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EC number: 269-924-1 | CAS number: 68391-05-9 This substance is identified by SDA Substance Name: C12-C18 dialkyl dimethyl ammonium chloride and SDA Reporting Number: 16-047-00.
- 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
Based on the results of the test and read across in vitro genotoxicity studies, the test substance is considered to be non-genotoxic.
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:
- From April 23, 1990 to May 10, 1990
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- not specified
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine: hisC3076, hisD3052 pKM101, hisG46, hisG46 pKM101
- Species / strain / cell type:
- S. typhimurium TA 1535
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Preliminary toxicity test:
1.0, 3.3, 10, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
Experiment 1
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate
Experiment 2
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate - Vehicle / solvent:
- Dimethylsulphoxide (DMSO) of spectroscopic quality
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA1535), 9-aminoacridine (TA1537), daunomycine (TA98), methylmethanesulfonate (TA100)
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (all 4)
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: until 10^9 cells/mL had been obtained
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): Histidine
NUMBER OF REPLICATIONS: Five different doses of the test substance have been tested in triplicate in each strain. An independentrepeat of the experiment was performed.
NUMBER OF CELLS EVALUATED: All colonies were counted.
DETERMINATION OF CYTOTOXICITY
Method: A preliminary toxicity test was performed with TA100 (with and without S9-mix), 9 concentrations were tested in duplicate. The survival of the TA100 culture was determined by comparing the number of colonies on the plate + test substance with those onthe solvent control plate. - Evaluation criteria:
- An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory
historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times
the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding
test with strain TA100 or should extend to 5 mg/plate (active ingredient).
A test substance was considered negative (not mutagenic) i n the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance was considered positive (mutagenic)in the Ames test if :
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent
control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two
concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision. - Statistics:
- Not reported
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (in preliminary test)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- 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
- Remarks:
- (in preliminary test)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (in preliminary test)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (in preliminary test)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
No data
RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100
was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data - Conclusions:
- Under the study conditions, the test substance was considered to be non mutagenic in the Ames test.
- Executive summary:
A study was conducted to evaluate the mutagenic potential of the test substance, C12 -18 DAQ (76.4% active in hydroalcoholic solution) in the Ames test, according to the OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. In the study S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains were used and S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats was used as metabolic activating system. A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100 was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix. Experiment 1: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. Experiment 2: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. Under the study conditions, the test substance was considered to be non mutagenic in the Ames test (Scheres, 1990).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From November 10, 1995 to March 06, 1996
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- cultured peripheral human lymphocytes
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's, The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (OOC) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer, The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).
Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.02 mg MgC12.6H20; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 umol HEPES. The above solution was filter (0.22 pm)-sterilized. To 0.5 ml S9-mix components 0.5 ml W-fraction (batch 95.7) was added (50% (v/v) S9-fraction). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to 5.3 ml exposition medium (4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) Phytohaemagglutinin). The concentration of the S9-fraction in the exposition medium was 1.8% (v/v). - Test concentrations with justification for top dose:
- First experiment: (a) Without S9-mix: 3-4.2 and 5.6 µg/mL (24 h) and 5.6 µg/mL (48h) (b) With S9-mix: 10-18 and 24 µg/mL (24 h) and 18 µg/mL (48 h)
Second experiment : (a) Without S9-mix: 3-4.2 and 7.5 µg/mL (24h) (b) With S9-mix: 3-18 and 24 µg/mL (24h) - Vehicle / solvent:
- - DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With S9 mix
- Details on test system and experimental conditions:
- Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix: Aroclor-1254 induced rat liver SS-mix from adult male Wistar rats, from Charles River Wiga, Sulzfeld, Germany.
CELL CULTURE
Blood samples
Blood samples were taken from a healthy adult male volunteer by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 h after blood collection lymphocyt cultures were started.
F10 complete culture medium
F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/1) and 30 U/ml heparin.
Cell culture conditions
Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (5 ml F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added.
Environmental conditions
All incubations were carried out in a humid atmosphere (80-95%) containing 5% co2 in air in the dark at 37°C. The temperature, humidity and CO2- percentage were monitored during the experiment.
Negative control:
The vehicle of the test article, being dimethylsulphoxide (DMSO)
Positive controls:
Solvent for positive controls
Hank's Balanced Salt Solution (HBSS) without calcium and magnesium
Without metabolic activation (-S9-mix):
Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.2 ug/ml (solvent: HBSS) for a 24 h treatment period and 0.1 ug/ml for a 48 h treatment period.
With metabolic activation (+S9-mix):
Cyclophosphamide (CP; CAS no. 50-18-O. Endoxan-Asta, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 15 ug/ml (solvent: HBSS) for a 3 h treatment period (24 h fixation time). - Rationale for test conditions:
- Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes. In combination with a mammalian metabolizing system (S9-mix) also indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, could be tested for clastogenic effects in vitro. Following treatment, cell division was arrested in the metaphase stage of the cell cycle by addition of the spindle poison colchicine. Structural chromosome changes such as breaks, gaps, minutes, dicentrics and exchange figures were examined microscopically in cultures treated with the test substance and the results were compared with those of the control (vehicle-treated) cultures. Chromosome aberrations were generally evaluated in the first post-treatment mitosis. The appearance of the first post-treatment mitosis could be considerably delayed, due to toxic insult of the cells. Therefore, cells were harvested at 24 hand 48 h after beginning of treatment to cover the interval in which maximum aberration frequency was expected. A test article which induced a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent.
- Evaluation criteria:
- Test was considered as acceptable, if it met the following criteria:
1. The Numbers of chromosome aberrations found in the solvent control cultures should reasonable be within the laboratory historical control.
2. The positive control substance should produce a statistically significant increase in the number of cells with chromosome aberrations
3. A homogeneous response between the replicate cultures is observed - Statistics:
- Chi square test for the dose related statistical significance
- Key result
- Species / strain:
- other: cultured human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to 24 µg/mL
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - In the first experiment an increase in the number of aberrant cells was found, only at the 24 h fixation time in the presence of S9-mix and at one concentration only (24 µg/mL).
- Since the type of aberrations abserved were only breaks, the increase was not observed at any other concentration and not confirmed in the second experiment, this finding was considered not to be of biological significance.
- Next to this the number of cells with aberrations were just at the historical control data range. - Conclusions:
- Under the study conditions, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation.
- Executive summary:
A study was conducted to determine the clastogenic potential of the test substance, DDAC (51.3% active) in cultured human lymphocytes, according to OECD Guideline 473 and EU method B.10, in compliance with GLP. After a preliminary toxicity test, test substance was tested in two independent experiments, with and without a metabolic activation system, the S9- mix. In the absence of S9 -mix test substance was tested up to 5.6 µg/mL for a 24 and 48 h fixation time in the first experiment. In the second experiment test substance was tested up to 7.5 µg/mL for a 24 h fixation time. In the presence of S9 -mix test substance was tested up to 24 µg/mL for 24 h fixation time and up to 18 µg/mL for a 48 h fixation time in the first experiment. In the second experiment it was tested up to 24 µg/mL for a 24 h fixation time. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since, the types of aberrations observed were only breaks, the increase was not dose related. Experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, therefore the increase was not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation (Van de waart, 1996).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From November 10, 1995 to March 06, 1996
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- other: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- None
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's, The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (OOC) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer, The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).
Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.02 mg MgC12.6H20; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 umol HEPES. The above solution was filter (0.22 pm)-sterilized. To 0.5 ml S9-mix components 0.5 ml W-fraction (batch 95.7) was added (50% (v/v) S9-fraction). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to 5.3 ml exposition medium (4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) Phytohaemagglutinin). The concentration of the S9-fraction in the exposition medium was 1.8% (v/v). - Test concentrations with justification for top dose:
- First experiment: (a) Without S9-mix: 3-4.2 and 5.6 µg/mL (24 h) and 5.6 µg/mL (48h) (b) With S9-mix: 10-18 and 24 µg/mL (24 h) and 18 µg/mL (48 h)
Second experiment : (a) Without S9-mix: 3-4.2 and 7.5 µg/mL (24h) (b) With S9-mix: 3-18 and 24 µg/mL (24h) - Vehicle / solvent:
- - DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With S9 mix
- Details on test system and experimental conditions:
- Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix: Aroclor-1254 induced rat liver SS-mix from adult male Wistar rats, from Charles River Wiga, Sulzfeld, Germany.
CELL CULTURE
Blood samples
Blood samples were taken from a healthy adult male volunteer by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 h after blood collection lymphocyt cultures were started.
F10 complete culture medium
F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/1) and 30 U/ml heparin.
Cell culture conditions
Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (5 ml F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added.
Environmental conditions
All incubations were carried out in a humid atmosphere (80-95%) containing 5% co2 in air in the dark at 37°C. The temperature, humidity and CO2- percentage were monitored during the experiment.
Negative control:
The vehicle of the test article, being dimethylsulphoxide (DMSO)
Positive controls:
Solvent for positive controls
Hank's Balanced Salt Solution (HBSS) without calcium and magnesium
Without metabolic activation (-S9-mix):
Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.2 ug/ml (solvent: HBSS) for a
24 h treatment period and 0.1 ug/ml for a 48 h treatment period.
With metabolic activation (+S9-mix):
Cyclophosphamide (CP; CAS no. 50-18-O. Endoxan-Asta, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 15 ug/ml (solvent: HBSS) for a 3 h treatment period (24 h fixation time). - Rationale for test conditions:
- Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes. In combination with a mammalian metabolizing system (S9-mix) also indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, could be tested for clastogenic effects in vitro. Following treatment, cell division was arrested in the metaphase stage of the cell cycle by addition of the spindle poison colchicine. Structural chromosome changes such as breaks, gaps, minutes, dicentrics and exchange figures were examined microscopically in cultures treated with the test substance and the results were compared with those of the control (vehicle-treated) cultures. Chromosome aberrations were generally evaluated in the first post-treatment mitosis. The appearance of the first post-treatment mitosis could be considerably delayed, due to toxic insult of the cells. Therefore, cells were harvested at 24 hand 48 h after beginning of treatment to cover the interval in which maximum aberration frequency was expected. A test article which induced a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent.
- Evaluation criteria:
- Test was considered as acceptable, if it met the following criteria:
1. The Numbers of chromosome aberrations found in the solvent control cultures should reasonable be within the laboratory historical control.
2. The positive control substance should produce a statistically significant increase in the number of cells with chromosome aberrations
3. A homogeneous response between the replicate cultures is observed - Statistics:
- Chi square test for the dose related statistical significance
- Key result
- Species / strain:
- other: cultured human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to 24 µg/mL
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - In the first experiment an increase in the number of aberrant cells was found, only at the 24 h fixation time in the presence of S9-mix and at one concentration only (24 µg/mL).
- Since the type of aberrations abserved were only breaks, the increase was not observed at any other concentration and not confirmed in the second experiment, this finding was considered not to be of biological significance.
- Next to this the number of cells with aberrations were just at the historical control data range. - Conclusions:
- Based on the results of the read across study, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation.
- Executive summary:
A study was conducted to determine the clastogenic potential of the read across substance, DDAC (51.3% active) in cultured human lymphocytes, according to OECD Guideline 473 and EU method B.10, in compliance with GLP. After a preliminary toxicity test, read across substance was tested in two independent experiments, with and without a metabolic activation system, the S9- mix. In the absence of S9 -mix read across substance was tested up to 5.6 µg/mL for a 24 and 48 h fixation time in the first experiment. In the second experiment read across substance was tested up to 7.5 µg/mL for a 24 h fixation time. In the presence of S9 -mix read across substance was tested up to 24 µg/mL for 24 h fixation time and up to 18 µg/mL for a 48 h fixation time in the first experiment. In the second experiment it was tested up to 24 µg/mL for a 24 h fixation time. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since, the types of aberrations observed were only breaks, the increase was not dose related. Experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, therefore the increase was not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix read across substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly (Van der waart, 1996). Based on the results of the read across study, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From November 27, 2001 to March 05, 2002
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Not applicable
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- TK (Thymidine kinase ) -/+, not able to grow in Trifluorothimidine medium
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix, from Aroclor 1254 induced (500 mg/kg i.p.) rat livers. The final concentration of S9 fraction in the culture medium was 2%.
- Test concentrations with justification for top dose:
- Experiments without S9 mix:
0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL (1st experiment),
0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL (2nd experiment)
Experiments with S9 mix:
0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL (1st experiment),
0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL (2nd experiment). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9 mix
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- Cells
L5178Y cells are an established cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test. They have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. The average cell cycle time is 12-14 hours and the TK phenotypic expression time is 2 days. L5178Y cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (77601, Marne-La-Vallée, France). The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethyl-sulfoxide (DMSO)) at -80°C or in liquid nitrogen. Each batch of frozen cells was purged of TK- mutants and checked for the absence of mycoplasma.
Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Annapolis, MD 21401, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use. The S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to culture medium.
Culture medium
RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10% v/v (RPMI 10) or 20% v/v (RPMI 20).
DURATION
- Exposure duration:
Without S9-mix: 1st experiment: 3 h; 2nd experiment: 24 h
With S9-mix: 1st experiment: 3 h; 2nd experiment: 3 h
NUMBER OF REPLICATIONS: Two plates/dose-level for test and four plates for control
NUMBER OF CELLS EVALUATED: 2000 cells/well - Rationale for test conditions:
- For details on treatment of results, kindly refer to attached background material section of the IUCLID.
- Evaluation criteria:
- Acceptance criteria
This study was considered valid since the following criteria were fulfilled:
• the cloning efficiency of the vehicle controls were between 0.6-1.4 for CE0 and between 0.7-1.3 for CE2,
• the mutation frequency of the vehicle controls were between 60-250E-6
• the mutation frequency of the positive controls were higher than that of the vehicle controls (more than two fold) and consistent with our historical data.
