 106 cells]
GEFe exceeded
Statistical Significant Increasef
Precipitate
Exp without S9
C1
0
100.0
100.0
64.1
/
/
/
-
C2
/
/
/
-
2
0.2
97.2
74.2
56.1
-7.9
-
-
3
0.5
84.1
64.8
63.2
-0.9
-
-
-
4
1.0
70.4
50.2
79.3
15.3
-
-
-
5
2.5
84.1
57.7
61.5
-2.5
-
-
-
6
5.0
85.4
53.5
71.5
7.5
-
-
-
7
7.5
92.5
53.4
69.7
5.6
-
-
-
8
10
82.8
46.3
57.5
-6.6
-
-
-
EMS
300 µg/mL
56.0
38.5
842.6
778.6
+
+
-
MMS
10 µg/mL
49.3
32.7
455.8
391.7
+
+
-
Exp with S9
C1
0
100.0
100.0
66.5
/
/
/
-
C2
/
/
/
-
2
0.2
103.8
108.7
74.6
8.1
-
-
-
3
0.5
103.8
107.4
57.7
-8.9
-
-
-
4
1.0
112.3
126.0
63.6
-2.9
-
-
-
5
2.5
117.8
112.7
66.6
0.1
-
-
-
6
5.0
121.8
119.8
64.2
-2.3
-
-
-
7
7.5
117.8
115.5
59.3
-7.2
-
-
-
8
10
123.9
119.1
78.6
12.1
-
-
-
B[a]P
2.5 µg/mL
86.9
58.8
864.1
797.5
+
+
-
C: Negative Controls
a: Relative Cloning Efficiency, RCE = [(CE test group / CE of corresponding controls) x 100]
Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)
b: Relative Total Growth, RTG = (RSG x RCE) / 100
c: Mutant Frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e: Global Evaluation Factor, GEF (126 mutants/106 cells); +: GEF exceeded, -: GEF not exceeded
f: statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test, p<0.05).
+: significant; -: not significant
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
B[a]P: Benzo[a]pyrene
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