Registration Dossier
Registration Dossier
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EC number: 810-161-6 | CAS number: 1229654-66-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial gene mutation (OECD 471): negative
Gene mutation in mammalian cells (OECD 476): negative
Cytogenicity/chromosome aberration in mammalian cells (OECD 473): negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07 - 24 May 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- adopted in 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- 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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone
- Test concentrations with justification for top dose:
- Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
5000 µg/plate was chosen as maximal concentration since minor toxic effects were observed in the pre-test/experiment I. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent DMSO was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
- Remarks:
- + S9: 2-AA (2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100; 10 µg/plate in TA 102) - S9: sodium azide (10 µg/plate in TA 1535, TA 100), 4-NOPD (10 µg/plate in TA 98; 50 µg/plate in TA 1537), methylmethanesulfonate (2.0 µL/plate in TA 102)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: other: number of revertants - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- Mean values and standard deviation were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 2500 and 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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
- Key result
- Species / strain:
- S. typhimurium TA 102
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-experiment/experiment 1 the test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 μg/plate. In experiment II precipitation of the test item was observed in the overlay agar in the test tubes and on the incubated agar plates from 2500 to 5000 μg/plate. The undissolved particles had no influence on the data recording.
RANGE-FINDING/SCREENING STUDIES: A pre-test (experiment I, plate incorporation method) was performed with all strains used to evaluate the toxicity of the test item and to choose a concentration range for experiment II. The test substance was tested with and without metabolic activation at eight concentrations ranging from 3 - 5000 µg/plate. A reduction in the number was observed in one strain only (TA 1537) at 2500 and 5000 µg/plate (precipitating concentrations) in the absence of metabolic activation. Thus, the same concentrations with the exception of 3 µg/plate were used in experiment II.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The historical control data represent approximately 550 experiments for TA 1535, TA 1537, TA 98 and TA 100 and approximately 200 experiments for TA 102 (from 2011; see table 1).
- Negative (solvent/vehicle) historical control data: The historical control data represent approximately 550 experiments for TA 1535, TA 1537, TA 98 and TA 100 and approximately 200 experiments for TA 102 (from 2011; see table 1). - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07 May - 20 Jun 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- adopted in 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- other: in vitro mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University, Darmstadt, Germany
- Suitability of cells: The cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype.
- Cell cycle length, doubling time or proliferation index: 12 - 16 h
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are propagated at 37 °C in 80 cm² plastic flasks. About 5×10E5 cells are seeded into each flask with 15 mL medium. The cells are sub-cultured twice weekly. The cell cultures are incubated at 37 °C in a 1.5% CO2 atmosphere (98.5% air).
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration: Minimal essential medium (MEM) containing Hank’s salts supplemented with 10% foetal bovine serum, 5 µg/mL neomycin and 1% amphotericin B in a 1.5% CO2 atmosphere. For selection of mutants the medium is supplemented with 11 μg/mL thioguanine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- Pre-test on toxicity: 10.2, 20.3, 40.6, 81.3, 162.5, 325, 650, 1300 µg/mL with and without metabolic activation
Experiment I: 81.3, 162.5, 325, 650, 1300 µg/mL without metabolic activation; 20.3, 40.6, 81.3, 162.5, 325 µg/mL with metabolic activation
Experiment II: 40.6, 81.3, 121.9, 162.5, 243.3 without metabolic activation; 81.3, 121.9, 162.5, 243.3, 325 µg/mL with metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- - S9: ethylmetanesulphonate (150 µg/mL) + S9: 7,12-dimethylbenzanthracene (1.1 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 1.5 x 10E6 (single culture), 500 (duplicate)
DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h (experiment I with and without metabolic activation, experiment II with metabolic activation), 24 h (experiment II without metabolic activation)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 19 - 20 days
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: duplicates in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- linear regression, p < 0.05
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- experiment I: 325 µg/mL with and 650 µg/mL without S9; experiment II: 325 µg/mL with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH of the medium even at the maximum concentration of the test item.
- Effects of osmolality: There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Precipitation occurred at 162.5 μg/mL and above with and without metabolic activation following 4 and 24 hours treatment.
RANGE-FINDING/SCREENING STUDIES: The range finding pre-experiment was performed using a concentration range of 20.3 to 2600 μg/mL (limited by the solubility of the test item in organic solvents and aqueous media) to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Strong toxic effects occurred in the presence of metabolic activation at 325, 650, and 1300 μg/mL (precipitating concentration range). Following 24 h treatment without metabolic activation cytotoxic effects occurred at 162.5 μg/mL and above. After 4 hour treatment without metabolic activation a single moderate cytotoxic effect occurred within the precipitating concentration range at 1300 μg/mL.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The historical control data represent the number of mutant colonies per 10E6 cells from 2011 - 2012 (see table 1).