Evaluation criteria
A reproducible noteworthy (at least two-fold) increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered. - Statistics:
- Not reported
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Without S9-mix: 5.56 µg/mL at 3 h treatment; 0.56 µg/mL at 24 h treatment; With S9-mix >5.56 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary toxicity test:
-The test item was freely soluble in the vehicle (DMSO) at 380 mg/mL (expressed as active item). In the culture medium, the dose-level of 3800 µg/mL showed a marked emulsion. At this dose-level, the pH was approximately 7.9 (7.4 for the vehicle control) and no increase in the osmolality due to the test item was noted. Consequently, with a treatment volume of 200 µL/20 mL (1% v/v) culture medium, the dose-levels for the preliminary toxicity test were: 7.6, 76, 380, 760, 1900 and 3800 µg/mL, both with and without S9.
-A slight to marked emulsion was observed at the end of the treatment period at dose-levels ≥ 760 µg/mL.
-Without S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 7.6 µg/mL (93-100% decrease in the cloning efficiency immediately after treatment (CE0) and in the relative survival (RS)).
-With S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 76 µg/mL (97-100% decrease in the CE0 and RS). - Conclusions:
- Under the study conditions, the test substance was considered to be non-mutagenic in mouse lymphoma assay with and without metabolic activation.
- Executive summary:
A study was conducted to determine the potential of the test substance, DDAC (40.37% active) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells, according to OECD guideline 476 and EU method B17, in compliance with GLP. After a preliminary toxicity test, test substance was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5E+6 (3-hour treatment: preliminary toxicity test and all experiments except for the second experiment without S9 mix) or 0.15E+6 (24-hour treatment: second experiment without S9 mix) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. The test substance was dissolved in dimethylsulfoxide (DMSO). All the dose-levels were expressed as active substance, taking into account the active material content of 40.37%. The selected dose-levels for test substance treatment without S9 mix were as follows: (a) 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL, for the first experiment (b) 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL, for the second experiment. The selected dose-levels for test substance treatment with S9 mix were as follows: (a) 0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL, for the first experiment (b) 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL, for the second experiment. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking. The dose-levels for the positive controls were as follows: (1) without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL (3-hour treatment) or 5 µg/mL (24-hour treatment) (2) with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL. Cytotoxicity was then determined using cloning efficiency (CE0) before expression of the mutant phenotype. Cell viability (using cloning efficiency CE2) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Other parameters such as cell concentration, relative survival (RS), relative suspension growth (RSG) and/or relative total growth (RTG) were also taken into consideration. In experiments without S9 mix. a slight to strong toxicity was induced, depending on the dose-levels and the treatment duration. No noteworthy increase in the mutation frequency was induced both experiments without S9 mix after the 3 and 24-hour treatments. In experiments with S9 mix, the test substance was moderately to strongly toxic, depending on the dose-levels. No noteworthy increase in the mutation frequency was induced in both experiments with S9 mix. The cloning efficiencies CE0 and CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Under the study conditions, the test substance was considered to be non-mutagenic in mouse lymphoma assay with and without metabolic activation (Haddouk, 2002).
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From November 27, 2001 to March 05, 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Not applicable
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- TK (Thymidine kinase ) -/+, not able to grow in Trifluorothimidine medium
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix, from Aroclor 1254 induced (500 mg/kg i.p.) rat livers. The final concentration of S9 fraction in the culture medium was 2%.
- Test concentrations with justification for top dose:
- Experiments without S9 mix:
0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL (1st experiment),
0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL (2nd experiment)
Experiments with S9 mix:
0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL (1st experiment),
0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL (2nd experiment). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9 mix
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- Cells
L5178Y cells are an established cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test. They have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. The average cell cycle time is 12-14 hours and the TK phenotypic expression time is 2 days. L5178Y cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (77601, Marne-La-Vallée, France). The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethyl-sulfoxide (DMSO)) at -80°C or in liquid nitrogen. Each batch of frozen cells was purged of TK- mutants and checked for the absence of mycoplasma.
Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Annapolis, MD 21401, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use. The S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to culture medium.
Culture medium
RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10% v/v (RPMI 10) or 20% v/v (RPMI 20).
DURATION
- Exposure duration:
Without S9-mix: 1st experiment: 3 h; 2nd experiment: 24 h
With S9-mix: 1st experiment: 3 h; 2nd experiment: 3 h
NUMBER OF REPLICATIONS: Two plates/dose-level for test and four plates for control
NUMBER OF CELLS EVALUATED: 2000 cells/well - Rationale for test conditions:
- For details on treatment of results, kindly refer to attached background material section of the IUCLID.
- Evaluation criteria:
- Acceptance criteria
This study was considered valid since the following criteria were fulfilled:
• the cloning efficiency of the vehicle controls were between 0.6-1.4 for CE0 and between 0.7-1.3 for CE2,
• the mutation frequency of the vehicle controls were between 60-250E-6
• the mutation frequency of the positive controls were higher than that of the vehicle controls (more than two fold) and consistent with our historical data.
Evaluation criteria
A reproducible noteworthy (at least two-fold) increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered. - Statistics:
- Not reported
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Without S9-mix: 5.56 µg/mL at 3 h treatment; 0.56 µg/mL at 24 h treatment; With S9-mix >5.56 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary toxicity test:
-The test item was freely soluble in the vehicle (DMSO) at 380 mg/mL (expressed as active item). In the culture medium, the dose-level of 3800 µg/mL showed a marked emulsion. At this dose-level, the pH was approximately 7.9 (7.4 for the vehicle control) and no increase in the osmolality due to the test item was noted. Consequently, with a treatment volume of 200 µL/20 mL (1% v/v) culture medium, the dose-levels for the preliminary toxicity test were: 7.6, 76, 380, 760, 1900 and 3800 µg/mL, both with and without S9.
-A slight to marked emulsion was observed at the end of the treatment period at dose-levels ≥ 760 µg/mL.
-Without S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 7.6 µg/mL (93-100% decrease in the cloning efficiency immediately after treatment (CE0) and in the relative survival (RS)).
-With S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 76 µg/mL (97-100% decrease in the CE0 and RS). - Conclusions:
- Based on results of the read across study, the test substance was considered to be non-mutagenic in mouse lymphoma assay with and without metabolic activation.
- Executive summary:
A study was conducted to determine the potential of the read across substance, DDAC (40.37% active) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells, according to OECD guideline 476 and EU method B17, in compliance with GLP. After a preliminary toxicity test, read across substance was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5E+6 (3-hour treatment: preliminary toxicity test and all experiments except for the second experiment without S9 mix) or 0.15E+6 (24-hour treatment: second experiment without S9 mix) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the read across or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. The read across substance was dissolved in dimethylsulfoxide (DMSO). All the dose-levels were expressed as active substance, taking into account the active material content of 40.37%. The selected dose-levels for read across substance treatment without S9 mix were as follows: (a) 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL, for the first experiment (b) 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL, for the second experiment. The selected dose-levels for read across substance treatment with S9 mix were as follows: (a) 0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL, for the first experiment (b) 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL, for the second experiment. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking. The dose-levels for the positive controls were as follows: (1) without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL (3-hour treatment) or 5 µg/mL (24-hour treatment) (2) with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL. Cytotoxicity was then determined using cloning efficiency (CE0) before expression of the mutant phenotype. Cell viability (using cloning efficiency CE2) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Other parameters such as cell concentration, relative survival (RS), relative suspension growth (RSG) and/or relative total growth (RTG) were also taken into consideration. In experiments without S9 mix. a slight to strong toxicity was induced, depending on the dose-levels and the treatment duration. No noteworthy increase in the mutation frequency was induced both experiments without S9 mix after the 3 and 24-hour treatments. In experiments with S9 mix, the read across substance was moderately to strongly toxic, depending on the dose-levels. No noteworthy increase in the mutation frequency was induced in both experiments with S9 mix. The cloning efficiencies CE0 and CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid (Haddouk, 2002). Based on results of the read across study, the test substance was considered to be non-mutagenic in mouse lymphoma assay with and without metabolic activation.
Referenceopen allclose all
All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
Results
Dose range finding test
In the dose range finding test blood cultures were treated with 3, 10, 33, 100 and 333 µg/ml culture medium with and without S9-mix. At a concentration of 333 µg/ml test substance precipitated in the culture medium. Therefore, a concentration of 333 µg/ml was used as the highest concentration of test substance.
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Experiment 1
Without S9-mix: (a) 3, 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (24 h fixation time) (b) 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (48 h fixation time)
With S9-mix: (a) 3, 10, 13, 18, 24 and 33 µg/ml culture medium (24 h fixation time) (b) 10, 13, 18, 24 and 33 µg/ml culture medium (48 h fixation time)
Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 3, 4.2 and 5.6 µg/ml cultuie medium (24 h fixation time), 5.6 µg/ml (48 h fixation time)
With S9- mix: 10, 18 and 24 µg/ml culture medium (24 h fixation time), 18 µg/ml (48 h fixation time)
The data of the dose range finding test and the first cytogenetic assay were used to determine the dose levels for the second cytogenetic assay.
Experiment 2
Without S9-mix: 1, 3, 4.2, 5.6 and 7.5 ug/ml culture medium (24 h fixation time)
With S9-mix: 3, 10, 13, 18, 21 and 24 µg/ml culture medium (24 h fixation time)
Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 3, 4.2 and 7 .5 µg/ml culture medium (24 h fixation time)
With S9-mix: 3, 18 and 24 µg/ml culture medium (24 h fi xati on time)
Cytogenetic assay
The ability of test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in two independent experiments. The results of duplicate cultures are indicated by A and B. The scores for the numbers of aberrant cells (inclusive and exclusive gaps) and the numbers of the various types of chromosome aberrations at the various concentrations of the test substance are presented. The criteria according to which the aberrations were classified are outlined. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps are included and excluded. Since, the types of aberrations observed were only breaks, the increase is not dose related, experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, the increase is not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {i.e.1.0 ± 3.3 (mean± three times the standard deviation) aberrant cells per 100 metaphases (without S9-mix; gaps excluded) and 0.8 ± 2.7 aberrant cells per 100 metaphases (with S9-mix; gaps excluded)}. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Finally, it is concluded that this test should be considered valid and that test substance is not clastogenic under the experimental conditions of this test.
Table 1. Table for Cytogenetic In-Vitro-Test: Chromosomal Analysis |
|||||
positive |
Dose in µg/ml |
||||
state mean and standard deviations below |
control |
||||
Experiment 1 |
|||||
Without S9-mix, 24 h fixation |
control |
3 |
4.2 |
5.6 |
|
Mitotic index |
64 |
100 |
82 |
73 |
47 |
Total aberrations with gaps: |
61 |
1 |
0 |
4 |
0 |
Total aberrations without gaps: |
63 |
1 |
0 |
2 |
0 |
With S9-mix, 24 h fixation |
pos. control |
control |
10 |
18 |
24 |
Mitotic index |
59 |
100 |
81 |
58 |
41 |
Total aberrations with gaps: |
53 |
1 |
6 |
0 |
9 |
Total aberrations without gaps: |
52 |
0 |
4 |
0 |
9 |
Without S9-mix, 48 h fixation |
pos. |
control |
5.6 |
||
Mitotic index |
78 |
100 |
52 |
||
Total aberrations with gaps: |
52 |
1 |
5 |
||
Total aberrations without gaps: |
52 |
0 |
3 |
||
With S9-mix, 48 h fixation |
pos. |
control |
18 |
||
Mitotic index |
n.a. |
100 |
49 |
||
Total aberrations with gaps: |
na |
1 |
6 |
||
Total aberrations without gaps: |
na |
1 |
4 |
||
Experiment 2 |
|||||
Without S9-mix, 24 h fixation |
pos. |
control |
3 |
4.2 |
7.5 |
Mitotic index |
58 |
100 |
83 |
61 |
45 |
Total aberrations with gaps: |
33 |
2 |
2 |
2 |
8 |
Total aberrations without gaps: |
33 |
1 |
1 |
2 |
4 |
With S9-mix, 24 h fixation |
pos. |
control |
3 |
18 |
24 |
Mitotic index |
51 |
100 |
79 |
61 |
49 |
Total aberrations with gaps: |
36 |
3 |
4 |
1 |
5 |
Total aberrations without gaps: |
36 |
3 |
2 |
1 |
3 |
Positve controls: the numbers are based on 150 instead 200 cells evaluated. |
Results
Dose range finding test
In the dose range finding test blood cultures were treated with 3, 10, 33, 100 and 333 µg/ml culture medium with and without S9-mix. At a concentration of 333 µg/ml test substance precipitated in the culture medium. Therefore, a concentration of 333 µg/ml was used as the highest concentration of test substance.
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Experiment 1
Without S9-mix: (a) 3, 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (24 h fixation time) (b) 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (48 h fixation time)
With S9-mix: (a) 3, 10, 13, 18, 24 and 33 µg/ml culture medium (24 h fixation time) (b) 10, 13, 18, 24 and 33 µg/ml culture medium (48 h fixation time)
Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 3, 4.2 and 5.6 µg/ml cultuie medium (24 h fixation time), 5.6 µg/ml (48 h fixation time)
With S9- mix: 10, 18 and 24 µg/ml culture medium (24 h fixation time), 18 µg/ml (48 h fixation time)
The data of the dose range finding test and the first cytogenetic assay were used to determine the dose levels for the second cytogenetic assay.
Experiment 2
Without S9-mix: 1, 3, 4.2, 5.6 and 7.5 ug/ml culture medium (24 h fixation time)
With S9-mix: 3, 10, 13, 18, 21 and 24 µg/ml culture medium (24 h fixation time)
Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 3, 4.2 and 7 .5 µg/ml culture medium (24 h fixation time)
With S9-mix: 3, 18 and 24 µg/ml culture medium (24 h fi xati on time)
Cytogenetic assay
The ability of test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in two independent experiments. The results of duplicate cultures are indicated by A and B. The scores for the numbers of aberrant cells (inclusive and exclusive gaps) and the numbers of the various types of chromosome aberrations at the various concentrations of the test substance are presented. The criteria according to which the aberrations were classified are outlined. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps are included and excluded. Since, the types of aberrations observed were only breaks, the increase is not dose related, experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, the increase is not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {i.e.1.0 ± 3.3 (mean± three times the standard deviation) aberrant cells per 100 metaphases (without S9-mix; gaps excluded) and 0.8 ± 2.7 aberrant cells per 100 metaphases (with S9-mix; gaps excluded)}. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Finally, it is concluded that this test should be considered valid and that test substance is not clastogenic under the experimental conditions of this test.
Table 1. Table for Cytogenetic In-Vitro-Test: Chromosomal Analysis |
|||||
positive |
Dose in µg/ml |
||||
state mean and standard deviations below |
control |
||||
Experiment 1 |
|||||
Without S9-mix, 24 h fixation |
control |
3 |
4.2 |
5.6 |
|
Mitotic index |
64 |
100 |
82 |
73 |
47 |
Total aberrations with gaps: |
61 |
1 |
0 |
4 |
0 |
Total aberrations without gaps: |
63 |
1 |
0 |
2 |
0 |
With S9-mix, 24 h fixation |
pos. control |
control |
10 |
18 |
24 |
Mitotic index |
59 |
100 |
81 |
58 |
41 |
Total aberrations with gaps: |
53 |
1 |
6 |
0 |
9 |
Total aberrations without gaps: |
52 |
0 |
4 |
0 |
9 |
Without S9-mix, 48 h fixation |
pos. |
control |
5.6 |
||
Mitotic index |
78 |
100 |
52 |
||
Total aberrations with gaps: |
52 |
1 |
5 |
||
Total aberrations without gaps: |
52 |
0 |
3 |
||
With S9-mix, 48 h fixation |
pos. |
control |
18 |
||
Mitotic index |
n.a. |
100 |
49 |
||
Total aberrations with gaps: |
na |
1 |
6 |
||
Total aberrations without gaps: |
na |
1 |
4 |
||
Experiment 2 |
|||||
Without S9-mix, 24 h fixation |
pos. |
control |
3 |
4.2 |
7.5 |
Mitotic index |
58 |
100 |
83 |
61 |
45 |
Total aberrations with gaps: |
33 |
2 |
2 |
2 |
8 |
Total aberrations without gaps: |
33 |
1 |
1 |
2 |
4 |
With S9-mix, 24 h fixation |
pos. |
control |
3 |
18 |
24 |
Mitotic index |
51 |
100 |
79 |
61 |
49 |
Total aberrations with gaps: |
36 |
3 |
4 |
1 |
5 |
Total aberrations without gaps: |
36 |
3 |
2 |
1 |
3 |
Positve controls: the numbers are based on 150 instead 200 cells evaluated. |
Results
Based on the preliminary toxicity test for the selection of
the doses, the selected dose-levels for treatment were as follow :
All the dose-levels were expressed as active substance,
taking into account the active material content of 40.37%.
Without S9 mix
- First experiment: 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL
- Second Experiment: 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL
After 3 h treatment (first experiment), a slight to
strong toxicity was showed by 39-69% decrease in the relative total
growth (RTG) at dose- levels >= 2.78 µg/mL and 96%
decrease in the RS at 5.56 µg/mL. After 24
h treatment (second experiment), the test substance
was moderately to strongly toxic at dose-levels >=0.56 µl/mL
(as shown mainly by 57 -100% decrease in the RS).
No noteworthy increase in the mutation frequency was induced
both after 3 and 24 h treatments.
With S9 mix
-First experiment: 0.21, 0.62, 1.85, 5.56 16.7 and 50 µg/mL
-Second Experiment: 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL
The test substance was moderately to markedly toxic at dose-levels between 5 and 7.5 µg/mL. At higher dose-levels, the test substance was strongly to completely toxic. No noteworthy increase in the mutation frequencies was induced in both experiments.
The cloning efficiencies
and the mutation
frequencies of the vehicle and positive controls were as
specified in acceptance criteria. The study was therefore
considered valid.
Table: First experiment without S9 mix, 3-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF 10-6 |
R |
|
40 |
|
|
11 |
|
|
74 |
18 |
4 |
|
|
0 |
24 |
0.7 |
100 |
18 |
1.0 |
100 |
79 |
12 |
5 |
121 |
1.0 |
|
28 |
|
|
25 |
|
|
76 |
14 |
6 |
|
|
|
38 |
|
|
26 |
|
|
74 |
16 |
6 |
|
|
0.07 |
25 |
0.8 |
121 |
14 |
0.9 |
88 |
79 |
13 |
4 |
105 |
0.9 |
|
27 |
|
|
34 |
|
|
81 |
10 |
5 |
|
|
0.21 |
36 |
0.6 |
96 |
8 |
1.0 |
103 |
72 |
17 |
7 |
119 |
1.0 |
|
32 |
|
|
30 |
|
|
79 |
12 |
5 |
|
|
0.62 |
35 |
0.6 |
93 |
11 |
1.0 |
102 |
73 |
16 |
7 |
137 |
1.1 |
|
35 |
|
|
28 |
|
|
73 |
21 |
2 |
|
|
1.85 |
29 |
0.7 |
109 |
6 |
1.2 |
118 |
80 |
6 |
6 |
84 |
0.7 |
|
30 |
|
|
24 |
|
|
78 |
12 |
6 |
|
|
2.78 |
24 |
0.8 |
124 |
7 |
1.0 |
100 |
81 |
8 |
7 |
68 |
0.6 |
|
26 |
|
|
33 |
|
|
87 |
6 |
3 |
|
|
5.56 |
31 |
0.8 |
122 |
32 |
0.7 |
72 |
71 |
17 |
10 |
179 |
1.5 |
|
20 |
|
|
30 |
|
|
78 |
7 |
11 |
|
|
MMS |
44 |
0.6 |
86 |
34 |
0.5 |
49 |
49 |
16 |
31 |
658 |
5.4 |
25 µg/ml |
32 |
|
|
55 |
|
|
53 |
21 |
23 |
|
|
0: vehicle control ( DMSO )
MMS:methylmethanesulfonate
LC: large colonies
CE0and CE2: cloning efficiency
SC: small colonies
RCE0and RCE2: relative cloning efficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
Table : First experiment without S9 mix, 3-hour treatment: cell growth during expression time
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
2.7 |
2.0 |
11.0 |
2.0 |
4.7 |
5.5 |
2.4 |
13.0 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
3.6 |
2.0 |
7.5 |
2.0 |
6.3 |
3.8 |
3.2 |
11.8 |
|
|
|
3.1 |
2.0 |
7.1 |
2.0 |
6.0 |
3.5 |
3.0 |
10.5 |
|
|
0.07 |
|
|
|
|
|
|
|
|
76 |
67 |
|
3.7 |
2.0 |
6.2 |
2.0 |
5.4 |
3.1 |
2.7 |
8.4 |
|
|
|
3.9 |
2.0 |
5.6 |
2.0 |
5.3 |
2.8 |
2.7 |
7.4 |
|
|
0.21 |
|
|
|
|
|
|
|
|
72 |
74 |
|
3.5 |
2.0 |
7.1 |
2.0 |
5.9 |
3.6 |
2.9 |
10.4 |
|
|
|
4.2 |
2.0 |
5.0 |
2.0 |
5.9 |
2.5 |
3.0 |
7.3 |
|
|
0.62 |
|
|
|
|
|
|
|
|
69 |
70 |
|
3.9 |
2.0 |
6.9 |
2.0 |
5.7 |
3.4 |
2.9 |
9.8 |
|
|
|
3.2 |
2.0 |
6.2 |
2.0 |
6.4 |
3.1 |
3.2 |
9.8 |
|
|
1.85 |
|
|
|
|
|
|
|
|
69 |
82 |
|
3.1 |
2.0 |
5.3 |
2.0 |
5.6 |
2.6 |
2.8 |
7.3 |
|
|
|
2.6 |
2.0 |
5.6 |
2.0 |
5.6 |
2.8 |
2.8 |
7.8 |
|
|
2.78 |
|
|
|
|
|
|
|
|
61 |
61 |
|
2.3 |
2.0 |
5.3 |
2.0 |
5.6 |
2.7 |
2.8 |
7.4 |
|
|
|
0.2 |
0.2 * |
0.3 |
0.3 * |
0.7 |
1.7 |
2.7 |
4.5 |
|
|
5.56 |
|
|
|
|
|
|
|
|
44 |
31 |
|
0.1 |
0.1 * |
0.1 |
0.1 * |
0.4 |
2.0 |
3.1 |
6.3 |
|
|
|
3.8 |
2.0 |
4.3 |
2.0 |
5.5 |
2.2 |
2.7 |
5.9 |
|
|
MMS |
|
|
|
|
|
|
|
|
60 |
29 |
25 µg/ml |
3.1 |
2.0 |
6.5 |
2.0 |
5.6 |
3.2 |
2.8 |
8.9 |
|
|
0: vehicle control ( DMSO )
conc.: cell concentration before being adjusted
adj.conc.:adjusted cell concentration when replating
SG: suspension growth
RSG: relative suspension growth
RTG: relative total growth
MMS: methylmethane sulfonate
*: cells were not replated as their density was < 2 x 105/ml
Table: First experiment without S9 mix, 3-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
2.7 |
|
|
|
|
0 |
|
1.0 |
0.7 |
0.7 |
100 |
|
3.6 |
|
|
|
|
|
3.1 |
|
|
|
|
0.07 |
|
1.1 |
0.8 |
0.9 |
130 |
|
3.7 |
|
|
|
|
|
3.9 |
|
|
|
|
0.21 |
|
1.2 |
0.6 |
0.8 |
115 |
|
3.5 |
|
|
|
|
|
4.2 |
|
|
|
|
0.62 |
|
1.3 |
0.6 |
0.8 |
121 |
|
3.9 |
|
|
|
|
|
3.2 |
|
|
|
|
1.85 |
|
1.0 |
0.7 |
0.7 |
110 |
|
3.1 |
|
|
|
|
|
2.6 |
|
|
|
|
2.78 |
|
0.8 |
0.8 |
0.7 |
97 |
|
2.3 |
|
|
|
|
|
0.2 |
|
|
|
|
5.56 |
|
0.0 |
0.8 |
0.0 |
4 |
|
0.1 |
|
|
|
|
|
3.8 |
|
|
|
|
MMS |
|
1.1 |
0.6 |
0.6 |
95 |
25 µg/ml |
3.1 |
|
|
|
|
0: vehicle control (DMSO )
CE0: cloning efficiency immediately after treatment
RS: relative survival
MMS: methylmethane sulfonate
Table : Second experiment without S9 mix, 24-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF10-6 |
R |
|
35 |
|
|
34 |
|
|
85 |
6 |
5 |
|
|
0 |
34 |
0.7 |
100 |
36 |
0.7 |
100 |
84 |