- Negative (solvent/vehicle) historical control data: The historical control data represent the number of mutant colonies per 10E6 cells from 2011 - 2012 (see table 1). - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15 May - 09 Jul 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- adopted in 1998
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, Darmstadt, Germany
- Suitability of cells: The high proliferation rate and a reasonable plating efficiency of untreated cells both necessary for the appropriate performance of the study, support the use of this cell line.
- Cell cycle length, doubling time or proliferation index: approximately 13 h
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37°C in 80 cm² plastic flasks. About 5 x 10E5 cells per flask were seeded in 15 mL medium. The cells were sub-cultured twice a week. Before treatment with trypsin-EDTA-solution at 37°C for approx. 5 min cells were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Then, by adding serum containing culture medium the enzymatic treatment was stopped and a single cell suspension was prepared. All incubations were done at 37°C in a humidified atmosphere with 1.5% CO2.
- Modal number of chromosomes: 22 ± 1
MEDIA USED
- Type and identity of media including CO2 concentration: minimal essential medium (MEM) containing Hank’s salts, glutamine and Hepes (25 mM) and supplemented with penicillin/streptomycin (100 U/mL/100 Kg/mL) and 10% (v/v) fetal bovine serum, 1.5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ beta-naphthoflavone
- Test concentrations with justification for top dose:
- 4 h and 18 h treatment without metabolic activation (experiment I and IIA): 5.1, 10.2, 20.3, 40.6, 81.3, 162.5, 325.0, 650.0, 1300.0 µg/mL
4 h treatment with metabolic activation (experiment I): 5.1, 10.2, 20.3, 40.6, 81.3, 162.5, 325.0, 650.0, 1300.0 µg/mL
4 h treatment with metabolic activation (experiment IIA): 25.0, 50.0, 100.0, 125.0, 150.0, 175.0, 200.0, 300.0, 600.0 µg/mL
4 h treatment with metabolic activation (experiment IIB): 50.0, 100.0, 120.0, 140.0, 150.0, 160.0, 180.0, 200.0, 600.0 µg/mL
The highest applied concentration in this study (1300.0 µg/mL) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Precipitation was observed at the end of treatment at 162.5 µg/mL and above. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- - S9: ethylmethanesulphonate (900.0 µg/mL in experiment I, 600.0 µg/mL in experiment IIA) + S9: cyclophosphamide (1.0 µg/mL in experiment I, 1.4 µg/mL in experiments IIA and IIB)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h
SPINDLE INHIBITOR (cytogenetic assays): colcemid (final concentration: 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: Duplicate cultures for every concentration/ control
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were seeded into Quadriperm dishes containing microscopic slides before treatment. Following incubation with the test substance the cells on the slides were treated with hypotonic solution (0.4 % KCl) for 20 min at 37°C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). The slides were stained with Giemsa, mounted after drying and covered with a slid.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): at least 100
DETERMINATION OF CYTOTOXICITY
- Method: other: mitotic index by counting 1000 cells per culture in duplicate, cell numbers by counting 10 defined fields per coded slide
OTHER EXAMINATIONS:
- Determination of polyploidy: yes (polyploid cells in 500 metaphase cells)
- Determination of endoreplication: yes - Evaluation criteria:
- A test item can be classified as non-clastogenic if the number of induced structural chromosomal aberrations in all evaluated dose groups is in the range of the historical laboratory control data and no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control. A test item can be classified as clastogenic if the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data and either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed. If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated. An increase in the number of polyploid cells or endoreduplicated cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations or to inhibit cell cycle progression.
- Statistics:
- Statistical significance is confirmed by the Fisher´s exact test (p < 0.05).
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 4 h and 18 h / - S9: at 1300 µg/mL 4 h / + S9: at 600 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant influence
- Effects of osmolality: no relevant influence
- Precipitation: Visible precipitation of the test item in the culture medium was observed at the end of treatment at 162.5 µg/mL and above in experiment I in the absence and presence of S9 mix and in experiment IIA in the absence of S9 mix, at 150.0 µg/mL and above in experiment IIA in the presence of S9 mix and at 180.0 µg/mL and above in experiment IIB in the presence of S9 mix.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 1
- Negative (solvent/vehicle) historical control data: see table 1
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index and cell number
Referenceopen allclose all
Table 1 Historical control data
Strain |
|
without S9 mix |
with S9 mix |
||||||
Mean |
SD |
Min |
Max |
Mean |
SD |
Min |
Max |
||
TA 1535 |
Solvent control |
14 |
2.37 |
9 |
23 |
20 |
3.75 |
11 |
35 |
Untreated control |
14 |
2.87 |
7 |
25 |
20 |
3.82 |
10 |
32 |
|
Positive control |
1751 |
226.44 |
710 |
2385 |
367 |
93.12 |
126 |
703 |
|
TA 1537 |
Solvent control |
12 |
3.31 |
5 |
26 |
16 |
4.34 |
7 |
30 |
Untreated control |
12 |
4.03 |
5 |
29 |
18 |
4.92 |
6 |
33 |
|
Positive control |
88 |
38.92 |
61 |
448 |
342 |
144.31 |
77 |
809 |
|
TA 98 |
Solvent control |
30 |
5.60 |
17 |
47 |
40 |
6.08 |
21 |
58 |
Untreated control |
31 |
6.36 |
17 |
55 |
42 |
6.83 |
24 |
68 |
|
Positive control |
372 |
78.05 |
158 |
595 |
2167 |
717.60 |
249 |
4089 |
|
TA 100 |
Solvent control |
142 |
29.42 |
86 |
243 |
156 |
29.4 |
99 |
249 |
Untreated control |
450 |
28.19 |
86 |
248 |
163 |
31.26 |
94 |
281 |
|
Positive control |
1741 |
488.75 |
569 |
3082 |
2642 |
796.59 |
825 |
4503 |
|
TA 102 |
Solvent control |
380 |
43.64 |
305 |
510 |
502 |
89.88 |
321 |
677 |
Untreated control |
372 |
41.71 |
300 |
479 |
511 |
87.27 |
315 |
659 |
|
Positive control |
2499 |
898.06 |
1080 |
4678 |
2329 |
598.00 |
1091 |
3972 |
|
Mean = mean value of revertants/plate; SD = standard deviation; Min = minimal value; Max = maximal value
Table 2. Test results of the pre-experiment/experiment I (plate incorporation)
With or without S9-Mix |
Test substance concentration [µg/plate] |
Mean number of revertant colonies per plate (average of 3 plates ± standard deviation) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
||
- |
0 (DMSO) |
13 ± 3 |
10 ± 4 |
33 ± 3 |
88 ± 4 |
355 ± 22 |
- |
0 |
14 ± 1 |
11 ± 2 |
37 ± 6 |
98 ± 6 |
329 ± 24 |
- |
3 |
15 ± 2 |
10 ± 5 |
34 ± 6 |
83 ± 2 |
325 ± 49 |
- |
10 |
11 ± 2 |
7 ± 2 |
30 ± 6 |
85 ± 4 |
366 ± 29 |
- |
33 |
12 ± 4 |
9 ± 1 |
24 ± 3 |
90 ± 6 |
366 ± 19 |
- |
100 |
16 ± 5 |
8 ± 0 |
35 ± 9 |
89 ± 23 |
311 ± 29 |
- |
333 |
13 ± 3 |
7 ± 2 |
30 ± 1 |
87 ± 12 |
317 ± 9 |
- |
1000 |
13 ± 8P |
8 ± 2P |
27 ± 6P |
90 ± 23P |
316 ± 2P |
- |
2500 |
11 ± 2P,M |
4 ± 2P,M |
28 ± 4P,M |
94 ± 9P,M |
224 ± 27P,M |
- |
5000 |
9 ± 2P,M |
3 ± 1P,M |
21 ± 1P,M |
92 ± 12P,M |
172 ± 9P,M |
Positive controls, - S9 |
Na-azide, 10 µg/plate |
1913 ± 54 |
|
|
1949 ± 122 |
|
4-NOPD, 10 µg/plate |
|
|
254 ± 69 |
|
|
|
4-NOPD, 50 µg/plate |
|
72 ± 6 |
|
|
|
|
MMS, 2.0 µL/plate |
|
|
|
|
3681 ± 508 |
|
+ |
0 (DMSO) |
20 ± 3 |
18 ± 4 |
29 ± 6B,M |
137 ± 10 |
484 ± 36 |
+ |
0 |
16 ± 5 |
16 ± 1 |
36 ± 6B,M |
147 ± 10 |
533 ± 17 |
+ |
3 |
19 ± 6 |
18 ± 3 |
27 ± 5B,M |
131 ± 10 |
460 ± 34 |
+ |
10 |
16 ± 4 |
16 ± 4 |
26 ± 6B,M |
137 ± 11 |
494 ± 26 |
+ |
33 |
21 ± 4 |
17 ± 5 |
23 ± 3B,M |
126 ± 8 |
512 ± 65 |
+ |
100 |
18 ± 7 |
15 ± 2 |
28 ± 4B,M |
140 ± 3 |
459 ± 47 |
+ |
333 |
17 ± 4 |
21 ± 2 |
29 ± 2B,M |
132 ± 11 |
439 ± 9 |
+ |
1000 |
18 ± 4 |
12 ± 3 |
31 ± 7B,M |
111 ± 8 |
485 ± 39 |
+ |
2500 |
16 ± 3P |
10 ± 1P,M |
31 ± 4B,P,M |
115 ± 11P |
514 ± 11P |
+ |
5000 |
12 ± 2P,M |
10 ±3P,M |
33 ± 8B,P,M |
119 ± 6P,M |
410 ± 1P,M |
Positive controls, + S9 |
2-AA, 2.5 µg/plate |
414 ± 23 |
373 ± 35 |
1905 ± 100B,M |
3106 ± 300 |
|
2-AA, 10 µg/plate |
|
|
|
|
3158 ± 70 |
|
Na-azide: sodium azide; 4-NOPD: 4-nitro-o-phenylene diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene
P = precipitate; M = manual count, B = extensive bacterial growth
Table 3. Test results of the experiment II (preincubation)
With or without S9-Mix |
Test substance concentration [µg/plate] |
Mean number of revertant colonies per plate (average of 3 plates ± standard deviation) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
||
- |
0 (DMSO) |
14 ± 5 |
9 ± 0 |
25 ± 9 |
90 ± 3 |
363 ± 4 |
- |
0 |
15 ± 4 |
8 ± 4 |
32 ± 12 |
98 ± 5 |
384 ± 19 |
- |
10 |
12 ± 6 |
9 ± 2 |
25 ± 7 |
101 ± 15 |
391 ± 18 |
- |
33 |
13 ± 3 |
8 ± 0 |
25 ± 4 |
81 ± 3 |
381 ± 20 |
- |
100 |
11 ± 3 |
9 ± 1 |
25 ± 3 |
98 ± 12 |
359 ± 27 |
- |
333 |
13 ± 5 |
9 ± 3 |
28 ± 2 |
85 ± 7 |
368 ± 34 |
- |
1000 |
12 ± 2 |
8 ± 1 |
22 ± 7 |
98 ± 19 |
398 ± 22 |
- |
2500 |
14 ± 1P |
8 ± 2P,M |
24 ± 6P |
87 ± 11P |
399 ± 10P |
- |
5000 |
11 ± 3P,M |
5 ± 3P,M |
20 ± 2P,M |
89 ± 11P,M |
330 ± 42P,M |
Positive controls, - S9 |
Na-azide, 10 µg/plate |
1939 ± 64 |
|
|
2144 ± 26 |
|
4-NOPD, 10 µg/plate |
|
|
342 ± 28 |
|
|
|
4-NOPD, 50 µg/plate |
|
68 ± 7 |
|
|
|
|
MMS, 2.0 µL/plate |
|
|
|
|
3761 ± 15 |
|
+ |
0 (DMSO) |
20 ± 3 |
18 ± 4 |
44 ± 8 |
129 ± 13 |
522 ± 27 |
+ |
0 |
18 ± 3 |
20 ± 5 |
45 ± 3 |
152 ± 10 |
493 ± 6 |
+ |
10 |
20 ± 4 |
17 ± 3 |
42 ± 5 |
133 ± 17 |
522 ± 25 |
+ |
33 |
22 ± 9 |
15 ± 7 |
37 ± 3 |
137 ± 21 |
527 ± 37 |
+ |
100 |
18 ± 4 |
19 ± 6 |
47 ± 15 |
137 ± 11 |
546 ± 63 |
+ |
333 |
19 ± 6 |
21 ± 2 |
41 ± 6 |
142 ± 14 |
476 ± 29 |
+ |
1000 |
23 ± 3 |
17 ± 2 |
50 ± 2 |
127 ± 9 |
509 ± 19 |
+ |
2500 |
20 ± 3P |
15 ± 5P |
61 ± 4P |
124 ± 4P |
557 ± 22P |
+ |
5000 |
13 ± 5P,M |
12 ± 4P,M |
46 ± 8P,M |
127 ± 9P,M |
534 ± 23P,M |
Positive controls, + S9 |
2-AA, 2.5 µg/plate |
415 ± 40 |
269 ± 8 |
2314 ± 458 |
3638 ± 20 |
|
2-AA, 10 µg/plate |
|
|
|
|
2217 ± 259 |
|
Na-azide: sodium azide; 4-NOPD: 4-nitro-o-phenylene diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene
P = precipitate; M = manual count
Table 1 Historical control data
Number of mutant colonies per 10^6 cells |
||
without metabolic activation (4 h treatment time) |
||
|
Positive control EMS 150 μg/mL |
Solvent control (DMSO) |
Range |
54.8 – 889.0 |
2.6 – 43.5 |
Mean value |
131.0 |
17.2 |
Standard deviation |
75.5 |
8.3 |
Number of studies |
75 |
75 |
with metabolic activation (4 h treatment time) |
||
|
Positive control DMBA 1.1 μg/mL |
Solvent control (DMSO) |
Range |
91.4 – 2666.3 |
3.4 – 44.2 |
Mean value |
723.8 |
15.5 |
Standard deviation |
342.9 |
6.9 |
Number of studies |
75 |
75 |
without metabolic activation (24 h treatment time) |
||
|
Positive control EMS 150 μg/mL |
Solvent control (DMSO) |
Range |
124.0 – 2746.9 |
2.4 – 40.5 |
Mean value |
357.6 |
16.0 |
Standard deviation |
275.0 |
8.4 |
Number of studies |
60 |
60 |
EMS = ethylmethanesulphonate, DMBA = 7,12-dimethylbenzanthracene
Table 2 Summary of results of experiment I and II
Concentration [µg/mL] |
Culture I |
Culture II |
||||||
Cloning efficiency [%] |