8 |
4 |
124 |
1.0 |
|
32 |
|
|
32 |
|
|
80 |
12 |
4 |
|
|
|
28 |
|
|
32 |
|
|
77 |
11 |
8 |
|
|
0.06 |
28 |
0.6 |
95 |
28 |
0.8 |
124 |
82 |
10 |
5 |
85 |
0.7 |
|
40 |
|
|
24 |
|
|
85 |
7 |
5 |
|
|
0.19 |
34 |
0.7 |
101 |
31 |
0.7 |
103 |
82 |
8 |
7 |
81 |
0.7 |
|
30 |
|
|
34 |
|
|
90 |
6 |
0 |
|
|
0.56 |
36 |
0.6 |
91 |
31 |
0.7 |
103 |
84 |
7 |
5 |
64 |
0.5 |
|
35 |
|
|
34 |
|
|
92 |
3 |
1 |
|
|
1.67 |
35 |
0.4 |
54 |
23 |
0.7 |
107 |
85 |
7 |
4 |
74 |
0.6 |
|
71 |
|
|
39 |
|
|
88 |
1 |
7 |
|
|
2.5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
MMS |
29 |
0.8 |
122 |
36 |
0.6 |
97 |
56 |
16 |
24 |
394 |
3.2 |
5 µg/ml |
22 |
|
|
33 |
|
|
60 |
23 |
15 |
|
|
0: vehicle control ( DMSO )
MMS: methylmethanesulfonate
LC: large colonies
CE0and CE2: cloning efficiency
SC: small colonies
RCE0and RCE2: relative cloning efficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
-: not evaluated due to severe toxicity observed
Table : Second experiment without S9 mix, 24-hour treatment: cell growthduring expressiontime
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
5.8 |
2.0 |
6.8 |
2.0 |
6.3 |
3.4 |
3.1 |
10.5 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
8.4 |
2.0 |
8.4 |
2.0 |
5.5 |
4.2 |
2.8 |
11.6 |
|
|
|
6.2 |
2.0 |
5.5 |
2.0 |
5.7 |
2.8 |
2.9 |
7.8 |
|
|
0.06 |
|
|
|
|
|
|
|
|
84 |
104 |
|
5.2 |
2.0 |
7.1 |
2.0 |
6.1 |
3.6 |
3.0 |
10.7 |
|
|
|
5.5 |
2.0 |
6.2 |
2.0 |
5.0 |
3.1 |
2.5 |
7.7 |
|
|
0.19 |
|
|
|
|
|
|
|
|
79 |
82 |
|
5.4 |
2.0 |
6.8 |
2.0 |
5.8 |
3.4 |
2.9 |
9.9 |
|
|
|
4.1 |
2.0 |
7.4 |
2.0 |
6.0 |
3.7 |
3.0 |
10.9 |
|
|
0.56 |
|
|
|
|
|
|
|
|
101 |
104 |
|
2.6 |
2.0 |
6.3 |
2.0 |
7.4 |
3.1 |
3.7 |
11.5 |
|
|
|
0.0 |
0.0 * |
0.2 |
0.2 * |
0.4 |
4.8 |
2.1 |
9.8 |
|
|
1.67 |
|
|
|
|
|
|
|
|
59 |
63 |
|
0.1 |
0.1 * |
0.1 |
0.1 * |
0.3 |
0.7 |
4.7 |
3.3 |
|
|
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
2.5 |
|
|
|
|
|
|
|
|
- |
- |
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
5 |
|
|
|
|
|
|
|
|
- |
- |
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
|
7.8 |
2.0 |
7.7 |
2.0 |
4.8 |
3.9 |
2.4 |
9.2 |
|
|
MMS |
|
|
|
|
|
|
|
|
82 |
80 |
5 µg/ml |
7.4 |
2.0 |
7.0 |
2.0 |
5.1 |
3.5 |
2.6 |
8.9 |
|
|
0:vehiclecontrol(DMSO)
conc.: cell concentration before being adjusted
adj.conc.:adjusted cell concentration when replating
SG: suspension growth
RSG: relative suspension growth
RTG: relative total growth
MMS: methylmethane sulfonate
*: cells were not replated as their density was < 2 x 105/ml
-: not evaluated due to severe toxicity observed
Table : Second experiment without S9 mix, 24-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
5.8 |
|
|
|
|
0 |
|
1.0 |
0.7 |
0.7 |
100 |
|
8.4 |
|
|
|
|
|
6.2 |
|
|
|
|
0.06 |
|
0.8 |
0.6 |
0.5 |
76 |
|
5.2 |
|
|
|
|
|
5.5 |
|
|
|
|
0.19 |
|
0.8 |
0.7 |
0.5 |
77 |
|
5.4 |
|
|
|
|
|
4.1 |
|
|
|
|
0.56 |
|
0.5 |
0.6 |
0.3 |
43 |
|
2.6 |
|
|
|
|
|
0.0 |
|
|
|
|
1.67 |
|
0.0 |
0.4 |
0.0 |
0 |
|
0.1 |
|
|
|
|
|
- |
|
|
|
|
2.5 |
|
- |
- |
- |
- |
|
- |
|
|
|
|
|
- |
|
|
|
|
5 |
|
- |
- |
- |
- |
|
- |
|
|
|
|
|
7.8 |
|
|
|
|
MMS |
|
1.1 |
0.8 |
0.9 |
130 |
5 µg/ml |
7.4 |
|
|
|
|
0: vehicle control ( DMSO )
CE0: cloning efficiency immediately after treatment
RS: relative survival
MMS: methylmethane sulfonate
-: not evaluated due to severe toxicity observed
Table: First experiment with S9 mix, 3-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF10-6 |
R |
|
8 |
|
|
10 |
|
|
77 |
11 |
8 |
|
|
0 |
6 |
1.4 |
100 |
14 |
1.3 |
100 |
82 |
9 |
5 |
75 |
1.0 |
|
17 |
|
|
21 |
|
|
80 |
11 |
5 |
|
|
|
12 |
|
|
4 |
|
|
78 |
9 |
9 |
|
|
0.21 |
5 |
2.1 |
151 |
5 |
1.8 |
139 |
83 |
9 |
4 |
46 |
0.6 |
|
2 |
|
|
6 |
|
|
80 |
11 |
5 |
|
|
0.62 |
5 |
1.7 |
123 |
11 |
1.4 |
112 |
77 |
15 |
4 |
52 |
0.7 |
|
8 |
|
|
8 |
|
|
88 |
6 |
2 |
|
|
1.85 |
3 |
2.1 |
151 |
4 |
1.6 |
127 |
76 |
9 |
11 |
56 |
0.7 |
|
4 |
|
|
10 |
|
|
84 |
2 |
10 |
|
|
5.56 |
13 |
1.4 |
106 |
16 |
1.0 |
80 |
85 |
2 |
9 |
45 |
0.6 |
|
6 |
|
|
21 |
|
|
90 |
2 |
4 |
|
|
16.7 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
CPA |
13 |
1.1 |
78 |
31 |
0.7 |
56 |
41 |
27 |
29 |
491 |
6.6 |
3 µg/ml |
22 |
|
|
30 |
|
|
54 |
13 |
30 |
|
|
0: vehicle control ( DMSO )
CPA:cyclophosphamide
LC: large colonies
CE0and CE2:cloningefficiency
SC: small colonies
RCE0and RCE2: relative cloningefficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
-: not evaluated due to severe toxicity observed
Table : First experiment with S9 mix, 3-hour treatment: cell growth during expression time
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
3.8 |
2.0 |
8.0 |
2.0 |
7.3 |
4.0 |
3.6 |
14.5 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
4.4 |
2.0 |
6.9 |
2.0 |
7.2 |
3.5 |
3.6 |
12.4 |
|
|
|
4.2 |
2.0 |
7.2 |
2.0 |
7.5 |
3.6 |
3.7 |
13.4 |
|
|
0.21 |
|
|
|
|
|
|
|
|
113 |
157 |
|
4.1 |
2.0 |
8.5 |
2.0 |
8.1 |
4.2 |
4.0 |
17.0 |
|
|
|
4.3 |
2.0 |
9.1 |
2.0 |
5.4 |
4.5 |
2.7 |
12.2 |
|
|
0.62 |
|
|
|
|
|
|
|
|
103 |
116 |
|
3.6 |
2.0 |
9.2 |
2.0 |
6.8 |
4.6 |
3.4 |
15.5 |
|
|
|
3.8 |
2.0 |
6.1 |
2.0 |
5.6 |
3.0 |
2.8 |
8.5 |
|
|
1.85 |
|
|
|
|
|
|
|
|
67 |
85 |
|
4.8 |
2.0 |
5.1 |
2.0 |
7.5 |
2.5 |
3.8 |
9.5 |
|
|
|
3.1 |
2.0 |
6.5 |
2.0 |
3.1 |
3.2 |
1.5 |
5.0 |
|
|
5.56 |
|
|
|
|
|
|
|
|
38 |
31 |
|
3.4 |
2.0 |
4.3 |
2.0 |
5.0 |
2.2 |
2.5 |
5.4 |
|
|
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
16.7 |
|
|
|
|
|
|
|
|
- |
- |
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
50 |
|
|
|
|
|
|
|
|
- |
- |
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
|
3.8 |
2.0 |
6.1 |
2.0 |
6.1 |
3.1 |
3.0 |
9.2 |
|
|
CPA |
|
|
|
|
|
|
|
|
82 |
46 |
3 µg/ml |
3.9 |
2.0 |
5.6 |
2.0 |
9.3 |
2.8 |
4.6 |
13.0 |
|
|
0: vehicle control ( DMSO )
conc.: cell concentration before being adjusted
adj.conc.:adjustedcellconcentrationwhenreplating SG: suspensiongrowth
RSG: relative suspension growth RTG: relative total growth
CPA: cyclophosphamide
-: not evaluated due to severe toxicity observed
Table : First experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
3.8 |
|
|
|
|
0 |
|
1.0 |
1.4 |
1.4 |
100 |
|
4.4 |
|
|
|
|
|
4.2 |
|
|
|
|
0.21 |
|
1.0 |
2.1 |
2.1 |
153 |
|
4.1 |
|
|
|
|
|
4.3 |
|
|
|
|
0.62 |
|
1.0 |
1.7 |
1.6 |
119 |
|
3.6 |
|
|
|
|
|
3.8 |
|
|
|
|
1.85 |
|
1.0 |
2.1 |
2.2 |
158 |
|
4.8 |
|
|
|
|
|
3.1 |
|
|
|
|
5.56 |
|
0.8 |
1.4 |
1.1 |
83 |
|
3.4 |
|
|
|
|
|
0.0 |
|
|
|
|
16.7 |
|
0.0 |
- |
- |
- |
|
0.0 |
|
|
|
|
|
0.0 |
|
|
|
|
50 |
|
0.0 |
- |
- |
- |
|
0.0 |
|
|
|
|
|
3.8 |
|
|
|
|
CPA |
|
0.9 |
1.1 |
1.0 |
73 |
3 µg/ml |
3.9 |
|
|
|
|
0: vehicle control ( DMSO )
CE0: cloning efficiency immediately after treatment RS: relative survival
CPA: cyclophosphamide
-: not evaluated due to severe toxicity observed
Table : Second experiment with S9 mix, 3-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF 10-6 |
R |
|
24 |
|
|
12 |
|
|
82 |
9 |
5 |
|
|
0 |
30 |
0.8 |
100 |
21 |
1.1 |
100 |
82 |
9 |
5 |
67 |
1.0 |
|
23 |
|
|
7 |
|
|
82 |
9 |
5 |
|
|
|
30 |
|
|
25 |
|
|
85 |
5 |
6 |
|
|
0.19 |
30 |
0.8 |
96 |
10 |
1.4 |
122 |
80 |
9 |
9 |
60 |
0.9 |
|
26 |
|
|
12 |
|
|
83 |
10 |
3 |
|
|
0.56 |
28 |
0.8 |
101 |
6 |
1.3 |
117 |
82 |
12 |
2 |
61 |
0.9 |
|
25 |
|
|
18 |
|
|
82 |
9 |
5 |
|
|
1.67 |
17 |
0.9 |
110 |
6 |
1.4 |
127 |
83 |
9 |
4 |
67 |
1.0 |
|
30 |
|
|
14 |
|
|
76 |
14 |
6 |
|
|
5 |
32 |
0.7 |
92 |
3 |
2.0 |
179 |
79 |
9 |
8 |
57 |
0.9 |
|
27 |
|
|
5 |
|
|
74 |
14 |
9 |
|
|
|
40 |
|
|
5 |
|
|
80 |
11 |
5 |
|
|
7.5 |
|
0.6 |
74 |
|
1.6 |
144 |
|
|
|
48 |
0.7 |
|
35 |
|
|
10 |
|
|
85 |
6 |
5 |
|
|
10 |
49 |
0.4 |
53 |
26 |
0.7 |
64 |
91 |
5 |
0 |
26 |
0.4 |
|
48 |
|
|
36 |
|
|
94 |
2 |
0 |
|
|
CPA |
38 |
0.5 |
65 |
26 |
0.7 |
63 |
60 |
15 |
22 |
440 |
6.6 |
3 µg/ml |
46 |
|
|
37 |
|
|
44 |
19 |
37 |
|
|
0: vehicle control ( DMSO )
CPA:cyclophosphamide
LC: largecolonies
CE0and CE2:cloning efficiency
SC: small colonies
RCE0and RCE2: relative cloningefficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
Table : Second experiment with S9 mix, 3-hour treatment: cell growth during expression time
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
5.7 |
2.0 |
6.9 |
2.0 |
9.6 |
3.5 |
4.8 |
16.5 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
5.6 |
2.0 |
7.0 |
2.0 |
7.9 |
3.5 |
4.0 |
13.7 |
|
|
|
5.2 |
2.0 |
8.3 |
2.0 |
6.3 |
4.1 |
3.2 |
13.0 |
|
|
0.19 |
|
|
|
|
|
|
|
|
84 |
102 |
|
5.1 |
2.0 |