Cell density [%] |
Mutant colonies/10^6 cells |
Induction factor |
Cloning efficiency [%] |
Cell density [%] |
Mutant colonies/10^6 cells |
Induction factor |
|
Experiment I / 4 h treatment without S9 |
||||||||
0 (DMSO) |
100.0 |
100.0 |
15.3 |
1.0 |
100.0 |
100.0 |
14.3 |
1.0 |
81.3 |
101.2 |
65.6 |
11.3 |
0.7 |
93.0 |
87.5 |
4.4 |
0.3 |
162.5 |
102.3 |
65.7 |
21.5 |
1.4 |
114.6 |
84.3 |
9.2 |
0.6 |
325.0P |
94.7 |
57.9 |
23.7 |
1.6 |
92.5 |
69.7 |
16.5 |
1.2 |
650.0P |
91.9 |
30.1 |
20.7 |
1.4 |
115.3 |
39.2 |
13.1 |
0.9 |
1300P |
97.8 |
53.0 |
14.4 |
0.9 |
86.1 |
84.5 |
9.9 |
0.7 |
Positive control (EMS, 150 µg/mL) |
85.9 |
71.2 |
141.5 |
9.3 |
84.1 |
77.0 |
96.8 |
6.8 |
Experiment I / 4 h treatment with S9 |
||||||||
0 (DMSO) |
100.0 |
100.0 |
11.5 |
1.0 |
100.0 |
100.0 |
11.6 |
1.0 |
20.3 |
103.1 |
83.6 |
11.4 |
1.0 |
103.1 |
143.6 |
12.9 |
1.1 |
40.6 |
105.0 |
121.2 |
25.4 |
2.2 |
94.1 |
139.4 |
22.4 |
1.9 |
81.3 |
101.4 |
122.8 |
12.7 |
1.1 |
98.9 |
137.4 |
13.7 |
1.2 |
162.5 |
102.8 |
163.6 |
11.6 |
1.0 |
98.6 |
130.5 |
21.3 |
1.8 |
325.0P |
103.6 |
17.0 |
23.1 |
2.0 |
95.1 |
19.0 |
36.0 |
3.1 |
Positive control (DMBA, 1.1 µg/mL) |
102.8 |
53.1 |
509.5 |
44.1 |
97.3 |
102.9 |
457.8 |
39.5 |
Experiment II / 24 h treatment without S9 |
||||||||
0 (DMSO) |
100.0 |
100.0 |
14.6 |
1.0 |
100.0 |
100.0 |
7.6 |
1.0 |
40.6 |
90.2 |
107.5 |
11.7 |
0.8 |
118.4 |
93.0 |
8.2 |
1.1 |
81.3 |
99.4 |
105.7 |
5.2 |
0.4 |
105.4 |
81.4 |
5.7 |
0.7 |
121.9 |
93.5 |
138.4 |
9.3 |
0.6 |
97.5 |
114.3 |
20.5 |
2.7 |
162.5 |
87.8 |
75.2 |
5.5 |
0.4 |
100.8 |
70.3 |
29.4 |
3.9 |
243.3 |
92.5 |
117.0 |
18.0 |
1.2 |
92.9 |
103.9 |
18.9 |
2.5 |
Positive control (EMS, 150 µg/mL) |
90.4 |
110.3 |
334.7 |
22.9 |
94.8 |
101.8 |
342.5 |
45.3 |
Experiment II / 4 h treatment with S9 |
||||||||
0 (DMSO) |
100.0 |
100.0 |
15.6 |
1.0 |
100.0 |
100.0 |
7.0 |
1.0 |
81.3 |
110.0 |
87.4 |
9.8 |
0.6 |
106.0 |
106.9 |
7.1 |
1.0 |
121.9 |
112.4 |
111.8 |
5.5 |
0.4 |
90.1 |
98.0 |
6.3 |
0.9 |
162.5P |
128.6 |
88.8 |
12.8 |
0.8 |
100.8 |
112.0 |
6.1 |
0.9 |
243.3P |
119.3 |
92.6 |
6.7 |
0.4 |
99.3 |
97.9 |
9.8 |
1.4 |
325.0P |
105.5 |
11.5 |
6.2 |
0.4 |
90.2 |
14.9 |
7.1 |
1.0 |
Positive control (DMBA, 1.1 µg/mL) |
97.3 |
94.6 |
357.6 |
23.0 |
91.1 |
73.7 |
398.8 |
57.2 |
EMS = ethylmethanesulphonate, DMBA = 7,12-dimethylbenzanthracene
P = precipitation
Table 1 Historical control data
ABERRANT CELLS (%) |
||||
|
No. of exp. |
Range |
Mean |
Standard deviation (±) |
Without S9 mix: preparation interval 18 h, treatment 4 h |
||||
Organic solvent1 |
33 |
0.0 – 3.0 |
1.5 |
0.7 |
PC: EMS 900 – 1000 µg/mL |
62 |
8.5 – 51.0 |
22.8 |
7.3 |
Without S9 mix: preparation interval 18 h, treatment 18 h |
||||
Organic solvent1 |
17 |
0.5 – 3.5 |
1.4 |
0.7 |
PC: EMS 500 – 1000 µg/mL |
42 |
8.0 – 51.0 |
24.4 |
8.6 |
With S9 mix: preparation interval 18 h, treatment 4 h |
||||
Organic solvent1 |
35 |
1.0 – 4.0 |
2.3 |
0.7 |
PC: CPA 1.0 – 2.0 µg/mL |
76 |
7.5 – 42.0 |
16.0 |
4.2 |
POLYPLOID CELLS (%) |
||||
|
No. of exp. |
Range |
Mean |
Standard deviation (±) |
Without S9 mix: preparation interval 18 h, treatment 4 h |
||||
Organic solvent1 |
14 |
0.0 – 4.2 |
2.6 |
0.8 |
Without S9 mix: preparation interval 18 h, treatment 18 h |
||||
Organic solvent1 |
7 |
1.5 – 4.2 |
3.0 |
0.7 |
With S9 mix: preparation interval 18 h, treatment 4 h |
||||
Organic solvent1 |
16 |
0.0 – 4.7 |
2.7 |
0.9 |
1Organic solvents: dimethyl sulfoxide, acetone, ethanol and tetrahydrofurane (0.5 % v/v)
PC = positive control, EMS = ethylmethanesulphonate, CPA = cyclophosphamide
Table 2 Summary of results of the chromosomal aberration study
Exp. |
Preparation interval |
Test item concentration [µg/mL] |
Polyploid cells [%] |
Endomitotic cells [%] |
Cell numbers [% of control] |
Mitotic indices [% of control] |
Aberrant cells [%] |