7.5 |
2.0 |
6.6 |
3.8 |
3.3 |
12.4 |
|
|
|
5.7 |
2.0 |
8.0 |
2.0 |
5.9 |
4.0 |
3.0 |
11.7 |
|
|
0.56 |
|
|
|
|
|
|
|
|
74 |
87 |
|
6.5 |
2.0 |
5.4 |
2.0 |
8.0 |
2.7 |
4.0 |
10.7 |
|
|
|
4.9 |
2.0 |
7.5 |
2.0 |
5.1 |
3.7 |
2.5 |
9.4 |
|
|
1.67 |
|
|
|
|
|
|
|
|
57 |
73 |
|
6.5 |
2.0 |
5.3 |
2.0 |
6.0 |
2.6 |
3.0 |
7.8 |
|
|
|
6.6 |
2.0 |
3.6 |
2.0 |
5.1 |
1.8 |
2.6 |
4.6 |
|
|
5 |
|
|
|
|
|
|
|
|
29 |
52 |
|
5.5 |
2.0 |
5.2 |
2.0 |
3.3 |
2.6 |
1.6 |
4.2 |
|
|
|
4.6 |
2.0 |
2.0 |
2.0 * |
2.0 |
1.0 |
1.0 |
1.0 |
|
|
7.5 |
|
|
|
|
|
|
|
|
9 |
13 |
|
5.1 |
2.0 |
2.4 |
2.0 |
2.7 |
1.2 |
1.3 |
1.6 |
|
|
|
2.7 |
2.0 |
2.0 |
2.0 * |
1.1 |
1.0 |
0.5 |
0.6 |
|
|
10 |
|
|
|
|
|
|
|
|
4 |
2 |
|
2.4 |
2.0 |
1.4 |
1.4 * |
1.1 |
0.7 |
0.8 |
0.5 |
|
|
|
6.4 |
2.0 |
5.5 |
2.0 |
7.1 |
2.8 |
3.5 |
9.7 |
|
|
CPA |
|
|
|
|
|
|
|
|
61 |
38 |
3 µg/ml |
6.7 |
2.0 |
6.2 |
2.0 |
5.7 |
3.1 |
2.9 |
8.8 |
|
|
0: vehicle control ( DMSO )
conc.: cell concentration before being adjusted
adj.conc.:adjusted cell concentration when replating
SG: suspensiongrowth
RSG: relative suspension growth
RTG: relative total growth
CPA: cyclophosphamide
*: cells were not replated as their density was < 2 x 105/ml
Table : Second experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
5.7 |
|
|
|
|
0 |
|
1.0 |
0.8 |
0.8 |
100 |
|
5.6 |
|
|
|
|
|
5.2 |
|
|
|
|
0.19 |
|
0.9 |
0.8 |
0.7 |
87 |
|
5.1 |
|
|
|
|
|
5.7 |
|
|
|
|
0.56 |
|
1.1 |
0.8 |
0.9 |
109 |
|
6.5 |
|
|
|
|
|
4.9 |
|
|
|
|
1.67 |
|
1.0 |
0.9 |
0.9 |
111 |
|
6.5 |
|
|
|
|
|
6.6 |
|
|
|
|
5 |
|
1.1 |
0.7 |
0.8 |
98 |
|
5.5 |
|
|
|
|
|
4.6 |
|
|
|
|
7.5 |
|
0.9 |
0.6 |
0.5 |
63 |
|
5.1 |
|
|
|
|
|
2.7 |
|
|
|
|
10 |
|
0.4 |
0.4 |
0.2 |
24 |
|
2.4 |
|
|
|
|
|
6.4 |
|
|
|
|
CPA |
|
1.2 |
0.5 |
0.6 |
75 |
3 µg/ml |
6.7 |
|
|
|
|
0: vehicle control ( DMSO )
CE0: cloning efficiency immediately after treatment
RS: relative survival
CPA: cyclophosphamide
Table: Historical data without S9 mix - Mouse lymphoma assay
|
3-hour treatment |
24-hour treatment |
||||||||||
assay No. |
vehicle |
MMS |
vehicle |
MMS |
||||||||
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
|
1 |
1.2 |
0.9 |
84 |
0.8 |
0.5 |
682 |
0.6 |
0.8 |
118 |
0.4 |
0.6 |
539 |
2 |
0.9 |
0.7 |
82 |
0.9 |
0.5 |
528 |
0.6 |
0.8 |
128 |
0.5 |
0.5 |
603 |
3 |
1.1 |
0.9 |
139 |
1 |
0.6 |
467 |
0.7 |
0.9 |
242 |
0.6 |
0.6 |
601 |
4 |
- |
- |
- |
- |
- |
- |
0.7 |
0.7 |
78 |
0.5 |
0.5 |
475 |
5 |
0.7 |
0.8 |
124 |
0.8 |
0.4 |
499 |
0.7 |
0.7 |
126 |
0.5 |
0.6 |
479 |
6 |
0.9 |
0.7 |
117 |
0.7 |
0.5 |
424 |
0.6 |
0.8 |
143 |
0.4 |
0.5 |
755 |
7 |
0.9 |
0.8 |
108 |
0.7 |
0.5 |
431 |
0.7 |
0.8 |
72 |
1.1 |
0.6 |
426 |
8 |
0.6 |
0.7 |
126 |
0.6 |
0.4 |
612 |
0.6 |
0.7 |
161 |
0.4 |
0.5 |
990 |
9 |
1.0 |
0.8 |
130 |
1.1 |
0.5 |
605 |
- |
- |
- |
- |
- |
- |
10 |
1.1 |
1.1 |
61 |
1.1 |
0.5 |
861 |
0.7 |
1.1 |
87 |
1.1 |
0.7 |
675 |
11 |
1.2 |
0.9 |
75 |
0.9 |
0.5 |
458 |
0.6 |
0.7 |
125 |
0.5 |
0.4 |
1298 |
12 |
1.3 |
0.9 |
58 |
1.8 |
0.7 |
462 |
1.0 |
1.3 |
94 |
0.8 |
0.5 |
823 |
13 |
1.0 |
1.3 |
77 |
1.2 |
0.6 |
533 |
1.0 |
1.3 |
94 |
0.8 |
0.5 |
823 |
minimum |
0.6 |
0.7 |
58 |
0.6 |
0.4 |
424 |
0.6 |
0.7 |
72 |
0.4 |
0.4 |
426 |
maximum |
1.3 |
1.3 |
139 |
1.8 |
0.7 |
861 |
1.0 |
1.3 |
242 |
1.1 |
0.7 |
1298 |
mean |
1.0 |
0.9 |
98 |
1.0 |
0.5 |
547 |
0.7 |
0.9 |
122 |
0.6 |
0.5 |
707 |
CE0: cloning efficiency at day 0
CE2: cloning efficiency at day 2
MF: mutation frequency
MMS: methylmethane sulfonate (3-hour treatment: 25µg/ml ; 24-hour treatment: 5µg/ml)
- : not performed
Table: Historical data with S9 mix Mouse lymphoma assay
assay No. |
vehicle |
CPA |
||||
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
|
1 |
0.9 |
0.8 |
83 |
0.5 |
0.4 |
1077 |
2 |
0.9 |
0.7 |
123 |
0.5 |
0.3 |
915 |
3 |
1.0 |
0.8 |
68 |
0.5 |
0.6 |
948 |
4 |
0.7 |
0.8 |
138 |
0.3 |
0.4 |
1177 |
5 |
0.7 |
1.0 |
80 |
0.5 |
0.4 |
1248 |
6 |
0.9 |
0.9 |
161 |
0.4 |
0.3 |
1200 |
7 |
0.7 |
0.8 |
186 |
0.4 |
0.4 |
958 |
8 |
0.8 |
0.7 |
137 |
0.4 |
0.3 |
1563 |
9 |
0.7 |
1.0 |
92 |
0.3 |
0.3 |
1200 |
10 |
0.6 |
1.0 |
136 |
0.4 |
0.5 |
643 |
11 |
0.9 |
0.8 |
246 |
0.7 |
0.8 |
618 |
12 |
0.6 |
0.8 |
199 |
0.4 |
0.4 |
1389 |
13 |
0.7 |
0.8 |
114 |
0.4 |
0.4 |
915 |
14 |
0.9 |
1.0 |
88 |
1.0 |
0.8 |
484 |
15 |
1.1 |
0.9 |
77 |
0.4 |
0.4 |
1077 |
16 |
0.7 |
0.8 |
86 |
0.4 |
0.7 |
675 |
17 |
0.8 |
0.7 |
113 |
0.5 |
0.5 |
754 |
18 |
0.7 |
0.9 |
91 |
0.5 |
0.4 |
1583 |
19 |
0.6 |
0.8 |
108 |
0.4 |
0.4 |
1126 |
20 |
1.2 |
1.0 |
79 |
1.4 |
0.7 |
544 |
21 |
0.7 |
1.2 |
72 |
0.6 |
0.8 |
636 |
22 |
0.9 |
1.2 |
86 |
0.7 |
0.9 |
721 |
23 |
1.0 |
0.8 |
108 |
0.6 |
0.3 |
1456 |
24 |
0.9 |
1.2 |
86 |
0.7 |
0.9 |
721 |
minimum |
0.6 |
0.7 |
68 |
0.3 |
0.3 |
484 |
maximum |
1.2 |
1.2 |
246 |
1.4 |
0.9 |
1583 |
mean |
0.8 |
0.9 |
115 |
0.5 |
0.5 |
985 |
CE0: cloning efficiency at day 0
CE2: cloning efficiency at day 2
MF: mutation frequency
CPA: cyclophosphamide (3µg/ml)
- : not performed
Results
Based on the preliminary toxicity test for the selection of
the doses, the selected dose-levels for treatment were as follow :
All the dose-levels were expressed as active substance,
taking into account the active material content of 40.37%.
Without S9 mix
- First experiment: 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL
- Second Experiment: 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL
After 3 h treatment (first experiment), a slight to
strong toxicity was showed by 39-69% decrease in the relative total
growth (RTG) at dose- levels >= 2.78 µg/mL and 96%
decrease in the RS at 5.56 µg/mL. After 24
h treatment (second experiment), the test substance
was moderately to strongly toxic at dose-levels >=0.56 µl/mL
(as shown mainly by 57 -100% decrease in the RS).
No noteworthy increase in the mutation frequency was induced
both after 3 and 24 h treatments.
With S9 mix
-First experiment: 0.21, 0.62, 1.85, 5.56 16.7 and 50 µg/mL
-Second Experiment: 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL
The test substance was moderately to markedly toxic at dose-levels between 5 and 7.5 µg/mL. At higher dose-levels, the test substance was strongly to completely toxic. No noteworthy increase in the mutation frequencies was induced in both experiments.
The cloning efficiencies
and the mutation
frequencies of the vehicle and positive controls were as
specified in acceptance criteria. The study was therefore
considered valid.
Table: First experiment without S9 mix, 3-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF 10-6 |
R |
|
40 |
|
|
11 |
|
|
74 |
18 |
4 |
|
|
0 |
24 |
0.7 |
100 |
18 |
1.0 |
100 |
79 |
12 |
5 |
121 |
1.0 |
|
28 |
|
|
25 |
|
|
76 |
14 |
6 |
|
|
|
38 |
|
|
26 |
|
|
74 |
16 |
6 |
|
|
0.07 |
25 |
0.8 |
121 |
14 |
0.9 |
88 |
79 |
13 |
4 |
105 |
0.9 |
|
27 |
|
|
34 |
|
|
81 |
10 |
5 |
|
|
0.21 |
36 |
0.6 |
96 |
8 |
1.0 |
103 |
72 |
17 |
7 |
119 |
1.0 |
|
32 |
|
|
30 |
|
|
79 |
12 |
5 |
|
|
0.62 |
35 |
0.6 |
93 |
11 |
1.0 |
102 |
73 |
16 |
7 |
137 |
1.1 |
|
35 |
|
|
28 |
|
|
73 |
21 |
2 |
|
|
1.85 |
29 |
0.7 |
109 |
6 |
1.2 |
118 |
80 |
6 |
6 |
84 |
0.7 |
|
30 |
|
|
24 |
|
|
78 |
12 |
6 |
|
|
2.78 |
24 |
0.8 |
124 |
7 |
1.0 |
100 |
81 |
8 |
7 |
68 |
0.6 |
|
26 |
|
|
33 |
|
|
87 |
6 |
3 |
|
|
5.56 |
31 |
0.8 |
122 |
32 |
0.7 |
72 |
71 |
17 |
10 |
179 |
1.5 |
|
20 |
|
|
30 |
|
|
78 |
7 |
11 |
|
|
MMS |
44 |
0.6 |
86 |
34 |
0.5 |
49 |
49 |
16 |
31 |
658 |
5.4 |
25 µg/ml |
32 |
|
|
55 |
|
|
53 |
21 |
23 |
|
|
0: vehicle control ( DMSO )
MMS:methylmethanesulfonate
LC: large colonies
CE0and CE2: cloning efficiency
SC: small colonies
RCE0and RCE2: relative cloning efficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
Table : First experiment without S9 mix, 3-hour treatment: cell growth during expression time
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
2.7 |
2.0 |
11.0 |
2.0 |
4.7 |
5.5 |
2.4 |
13.0 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
3.6 |
2.0 |
7.5 |
2.0 |
6.3 |
3.8 |
3.2 |
11.8 |
|
|
|
3.1 |
2.0 |
7.1 |
2.0 |
6.0 |
3.5 |
3.0 |
10.5 |
|
|
0.07 |
|
|
|
|
|
|
|
|
76 |
67 |
|
3.7 |
2.0 |
6.2 |
2.0 |
5.4 |
3.1 |
2.7 |
8.4 |
|
|
|
3.9 |
2.0 |
5.6 |
2.0 |
5.3 |
2.8 |
2.7 |
7.4 |
|
|
0.21 |
|
|
|
|
|
|
|
|
72 |
74 |
|
3.5 |
2.0 |
7.1 |
2.0 |
5.9 |
3.6 |
2.9 |
10.4 |
|
|
|
4.2 |
2.0 |
5.0 |
2.0 |
5.9 |
2.5 |
3.0 |
7.3 |
|
|
0.62 |
|
|
|
|
|
|
|
|
69 |
70 |
|
3.9 |
2.0 |
6.9 |
2.0 |