||
|
|
|
|
|
|
|
incl. gaps* |
excl. gaps* |
with exchanges |
Exposure period 4 h without S9 mix |
|||||||||
I |
18 h |
Solvent control1 |
5.5 |
0.0 |
100.0 |
100.0 |
2.0 |
2.0 |
0.0 |
|
|
Positive control2 |
n.d. |
n.d. |
n.d. |
82.4 |
16.0 |
16.0S |
9.5 |
|
|
81.3 |
5.0 |
0.0 |
87.7 |
111.0 |
1.5 |
1.0 |
0.0 |
|
|
162.5P |
4.3 |
0.0 |
85.6 |
118.6 |
2.5 |
2.0 |
0.0 |
|
|
325.0P |
2.9 |
0.0 |
42.1 |
83.8 |
2.0 |
2.0 |
0.5 |
|
|
1300.0P |
1.9 |
0.0 |
64.8 |
41.7 |
2.5 |
2.0 |
0.0 |
Exposure period 18 hrs without S9 mix |
|||||||||
IIA |
18 hrs |
Solvent control1 |
2.0 |
0.0 |
100.0 |
100.0 |
2.0 |
1.5 |
0.0 |
|
|
Positive control3 |
n.d. |
n.d. |
n.d. |
67.2 |
28.0 |
27.5S |
11.0 |
|
|
40.6 |
3.0 |
0.0 |
96.2 |
97.6 |
2.5 |
2.0 |
0.5 |
|
|
81.3 |
2.3 |
0.1 |
91.9 |
71.6 |
3.5 |
2.5 |
0.0 |
|
|
162.5P# |
1.4 |
0.0 |
63.9 |
49.6 |
2.3 |
2.3 |
0.3 |
Exposure period 4 h with S9 mix |
|||||||||
I |
18 h |
Solvent control1 |
5.4 |
0.0 |
100.0 |
100.0 |
1.5 |
1.5 |
0.0 |
|
|
Positive control4 |
n.d. |
n.d. |
n.d. |
41.9 |
21.0 |
21.0S |
8.5 |
|
|
40.6 |
3.2 |
0.0 |
86.2 |
99.4 |
1.5 |
1.5 |
0.0 |
|
|
81.3# |
5.1 |
0.0 |
89.5 |
101.5 |
3.8 |
3.5 |
0.3 |
|
|
162.5P |
4.7 |
0.0 |
52.0 |
98.2 |
3.0 |
2.0 |
0.5 |
IIA |
18 h |
Solvent control1 |
3.3 |
0.1 |
100.0 |
100.0 |
0.5 |
0.5 |
0.0 |
|
|
Positive control5 |
n.d. |
n.d. |
n.d. |
66.9 |
26.0 |
26.0S |
8.5 |
|
|
125.0 |
3.1 |
0.2 |
98.6 |
103.3 |
2.0 |
2.0 |
0.5 |
|
|
150.0#P |
2.3 |
0.2 |
84.8 |
84.5 |
5.3 |
4.5S |
1.8 |
|
|
175.0P |
3.7 |
0.6 |
76.7 |
72.0 |
3.5 |
3.5S |
0.5 |
|
|
200.0P |
2.3 |
0.2 |
57.6 |
44.1 |
3.5 |
3.5S |
0.5 |
IIB |
18 h |
Solvent control1 |
4.9 |
0.5 |
100.0 |
100.0 |
1.5 |
1.5 |
0.5 |
|
|
Positive control5 |
n.d. |
n.d. |
n.d. |
83.7 |
15.0 |
14.5S |
5.5 |
|
|
140.0 |
2.5 |
0.4 |
114.3 |
101.9 |
3.0 |
3.0 |
0.5 |
|
|
150.0 |
2.4 |
0.0 |
78.6 |
60.4 |
1.5 |
1.0 |
1.0 |
|
|
160.0 |
2.0 |
0.0 |
73.3 |
66.7 |
2.5 |
2.5 |
0.0 |
|
|
180.0P |
3.1 |
0.0 |
106.7 |
47.4 |
1.5 |
1.5 |
0.0 |
* Including cells carrying exchanges
# Evaluation of 200 metaphases per culture
n.d. Not determined
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significant higher than corresponding control values
1 DMSO 0.5 % (v/v)
2 Ethylmethanesulphonate 900.0 µg/mL
3 Ethylmethanesulphonate 600.0 µg/mL
4 Cyclophosphamide 1.0 µg/mL
5 Cyclophosphamide 1.4 µg/mL
The statistically significant increases in chromosomal aberrations observed were not considered treatment-related since they did not occur in a dose-related manner and/or were within the range of historical control data. Additionally, in the confirmatory experiment IIB with a top dose of 600.0 µg/mL and narrow concentration spacing, no increases in chromosomal aberrations were observed.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Erythrocyte micronucleus study in mice (OECD 474): negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 Sep - 30 Oct 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted in 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Version / remarks:
- adopted in 1998
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- other: in vivo micronucleus test in mice
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 35.0 ± 1.7 g (males), 28.5 ± 1.2 g (females)
- Assigned to test groups randomly: yes
- Housing: individual in Makrolon Type II (pre-test) / III (main study) cages with wire mesh top, granulated soft wood bedding
- Diet: certified standard diet, pelleted, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 42.8 - 65
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Corn oil was chosen due to its relative non-toxicity for the animals and as a smooth homogeneous suspension was attainable, which was not the case for several aqueous solvents/vehicles tested.