5.7 |
3.4 |
2.9 |
9.8 |
|
|
|
3.2 |
2.0 |
6.2 |
2.0 |
6.4 |
3.1 |
3.2 |
9.8 |
|
|
1.85 |
|
|
|
|
|
|
|
|
69 |
82 |
|
3.1 |
2.0 |
5.3 |
2.0 |
5.6 |
2.6 |
2.8 |
7.3 |
|
|
|
2.6 |
2.0 |
5.6 |
2.0 |
5.6 |
2.8 |
2.8 |
7.8 |
|
|
2.78 |
|
|
|
|
|
|
|
|
61 |
61 |
|
2.3 |
2.0 |
5.3 |
2.0 |
5.6 |
2.7 |
2.8 |
7.4 |
|
|
|
0.2 |
0.2 * |
0.3 |
0.3 * |
0.7 |
1.7 |
2.7 |
4.5 |
|
|
5.56 |
|
|
|
|
|
|
|
|
44 |
31 |
|
0.1 |
0.1 * |
0.1 |
0.1 * |
0.4 |
2.0 |
3.1 |
6.3 |
|
|
|
3.8 |
2.0 |
4.3 |
2.0 |
5.5 |
2.2 |
2.7 |
5.9 |
|
|
MMS |
|
|
|
|
|
|
|
|
60 |
29 |
25 µg/ml |
3.1 |
2.0 |
6.5 |
2.0 |
5.6 |
3.2 |
2.8 |
8.9 |
|
|
0: vehicle control ( DMSO )
conc.: cell concentration before being adjusted
adj.conc.:adjusted cell concentration when replating
SG: suspension growth
RSG: relative suspension growth
RTG: relative total growth
MMS: methylmethane sulfonate
*: cells were not replated as their density was < 2 x 105/ml
Table: First experiment without S9 mix, 3-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
2.7 |
|
|
|
|
0 |
|
1.0 |
0.7 |
0.7 |
100 |
|
3.6 |
|
|
|
|
|
3.1 |
|
|
|
|
0.07 |
|
1.1 |
0.8 |
0.9 |
130 |
|
3.7 |
|
|
|
|
|
3.9 |
|
|
|
|
0.21 |
|
1.2 |
0.6 |
0.8 |
115 |
|
3.5 |
|
|
|
|
|
4.2 |
|
|
|
|
0.62 |
|
1.3 |
0.6 |
0.8 |
121 |
|
3.9 |
|
|
|
|
|
3.2 |
|
|
|
|
1.85 |
|
1.0 |
0.7 |
0.7 |
110 |
|
3.1 |
|
|
|
|
|
2.6 |
|
|
|
|
2.78 |
|
0.8 |
0.8 |
0.7 |
97 |
|
2.3 |
|
|
|
|
|
0.2 |
|
|
|
|
5.56 |
|
0.0 |
0.8 |
0.0 |
4 |
|
0.1 |
|
|
|
|
|
3.8 |
|
|
|
|
MMS |
|
1.1 |
0.6 |
0.6 |
95 |
25 µg/ml |
3.1 |
|
|
|
|
0: vehicle control (DMSO )
CE0: cloning efficiency immediately after treatment
RS: relative survival
MMS: methylmethane sulfonate
Table : Second experiment without S9 mix, 24-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF10-6 |
R |
|
35 |
|
|
34 |
|
|
85 |
6 |
5 |
|
|
0 |
34 |
0.7 |
100 |
36 |
0.7 |
100 |
84 |
8 |
4 |
124 |
1.0 |
|
32 |
|
|
32 |
|
|
80 |
12 |
4 |
|
|
|
28 |
|
|
32 |
|
|
77 |
11 |
8 |
|
|
0.06 |
28 |
0.6 |
95 |
28 |
0.8 |
124 |
82 |
10 |
5 |
85 |
0.7 |
|
40 |
|
|
24 |
|
|
85 |
7 |
5 |
|
|
0.19 |
34 |
0.7 |
101 |
31 |
0.7 |
103 |
82 |
8 |
7 |
81 |
0.7 |
|
30 |
|
|
34 |
|
|
90 |
6 |
0 |
|
|
0.56 |
36 |
0.6 |
91 |
31 |
0.7 |
103 |
84 |
7 |
5 |
64 |
0.5 |
|
35 |
|
|
34 |
|
|
92 |
3 |
1 |
|
|
1.67 |
35 |
0.4 |
54 |
23 |
0.7 |
107 |
85 |
7 |
4 |
74 |
0.6 |
|
71 |
|
|
39 |
|
|
88 |
1 |
7 |
|
|
2.5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
MMS |
29 |
0.8 |
122 |
36 |
0.6 |
97 |
56 |
16 |
24 |
394 |
3.2 |
5 µg/ml |
22 |
|
|
33 |
|
|
60 |
23 |
15 |
|
|
0: vehicle control ( DMSO )
MMS: methylmethanesulfonate
LC: large colonies
CE0and CE2: cloning efficiency
SC: small colonies
RCE0and RCE2: relative cloning efficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
-: not evaluated due to severe toxicity observed
Table : Second experiment without S9 mix, 24-hour treatment: cell growthduring expressiontime
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
5.8 |
2.0 |
6.8 |
2.0 |
6.3 |
3.4 |
3.1 |
10.5 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
8.4 |
2.0 |
8.4 |
2.0 |
5.5 |
4.2 |
2.8 |
11.6 |
|
|
|
6.2 |
2.0 |
5.5 |
2.0 |
5.7 |
2.8 |
2.9 |
7.8 |
|
|
0.06 |
|
|
|
|
|
|
|
|
84 |
104 |
|
5.2 |
2.0 |
7.1 |
2.0 |
6.1 |
3.6 |
3.0 |
10.7 |
|
|
|
5.5 |
2.0 |
6.2 |
2.0 |
5.0 |
3.1 |
2.5 |
7.7 |
|
|
0.19 |
|
|
|
|
|
|
|
|
79 |
82 |
|
5.4 |
2.0 |
6.8 |
2.0 |
5.8 |
3.4 |
2.9 |
9.9 |
|
|
|
4.1 |
2.0 |
7.4 |
2.0 |
6.0 |
3.7 |
3.0 |
10.9 |
|
|
0.56 |
|
|
|
|
|
|
|
|
101 |
104 |
|
2.6 |
2.0 |
6.3 |
2.0 |
7.4 |
3.1 |
3.7 |
11.5 |
|
|
|
0.0 |
0.0 * |
0.2 |
0.2 * |
0.4 |
4.8 |
2.1 |
9.8 |
|
|
1.67 |
|
|
|
|
|
|
|
|
59 |
63 |
|
0.1 |
0.1 * |
0.1 |
0.1 * |
0.3 |
0.7 |
4.7 |
3.3 |
|
|
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
2.5 |
|
|
|
|
|
|
|
|
- |
- |
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
5 |
|
|
|
|
|
|
|
|
- |
- |
|
- |
- |
- |
- |
- |
- |
- |
- |
|
|
|
7.8 |
2.0 |
7.7 |
2.0 |
4.8 |
3.9 |
2.4 |
9.2 |
|
|
MMS |
|
|
|
|
|
|
|
|
82 |
80 |
5 µg/ml |
7.4 |
2.0 |
7.0 |
2.0 |
5.1 |
3.5 |
2.6 |
8.9 |
|
|
0:vehiclecontrol(DMSO)
conc.: cell concentration before being adjusted
adj.conc.:adjusted cell concentration when replating
SG: suspension growth
RSG: relative suspension growth
RTG: relative total growth
MMS: methylmethane sulfonate
*: cells were not replated as their density was < 2 x 105/ml
-: not evaluated due to severe toxicity observed
Table : Second experiment without S9 mix, 24-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
5.8 |
|
|
|
|
0 |
|
1.0 |
0.7 |
0.7 |
100 |
|
8.4 |
|
|
|
|
|
6.2 |
|
|
|
|
0.06 |
|
0.8 |
0.6 |
0.5 |
76 |
|
5.2 |
|
|
|
|
|
5.5 |
|
|
|
|
0.19 |
|
0.8 |
0.7 |
0.5 |
77 |
|
5.4 |
|
|
|
|
|
4.1 |
|
|
|
|
0.56 |
|
0.5 |
0.6 |
0.3 |
43 |
|
2.6 |
|
|
|
|
|
0.0 |
|
|
|
|
1.67 |
|
0.0 |
0.4 |
0.0 |
0 |
|
0.1 |
|
|
|
|
|
- |
|
|
|
|
2.5 |
|
- |
- |
- |
- |
|
- |
|
|
|
|
|
- |
|
|
|
|
5 |
|
- |
- |
- |
- |
|
- |
|
|
|
|
|
7.8 |
|
|
|
|
MMS |
|
1.1 |
0.8 |
0.9 |
130 |
5 µg/ml |
7.4 |
|
|
|
|
0: vehicle control ( DMSO )
CE0: cloning efficiency immediately after treatment
RS: relative survival
MMS: methylmethane sulfonate
-: not evaluated due to severe toxicity observed
Table: First experiment with S9 mix, 3-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF10-6 |
R |
|
8 |
|
|
10 |
|
|
77 |
11 |
8 |
|
|
0 |
6 |
1.4 |
100 |
14 |
1.3 |
100 |
82 |
9 |
5 |
75 |
1.0 |
|
17 |
|
|
21 |
|
|
80 |
11 |
5 |
|
|
|
12 |
|
|
4 |
|
|
78 |
9 |
9 |
|
|
0.21 |
5 |
2.1 |
151 |
5 |
1.8 |
139 |
83 |
9 |
4 |
46 |
0.6 |
|
2 |
|
|
6 |
|
|
80 |
11 |
5 |
|
|
0.62 |
5 |
1.7 |
123 |
11 |
1.4 |
112 |
77 |
15 |
4 |
52 |
0.7 |
|
8 |
|
|
8 |
|
|
88 |
6 |
2 |
|
|
1.85 |
3 |
2.1 |
151 |
4 |
1.6 |
127 |
76 |
9 |
11 |
56 |
0.7 |
|
4 |
|
|
10 |
|
|
84 |
2 |
10 |
|
|
5.56 |
13 |
1.4 |
106 |
16 |
1.0 |
80 |
85 |
2 |
9 |
45 |
0.6 |
|
6 |
|
|
21 |
|
|
90 |
2 |
4 |
|
|
16.7 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
|
|
- |
|
|
- |
- |
- |
|
|
CPA |
13 |
1.1 |
78 |
31 |
0.7 |
56 |
41 |
27 |
29 |
491 |
6.6 |
3 µg/ml |
22 |
|
|
30 |
|
|
54 |
13 |
30 |
|
|
0: vehicle control ( DMSO )
CPA:cyclophosphamide
LC: large colonies
CE0and CE2:cloningefficiency
SC: small colonies
RCE0and RCE2: relative cloningefficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
-: not evaluated due to severe toxicity observed
Table : First experiment with S9 mix, 3-hour treatment: cell growth during expression time
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
3.8 |
2.0 |
8.0 |
2.0 |
7.3 |
4.0 |
3.6 |
14.5 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
4.4 |
2.0 |
6.9 |
2.0 |
7.2 |
3.5 |
3.6 |
12.4 |
|
|
|
4.2 |
2.0 |
7.2 |
2.0 |
7.5 |
3.6 |
3.7 |
13.4 |
|
|
0.21 |
|
|
|
|
|
|
|
|
113 |
157 |
|
4.1 |
2.0 |
8.5 |
2.0 |
8.1 |
4.2 |
4.0 |
17.0 |
|
|
|
4.3 |
2.0 |
9.1 |
2.0 |
5.4 |
4.5 |
2.7 |
12.2 |
|
|
0.62 |
|
|
|
|
|
|
|
|
103 |
116 |
|
3.6 |
2.0 |
9.2 |
2.0 |
6.8 |
4.6 |
3.4 |
15.5 |
|
|
|
3.8 |
2.0 |
6.1 |
2.0 |
5.6 |
3.0 |
2.8 |
8.5 |
|
|
1.85 |
|
|
|
|
|
|
|
|
67 |
85 |
|
4.8 |
2.0 |
5.1 |
2.0 |
7.5 |
2.5 |
3.8 |
9.5 |
|
|
|
3.1 |
2.0 |
6.5 |
2.0 |
3.1 |
3.2 |
1.5 |
5.0 |
|
|
5.56 |
|
|
|
|
|
|
|
|
38 |
31 |
|
3.4 |
2.0 |
4.3 |
2.0 |
5.0 |
2.2 |
2.5 |
5.4 |
|
|
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
16.7 |
|
|
|
|
|
|
|
|
- |
- |
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
50 |
|
|
|
|
|
|
|
|
- |
- |
|
0.0 |
- |
- |
- |
- |
- |
- |
- |
|
|
|
3.8 |
2.0 |
6.1 |
2.0 |
6.1 |
3.1 |
3.0 |
9.2 |
|
|
CPA |
|
|
|
|
|
|
|
|
82 |
46 |
3 µg/ml |
3.9 |
2.0 |
5.6 |
2.0 |
9.3 |
2.8 |
4.6 |
13.0 |
|
|
0: vehicle control ( DMSO )
conc.: cell concentration before being adjusted
adj.conc.:adjustedcellconcentrationwhenreplating SG: suspensiongrowth
RSG: relative suspension growth RTG: relative total growth
CPA: cyclophosphamide
-: not evaluated due to severe toxicity observed
Table : First experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
3.8 |
|
|
|
|
0 |
|
1.0 |
1.4 |
1.4 |
100 |
|
4.4 |
|
|
|
|
|
4.2 |
|
|
|
|
0.21 |
|
1.0 |
2.1 |
2.1 |
153 |
|
4.1 |
|
|
|
|
|
4.3 |
|
|
|
|
0.62 |
|
1.0 |
1.7 |
1.6 |
119 |
|
3.6 |
|
|
|
|
|
3.8 |
|
|
|
|
1.85 |
|
1.0 |
2.1 |
2.2 |
158 |
|
4.8 |
|
|
|
|
|
3.1 |
|
|
|
|
5.56 |
|
0.8 |
1.4 |
1.1 |
83 |
|
3.4 |
|
|
|
|
|
0.0 |
|
|
|
|
16.7 |
|
0.0 |
- |
- |
- |
|
0.0 |
|
|
|
|
|
0.0 |
|
|
|
|
50 |
|
0.0 |
- |
- |
- |
|
0.0 |
|
|
|
|
|
3.8 |
|
|
|
|
CPA |
|