- Amount of vehicle: 10 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was suspended in corn oil. Grinding of the test item in a mortar was used to formulate the test item.
- Duration of treatment / exposure:
- not applicable
- Frequency of treatment:
- single treatment
- Post exposure period:
- 24 and 48 h after last treatment
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 2 (pre-experiment on toxicity)
6 (main study) - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: orally once
- Doses / concentrations: 40 mg/kg bw - Tissues and cell types examined:
- Cell type: bone marrow erythroblasts
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Since no signs of toxicity were observed in a pre-experiment on toxicity a limit test was performed in two sexes using the maximum dose of 2000 mg/kg b.w.
TREATMENT AND SAMPLING TIMES: The animals of all dose groups, except the positive control, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.
DETAILS OF SLIDE PREPARATION: Following sacrifice the femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal bovine serum using a syringe. The cell suspension was centrifuged and a small drop of the re-suspended cell pellet was spread on a slide. The smear was airdried, stained with Giemsa and cover slips were mounted. One slide was made from each bone marrow sample.
METHOD OF ANALYSIS: Evaluation of the slides was performed using light microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. - Evaluation criteria:
- A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group above the laboratory’s historical solvent control data range. The primary point of consideration is the biological relevance of the results and statistical analysis was used as an aid in evaluating the results, if necessary. A test item that fails to produce a biological relevant increase in the number of micronucleated
polychromatic erythrocytes is considered non-mutagenic in this system. - Statistics:
- Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the nonparametric Mann-Whitney test.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: The animals treated with the test item did not express any clinical symptoms.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and gender after administration of the test item.
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of polychromatic erythrocytes of the vehicle control.
Reference
Table 1: Results of the in vivo micronucleus assay in male animals
|
Mean PCEs / 2000 erythrocytes at sampling time
|
Total micronuclei per 1000 PCEs at sampling time (range) |
PCEs with micronuclei (%) |
|||||
Test group |
Number of animals |
Dose [mg/kg bw] |
24 h |
48 h |
24 h |
48 h |
24 h |
48 h |
Vehicle control (corn oil) |
6 |
0 |
1160 |
n.d. |
1 - 6 |
n.d. |
0.150 |
n.d. |
Positive control (cyclophosphamide) |
6 |
40 |
1163 |
n.d. |
12 – 60* |
n.d. |
1.900* |
n.d. |
Test substance |
6 |
2000 |
1173 |
1178 |
1 - 4 |
1 - 4 |
0.117 |
0.117 |
n.d. = not determined; *statistically significant (p<0.001); PCE = polychromatic erythrocytes
Table 2: Results of the in vivo micronucleus assay in female animals
|
Mean PCEs / 2000 erythrocytes at sampling time
|
Total micronuclei per 1000 PCEs at sampling time (range) |
PCEs with micronuclei (%) |
|||||
Test group |
Number of animals |
Dose [mg/kg bw] |
24 h |
48 h |
24 h |
48 h |
24 h |
48 h |
Vehicle control (corn oil) |
6 |
0 |
1149 |
n.d. |
0 - 5 |
n.d. |
0.083 |
n.d. |
Positive control (cyclophosphamide) |
6 |
40 |
1163 |
n.d. |
33 – 89* |
n.d. |
2.625* |
n.d. |
Test substance |
6 |
2000 |
1271 |
1212 |
0 - 3 |
0 - 4 |
0.108 |
0.083 |
n.d. = not determined; *statistically significant (p<0.001); PCE = polychromatic erythrocytes
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Reliable studies regarding genetic toxicity are available for the test substance.
In vitro:
- Gene mutation in bacteria:
The potential of the test substance to induce gene mutations was assessed in a study performed according to OECD Guideline 471 and in compliance with GLP (M-461517-01-1). The test item was suspended in DMSO, which was also used as vehicle control. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with the test material for 48 h using the Ames plate incorporation (experiment I) and the pre-incubation (experiment II) method at concentrations ranging from 3 to 5000 µg/plate, in triplicate, both with and without liver microsomal activation. In the plate incorporation test the test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 µg/plate. Using the pre-incubation method precipitation of the test item was observed in the overlay agar in the test tubes and on the incubated agar plates from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Only in experiment I a reduction in the number of revertants (below the indication factor of 0.5) was observed in strain TA 1537 at 2500 and 5000 µg/plate in the absence of metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.
- Gene mutation in mammalian cells:
The potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was assessed in a study performed according to OECD Guideline 476 and in compliance with GLP (M-462519-01-1).The test item was suspended in DMSO, which was also used as vehicle control. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h at concentrations ranging from 10.2 to 1300 µg/mL.Precipitation of the test item was observed at 325 µg/mL and above. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation at concentrations ranging from 10.2 to 325 µg/mL. Precipitation of the test item was observed at 325 and at 162.5 µg/mL and above with and without metabolic activation, respectively. Cytotoxicty, evident as a reduction of the cloning efficiency, was observed at 325 µg/mL with and 650 µg/mL without metabolic activation in the first experiment and at 325 µg/mL with metabolic activation in the second experiment. No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the two main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.
- Cytogenicity / chromosome aberration in mammalian cells:
The potential of the test substance to induce structural chromosomal aberrations in V79 cells of the Chinese hamster was assessed in a study performed according to OECD Guideline 473 and in compliance with GLP (M-465032-01-1). The test item was suspended in DMSO, which was also used as vehicle control. Cells were treated with the test material both with and without metabolic activation. In the first experiment without metabolic activation (experiment I, -S9) cells were exposed for 4 h followed by a recovery interval of 14 h. In the second experiment without metabolic activation (experiment IIA (-S9)) cells were exposed for 18 h without recovery. Three experiments with an exposure period of 4 h and a 14 h-recovery interval were performed with metabolic activation (experiment I, IIA and IIB (+S9)). In experiments I with and without S9 mix and in experiment IIA without S9 mix concentrations of the test substance ranged from 5.1 to 1300.0 µg/mL. Precipitation was observed at 162.5 µg/mL and above. In experiments IIA and IIB (+S9) cells were treated with concentrations ranging from 25.0 to 600.0 µg/mL using narrow concentration intervals between 100.0 and 200.0 µg/mL in experiment IIB. Precipitation was observed at 150.0 and 180.0 µg/mL and above in experiment IIA and IIB (+S9), respectively. Cytotoxicity indicated as a reduced mitotic index was observed in experiment I and IIA (-S9) and in experiment IIA and IIB (+S9) at the highest evaluated concentration. In experiment I (+S9) cytotoxicity indicated as a reduced cell number was observed at the highest evaluated concentration. No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. In experiment IIA (+S9) statistically significant increases in chromosomal aberrations were observed at 150.0, 175.0 and 200.0 µg/mL (4.5, 3.5, 3.5 % aberrant cells, excluding gaps), however, the increases did not occur in a dose-related manner. The first value (4.5 %) slightly exceeded the range of the laboratory historical control data (0.0 - 4.0 % aberrant cells, excluding gaps), whilst the value (3.5 %) at the two higher concentrations of 175.0 and 200.0 µg/mL, was within this range. In the confirmatory experiment IIB, with a top dose of 600.0 µg/mL and narrow concentration spacing, no increases in chromosomal aberrations were observed. Thus, the increases in chromosomal aberrations in experiment IIA are not considered treatment-related. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. These substances induced statistically significant increases in cells with structural chromosome aberrations. Thus, under the conditions of this study, the test substance is considered to be non-clastogenic.
In vivo:
- Cytogenicity / micronucleus test in mice:
The potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was assessed in a study performed according to OECD Guideline 474 and in compliance with GLP (M-473874-01-1). The test item was suspended in corn oil, which was also used as vehicle control. Six male and six female C57BL/6J mice per test group were administered 2000 mg/kg bw. 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any gender used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.
Justification for classification or non-classification
The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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