0.9 |
1.1 |
1.0 |
73 |
3 µg/ml |
3.9 |
|
|
|
|
0: vehicle control ( DMSO )
CE0: cloning efficiency immediately after treatment RS: relative survival
CPA: cyclophosphamide
-: not evaluated due to severe toxicity observed
Table : Second experiment with S9 mix, 3-hour treatment: mutagenicity results
Doses µg/ml (a.i.) |
Empty wells* |
CE0 |
RCE0 % |
Empty wells* |
CE2 |
RCE2 % |
Empty wells* |
LC |
SC |
MF 10-6 |
R |
|
24 |
|
|
12 |
|
|
82 |
9 |
5 |
|
|
0 |
30 |
0.8 |
100 |
21 |
1.1 |
100 |
82 |
9 |
5 |
67 |
1.0 |
|
23 |
|
|
7 |
|
|
82 |
9 |
5 |
|
|
|
30 |
|
|
25 |
|
|
85 |
5 |
6 |
|
|
0.19 |
30 |
0.8 |
96 |
10 |
1.4 |
122 |
80 |
9 |
9 |
60 |
0.9 |
|
26 |
|
|
12 |
|
|
83 |
10 |
3 |
|
|
0.56 |
28 |
0.8 |
101 |
6 |
1.3 |
117 |
82 |
12 |
2 |
61 |
0.9 |
|
25 |
|
|
18 |
|
|
82 |
9 |
5 |
|
|
1.67 |
17 |
0.9 |
110 |
6 |
1.4 |
127 |
83 |
9 |
4 |
67 |
1.0 |
|
30 |
|
|
14 |
|
|
76 |
14 |
6 |
|
|
5 |
32 |
0.7 |
92 |
3 |
2.0 |
179 |
79 |
9 |
8 |
57 |
0.9 |
|
27 |
|
|
5 |
|
|
74 |
14 |
9 |
|
|
|
40 |
|
|
5 |
|
|
80 |
11 |
5 |
|
|
7.5 |
|
0.6 |
74 |
|
1.6 |
144 |
|
|
|
48 |
0.7 |
|
35 |
|
|
10 |
|
|
85 |
6 |
5 |
|
|
10 |
49 |
0.4 |
53 |
26 |
0.7 |
64 |
91 |
5 |
0 |
26 |
0.4 |
|
48 |
|
|
36 |
|
|
94 |
2 |
0 |
|
|
CPA |
38 |
0.5 |
65 |
26 |
0.7 |
63 |
60 |
15 |
22 |
440 |
6.6 |
3 µg/ml |
46 |
|
|
37 |
|
|
44 |
19 |
37 |
|
|
0: vehicle control ( DMSO )
CPA:cyclophosphamide
LC: largecolonies
CE0and CE2:cloning efficiency
SC: small colonies
RCE0and RCE2: relative cloningefficiency
R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells
*: empty wells are counted on a total number of 96 wells
Table : Second experiment with S9 mix, 3-hour treatment: cell growth during expression time
Doses µg/ml (a.i.) |
Cell concentration (x 10+5/ml) |
Daily cell growth |
RSG % |
RTG % |
||||||
Day 0 |
Day 1 |
Day 2 |
Day 1 |
Day 2 |
Day 1x Day 2 (SG) |
|||||
Conc. |
Adj. Conc. |
Conc. |
Adj. Conc. |
Conc. |
||||||
|
5.7 |
2.0 |
6.9 |
2.0 |
9.6 |
3.5 |
4.8 |
16.5 |
|
|
0 |
|
|
|
|
|
|
|
|
100 |
100 |
|
5.6 |
2.0 |
7.0 |
2.0 |
7.9 |
3.5 |
4.0 |
13.7 |
|
|
|
5.2 |
2.0 |
8.3 |
2.0 |
6.3 |
4.1 |
3.2 |
13.0 |
|
|
0.19 |
|
|
|
|
|
|
|
|
84 |
102 |
|
5.1 |
2.0 |
7.5 |
2.0 |
6.6 |
3.8 |
3.3 |
12.4 |
|
|
|
5.7 |
2.0 |
8.0 |
2.0 |
5.9 |
4.0 |
3.0 |
11.7 |
|
|
0.56 |
|
|
|
|
|
|
|
|
74 |
87 |
|
6.5 |
2.0 |
5.4 |
2.0 |
8.0 |
2.7 |
4.0 |
10.7 |
|
|
|
4.9 |
2.0 |
7.5 |
2.0 |
5.1 |
3.7 |
2.5 |
9.4 |
|
|
1.67 |
|
|
|
|
|
|
|
|
57 |
73 |
|
6.5 |
2.0 |
5.3 |
2.0 |
6.0 |
2.6 |
3.0 |
7.8 |
|
|
|
6.6 |
2.0 |
3.6 |
2.0 |
5.1 |
1.8 |
2.6 |
4.6 |
|
|
5 |
|
|
|
|
|
|
|
|
29 |
52 |
|
5.5 |
2.0 |
5.2 |
2.0 |
3.3 |
2.6 |
1.6 |
4.2 |
|
|
|
4.6 |
2.0 |
2.0 |
2.0 * |
2.0 |
1.0 |
1.0 |
1.0 |
|
|
7.5 |
|
|
|
|
|
|
|
|
9 |
13 |
|
5.1 |
2.0 |
2.4 |
2.0 |
2.7 |
1.2 |
1.3 |
1.6 |
|
|
|
2.7 |
2.0 |
2.0 |
2.0 * |
1.1 |
1.0 |
0.5 |
0.6 |
|
|
10 |
|
|
|
|
|
|
|
|
4 |
2 |
|
2.4 |
2.0 |
1.4 |
1.4 * |
1.1 |
0.7 |
0.8 |
0.5 |
|
|
|
6.4 |
2.0 |
5.5 |
2.0 |
7.1 |
2.8 |
3.5 |
9.7 |
|
|
CPA |
|
|
|
|
|
|
|
|
61 |
38 |
3 µg/ml |
6.7 |
2.0 |
6.2 |
2.0 |
5.7 |
3.1 |
2.9 |
8.8 |
|
|
0: vehicle control ( DMSO )
conc.: cell concentration before being adjusted
adj.conc.:adjusted cell concentration when replating
SG: suspensiongrowth
RSG: relative suspension growth
RTG: relative total growth
CPA: cyclophosphamide
*: cells were not replated as their density was < 2 x 105/ml
Table : Second experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment
Doses µg/ml (a.i.) |
post-treatment cell count (x 105cells/ml) |
Cell count factor |
CE0 |
Survival |
RS % |
|
5.7 |
|
|
|
|
0 |
|
1.0 |
0.8 |
0.8 |
100 |
|
5.6 |
|
|
|
|
|
5.2 |
|
|
|
|
0.19 |
|
0.9 |
0.8 |
0.7 |
87 |
|
5.1 |
|
|
|
|
|
5.7 |
|
|
|
|
0.56 |
|
1.1 |
0.8 |
0.9 |
109 |
|
6.5 |
|
|
|
|
|
4.9 |
|
|
|
|
1.67 |
|
1.0 |
0.9 |
0.9 |
111 |
|
6.5 |
|
|
|
|
|
6.6 |
|
|
|
|
5 |
|
1.1 |
0.7 |
0.8 |
98 |
|
5.5 |
|
|
|
|
|
4.6 |
|
|
|
|
7.5 |
|
0.9 |
0.6 |
0.5 |
63 |
|
5.1 |
|
|
|
|
|
2.7 |
|
|
|
|
10 |
|
0.4 |
0.4 |
0.2 |
24 |
|
2.4 |
|
|
|
|
|
6.4 |
|
|
|
|
CPA |
|
1.2 |
0.5 |
0.6 |
75 |
3 µg/ml |
6.7 |
|
|
|
|
0: vehicle control ( DMSO )
CE0: cloning efficiency immediately after treatment
RS: relative survival
CPA: cyclophosphamide
Table: Historical data without S9 mix - Mouse lymphoma assay
|
3-hour treatment |
24-hour treatment |
||||||||||
assay No. |
vehicle |
MMS |
vehicle |
MMS |
||||||||
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
|
1 |
1.2 |
0.9 |
84 |
0.8 |
0.5 |
682 |
0.6 |
0.8 |
118 |
0.4 |
0.6 |
539 |
2 |
0.9 |
0.7 |
82 |
0.9 |
0.5 |
528 |
0.6 |
0.8 |
128 |
0.5 |
0.5 |
603 |
3 |
1.1 |
0.9 |
139 |
1 |
0.6 |
467 |
0.7 |
0.9 |
242 |
0.6 |
0.6 |
601 |
4 |
- |
- |
- |
- |
- |
- |
0.7 |
0.7 |
78 |
0.5 |
0.5 |
475 |
5 |
0.7 |
0.8 |
124 |
0.8 |
0.4 |
499 |
0.7 |
0.7 |
126 |
0.5 |
0.6 |
479 |
6 |
0.9 |
0.7 |
117 |
0.7 |
0.5 |
424 |
0.6 |
0.8 |
143 |
0.4 |
0.5 |
755 |
7 |
0.9 |
0.8 |
108 |
0.7 |
0.5 |
431 |
0.7 |
0.8 |
72 |
1.1 |
0.6 |
426 |
8 |
0.6 |
0.7 |
126 |
0.6 |
0.4 |
612 |
0.6 |
0.7 |
161 |
0.4 |
0.5 |
990 |
9 |
1.0 |
0.8 |
130 |
1.1 |
0.5 |
605 |
- |
- |
- |
- |
- |
- |
10 |
1.1 |
1.1 |
61 |
1.1 |
0.5 |
861 |
0.7 |
1.1 |
87 |
1.1 |
0.7 |
675 |
11 |
1.2 |
0.9 |
75 |
0.9 |
0.5 |
458 |
0.6 |
0.7 |
125 |
0.5 |
0.4 |
1298 |
12 |
1.3 |
0.9 |
58 |
1.8 |
0.7 |
462 |
1.0 |
1.3 |
94 |
0.8 |
0.5 |
823 |
13 |
1.0 |
1.3 |
77 |
1.2 |
0.6 |
533 |
1.0 |
1.3 |
94 |
0.8 |
0.5 |
823 |
minimum |
0.6 |
0.7 |
58 |
0.6 |
0.4 |
424 |
0.6 |
0.7 |
72 |
0.4 |
0.4 |
426 |
maximum |
1.3 |
1.3 |
139 |
1.8 |
0.7 |
861 |
1.0 |
1.3 |
242 |
1.1 |
0.7 |
1298 |
mean |
1.0 |
0.9 |
98 |
1.0 |
0.5 |
547 |
0.7 |
0.9 |
122 |
0.6 |
0.5 |
707 |
CE0: cloning efficiency at day 0
CE2: cloning efficiency at day 2
MF: mutation frequency
MMS: methylmethane sulfonate (3-hour treatment: 25µg/ml ; 24-hour treatment: 5µg/ml)
- : not performed
Table: Historical data with S9 mix Mouse lymphoma assay
assay No. |
vehicle |
CPA |
||||
CE0 |
CE2 |
MF |
CE0 |
CE2 |
MF |
|
1 |
0.9 |
0.8 |
83 |
0.5 |
0.4 |
1077 |
2 |
0.9 |
0.7 |
123 |
0.5 |
0.3 |
915 |
3 |
1.0 |
0.8 |
68 |
0.5 |
0.6 |
948 |
4 |
0.7 |
0.8 |
138 |
0.3 |
0.4 |
1177 |
5 |
0.7 |
1.0 |
80 |
0.5 |
0.4 |
1248 |
6 |
0.9 |
0.9 |
161 |
0.4 |
0.3 |
1200 |
7 |
0.7 |
0.8 |
186 |
0.4 |
0.4 |
958 |
8 |
0.8 |
0.7 |
137 |
0.4 |
0.3 |
1563 |
9 |
0.7 |
1.0 |
92 |
0.3 |
0.3 |
1200 |
10 |
0.6 |
1.0 |
136 |
0.4 |
0.5 |
643 |
11 |
0.9 |
0.8 |
246 |
0.7 |
0.8 |
618 |
12 |
0.6 |
0.8 |
199 |
0.4 |
0.4 |
1389 |
13 |
0.7 |
0.8 |
114 |
0.4 |
0.4 |
915 |
14 |
0.9 |
1.0 |
88 |
1.0 |
0.8 |
484 |
15 |
1.1 |
0.9 |
77 |
0.4 |
0.4 |
1077 |
16 |
0.7 |
0.8 |
86 |
0.4 |
0.7 |
675 |
17 |
0.8 |
0.7 |
113 |
0.5 |
0.5 |
754 |
18 |
0.7 |
0.9 |
91 |
0.5 |
0.4 |
1583 |
19 |
0.6 |
0.8 |
108 |
0.4 |
0.4 |
1126 |
20 |
1.2 |
1.0 |
79 |
1.4 |
0.7 |
544 |
21 |
0.7 |
1.2 |
72 |
0.6 |
0.8 |
636 |
22 |
0.9 |
1.2 |
86 |
0.7 |
0.9 |
721 |
23 |
1.0 |
0.8 |
108 |
0.6 |
0.3 |
1456 |
24 |
0.9 |
1.2 |
86 |
0.7 |
0.9 |
721 |
minimum |
0.6 |
0.7 |
68 |
0.3 |
0.3 |
484 |
maximum |
1.2 |
1.2 |
246 |
1.4 |
0.9 |
1583 |
mean |
0.8 |
0.9 |
115 |
0.5 |
0.5 |
985 |
CE0: cloning efficiency at day 0
CE2: cloning efficiency at day 2
MF: mutation frequency
CPA: cyclophosphamide (3µg/ml)
- : not performed
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Based on the results of the test and read across in vivo genotoxicity studies, the test substance is considered to be non-genotoxic.
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo, OECD 486
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07.07.2006 - 29.05.2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- Version / remarks:
- circa. 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hanlbm: WIST (SPF)
- Details on species / strain selection:
- The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the I-JDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
Number of animals: 32 (males)
Acclimatisation and quarantine: minimum 5 days
Age of the animals: 6 - 10 weeks
Initial body weight at start of treatment: Mean value 179.4 g (SD* ± 14.8 g)
* SD = Standard deviation
According to the supplier's assurance the animals were in healthy condition. The animals underwent quarantine in the animal house of RCC-CCR for at least five days after their arrival. During this period the animals did not show signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.
Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage type: Makrolon Type Il, with wire mesh top (Ehret, D-79312 Emmendingen)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
Drinking water: tap water, ad libitum (Gemeindewerke Roßdorf, D-64380 Roßdorf)
Environment:
temperature 21 ± 30 C
relative humidity 30 - 72 %
artificial light 6.00 a.m. - 6.00 p.m. - Route of administration:
- oral: gavage
- Vehicle:
- 30% DMSO/70% PEG 400
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 30% DMSO /70% PEG 400 and heated to 37C. The vehicle was chosen to its non-toxicity for the animals. The animals received a single standar volume of 10 ml/kg bw orally. - Duration of treatment / exposure:
- Test subsance administered in a single treatment.
- Frequency of treatment:
- Test subsance administered in a single treatment.
- Post exposure period:
- 1, 2-4, 6 and 24 h after administrationof the test item
- No. of animals per sex per dose:
- Preliminary experiments: 2 animals per sex per dose group per dose per timepoint.
Main experiment: 4 male sex per dose per timepoint - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Positive control 4hr: DMH; N,N'-dimethylhydrazinedihydrochloride 80 mg/kg bw
Positive control 16hr: 2-AAF; 2-acetylaminofluorene 100 mg/kg bw - Tissues and cell types examined:
- Primary Hepatocytes
- Details of tissue and slide preparation:
- Isolation of the Primary Hepatocytes
8.4.5.
After anaesthetising the rats with 46% Ketamin (Ketavet 100, Pharmacia GmbH, D-76139 Karlsruhe), 23% Xylazin (Rompun 2%, Bayer HealthCare, D-51368 Leverkusen) and 31% Midazolam (Dormicum, Hoffmann LaRoche, D-79639 Grenzach-Wyhlen) (approx. 2 mL/kg b.w.) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Invitrogen, D-76344 Eggenstein) supplemented with collagenase (0.05 % (w/v), Roche Diagnostics, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 370 C (8).
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh (94 pm) to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.
8.4.6.
Culture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME, Invitrogen, D-76344 Eggenstein) supplemented (I) with:
Hepes 2.38 mg/ml
Penicillin 100 units/ml
Streptomycin 0.10 mg/ml
L-GIutamine 0.29 mg/ml
Insulin 0.50 ug/ml
Fetal calf serum (FCS) 100 ul/ml
This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 ml with freshly isolated hepatocytes in complete culture medium (2.0 x 105 viable cells/ml) were added to 35 mm six-well dishes (Greiner, D-72603 Nürtingen) containing one 25 mm round plastic coverslip (Thermanox, Nunc, D-65203 Wiesbaden) per well coated with gelatine. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % C02 humidified incubator at 370 C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells (9). Subsequently, 3HTdR (5 pCi/ml, specific activity 20 Ci/mmol; New England Nuclear, D-63033 Dreieich) in 2.0 ml culture medium
(WME, I % (v/v) FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with I % (wv) FCS and 0.25 rnM unlabelled thymidine. Cultures were incubated overnight using the same medium (2). To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection (9). The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 wv) for 20 minutes each, rinsed with 96 % (VIV) ethanol, and air-dried.
8.4.7. Autoradiographic Processing
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK N TB (Tecnomara, D-35463 Fernwald) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 40 C. The photographic emulsion was then developed with Ilford Phenisol (Ilford Imaging GmbH, 63265 Dreieich) at room temperature, fixed in Rapid Fixer (llford Imaging GmbH, 63265 Dreieich) and stained with hematoxylin/eosin.
8.4.8. Quantification of I-JDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer I-JDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labelled nuclear-sized cytoplasm area adjacent to the nucleus was counted (2). At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabelled cells undergoing replicative DNA synthesis were excluded from counting.
Three animals per group were evaluated as described above.
8.4.9. Data Recording
The data generated were recorded in the raw data. The results were presented in tabular form, including experimental groups with the test item, vehicle and positive controls.
The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) are reported separately (5). Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively. - Evaluation criteria:
- Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test (4).
A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- ruffled fur, reduction in spontaneous activity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- ruffled fur, reductions in spontaneous activity, eye lid closure
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- BIOMETRY
- RESULTS
- Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
Therefore, Arquad 2C is considered to be non-genotoxic in this in vivo/ in vitro I-JDS test system. - Executive summary:
The test item Arquad 2C was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats with doses of 500 mg/kg b.w. and 1000 mg/kg b.w. (4 h and 16 h preparation interval).
The test item was formulated in 30% DMSO / 70% PEG 400, which was used as vehicle control. The volume administered orally was 10 ml/kg body weight. After a treatment period of 4 and 16 hours, respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR (methyl-3H-thymidine) which is incorporated if UDS occurs (2).
The highest dose was estimated in a pre-experiment to be the maximum applicable dose, at which clinical signs of toxicity occurred. In the main experiment one animal died during the 16 h preparation interval.
The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of our historical laboratory control (3).
No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 4 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative.
In addition, the obtained percentage of cells in repair were within the historical control range (up to 12%). Due to a great variation in the percentages of cells (26 and 4% for slides 28a and 28b, respectively) in repair observed in the two slides scored for animal no. 28, a third slide was additionally scored. Thus, for animal no. 28 a total of 150 cells were scored instead of 100. The data from the third slide (2% cells in repair) confirmed the value obtained from slide 28b (4% cells in repair). Thus, the observed increase in the percentage of cells in repair in slide 28a is not reproducible and, therefore, not biologically relevant.
Appropriate reference mutagens [DMH (10), 80 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.] were used as positive controls. In vivo treatment with DMA or 2-AAF revealed distinct increases in the number of nuclear and net grain counts.
Reference
8.6. Historical Controls (1999-2005)
59 finalised studies
Test group | Net grains | Treatment period | Number of animals | |
Range | Mean | |||
Vehicle control* | -14.3 to -3.2 | -6.7±2.2 | 2 or 16 hrs | 352 |
Positive controls (DMH) | 3.1 to 39.1 | 13.4± 7.5 | 2 hrs | 176 |
Positive controls (2-AAF) | 2.9 to 35.9 | 15.1 ± 7.3 | 16 hrs | 175 |
* : Vehicles: deionised water, sesame oil, corn oil, PEG 400, CMC (0.5-1.0%).
A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.
10.1. Pre-Experiment
In the first pre-experiment 2 male and 2 females rats received orally a single dose of 200 mg/kg b.w. of Arquad 2C formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 ml/kg b.w..
10.1.1 The treated animals expressed toxic reactions as shown in the table:
toxic reactions | hours post-treatment male / female | |||
1h | 2-4h | 6h | 24h | |
ruffled fur | 0/0 | 2/0 | 1/0 | 0/0 |
In the second pre-experiment 2 male and 2 females rats received orally a single dose of 1000 mg/kg b.w. of Arquad 2C formulated in DMSO / PEG 400. The volume administered was 10 ml/kg b.w..
10.1.2 The treated animals expressed toxic reactions as shown in the table:
toxic reactions | hours post-treatment male / female | |||
1h | 2-4h | 6h | 24h | |
reduction of spontaneous activity | 2/1 | 2/1 | -/- | 2/0 |
abdominal position | 0/0 | 1/0 | -/- | 2/0 |
eyelid closure | 2/0 | 2/0 | -/- | 0/0 |
ruffled fur | 2/2 | 2/2 | -/- | 2/2 |
breathing difficulty | 0/0 | 0/0 | -/- | 2/0 |
bloody mouth | 0/0 | 0/0 | -/- | 2/0 |
diarrhoea | 0/0 | 0/0 | -/- | 2/0 |
-/-: no observation made.
In the third pre-experiment 2 male and 2 females rats received orally a single dose of 1250 mg/kg b.w. of Arquad 2C formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 ml/kg b.w..
10.1.3 The treated animals expressed toxic reactions as shown in the table:
toxic reactions | hours post-treatment male / female | |||
1h | 2-4h | 6h | 24h | |
reduction of spontaneous activity | 2/2 | 2/2 | 2/2 | 1/1 |
eyelid closure | 2/0 | 2/0 | 2/0 | 1/0 |
ruffled fur | 2/2 | 2/2 | 2/2 | 1/1 |
breathing difficulty | 0/0 | 1/1 | 1/1 | 0/0 |
death | 0/0 | 0/0 | 0/0 | 1/1 |
On the basis of these data the dose of 1000 mg/kg b.w. was estimated to be suitable as the high dose
10.2. Toxic symptoms in the Main Experiment
In the main experiment with the 4 h treatment period 4 animals received orally a single dose of 500 and 1000 mg/kg b.w.. Arquad 2C formulated in 30% DMSO / 70% PEG 400.
The volume administered was 10 ml/kg b.w..
10.2.1 The treated animals expressed toxic reactions as shown in the table:
toxic reactions | hours post-treatment 500 / 1000 mg/kg b.w. | ||
1h | 2h | 4h | |
reduction of spontaneous activity | 2/3 | 2/4 | 4/4 |
eyelid closure | 2/4 | 2/4 | 4/4 |
In the main experiment with the 16 h treatment period 4 animals received orally a single dose of 500 and 1000 mg/kg b.w.. Arquad 2C formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 ml/kg b.w..
10.2.2 The treated animals expressed toxic reactions as shown in the table:
toxic reactions | hours post-treatment 500 / 1000 mg/kg b.w. |
|
|
| 1h | 2-4h | 16h |
reduction of spontaneous activity | 4/4 | -/- | 4/3 |
ruffled fur | 2/4 | -/- | 4/3 |
breathing difficulty | 0/1 | -/- | 0/1 |
diarrhoea | 0/0 | -/- | 4/3 |
death | 0/0 | -/- | 0/1 |
-/-: no observation made.
10.3 Viability of Hepatocytes
Treatment | Animal no. | Viability* [%] | Number of isolated cells [x10^6] | |
Vehicle control 30% DMSO / 70% PEG 400 | 4 h | 1 | 78 | 452 |
2 | 88 | 350 | ||
3 | 73 | 297 | ||
4 | 74 | 275 | ||
500 mg/kg b.w. Arquad 2C | 4 h | 5 | 83 | 314 |
6 | Perfusion failed | |||
7 | 86 | 306 | ||
8 | 78 | 359 | ||
1000 mg/kg b.w. Arquad 2C | 4 h | 9 | 84 | 299 |
10 | 80 | 177 | ||
11 | 78 | 227 | ||
12 | 78 | 234 | ||
Positive control DMH 80 mg/kg b.w. | 4 h | 13 | 76 | 396 |
14 | 87 | 375 | ||
15 | 88 | 303 | ||
16 | 83 | 381 | ||
Vehicle control 30% DMSO / 70% PEG 400 | 16 h | 17 | 93 | 498 |
18 | 75 | 377 | ||
19 | Perfusion failed | |||
20 | 96 | 251 | ||
500 mg/kg b.w. Arquad 2C | 16 h | 21 | 76 | 186 |
22 | Perfusion failed | |||
23 | 82 | 453 | ||
24 | 80 | 314 | ||
1000 mg/kg b.w. Arquad 2C | 16 h | 25 | 72 | 351 |
26 | 77 | 254 | ||
27 | Animal died | |||
28 | 91 | |||
Positive control 2-AAF 100 mg/kg b.w. | 16 h | 29 | Animal died# | |
30 | 79 | 346 | ||
31 | 79 | 249 | ||
32 | 85 | 391 |
*Viability determined by means of trypan blue dye exclusion assay
#application failure
One spare animal was included for each dose group.
Table 10.4.1 Mean nucleus, cytoplasmic area, and net grains at 4 hr
Test group | Animal No. | Mean Nuclear Grain Count | Mean Cytoplasmic grain count§ | Mean Net Grain Counts | Mean Nuclear Grains of Cells in Repair | % Cells in Repair | ||||
Mean | S.D. | Mean | S.D. | Mean | S.D. | Mean | S.D. | |||
Vehicle control 30% DMSO / 70% PEG 400 | 1 | 8.14 | 3.73 | 14.82 | 5.55 | -6.68 | 5.2 | 5 | 0 | 1 |
2 | 13.43 | 5.95 | 18.71 | 7.67 | -5.28 | 7 | 6.75 | 1.71 | 4 | |
4 | 6.83 | 3.85 | 12.4 | 4.35 | -5.57 | 4 | 0 | 0 | 0 | |
Mean | 9.47 | 3.49 | 15.31 | 3.18 | -5.84 | 0.74 | 3.92 | 3.5 | 2 | |
500 mg/kg b.w. Arquad 2C | 5 | 5.41 | 2.63 | 9.86 | 2.59 | -4.45 | 3.51 | 0 | 0 | 0 |
7 | 6.01 | 2.85 | 10.24 | 2.92 | 4.23 | 3.09 | 0 | 0 | 0 | |
8 | 5.27 | 2.55 | 9.07 | 2.59 | -3.8 | 3.1 | 6 | 0 | 1 | |
Mean | 5.56 | 0.39 | 9.72 | 0.6 | 416 | 0.33 | 2 | 3.46 | 0 | |
1000 mg/kg b.w. Arquad 2C | 9 | 7.93 | 3.73 | 16.13 | 4.34 | -8.2 | 4.42 | 5 | 0 | 1 |
11 | 8.93 | 4.44 | 16.8 | 6.53 | -7.87 | 6.73 | 8 | 0 | 1 | |
12 | 6.02 | 3.02 | 12.77 | 3.71 | -6.75 | 4.03 | 0 | 0 | 0 | |
Mean | 7.63 | 1.48 | 15.23 | 2.16 | -7.61 | 0.76 | 433 | 4.04 | 1 | |
Positive control DMH 80 mg/kg b.w. | 13 | 39.6 | 12.08 | 14.5 | 4.98 | 25.1 | 10.58 | 25.75 | 10.05 | 97 |
14 | 43.93 | 11.69 | 15.89 | 6.32 | 28.04 | 11.06 | 28.32 | 10.75 | 99 | |
15 | 31.94 | 10.34 | 11.38 | 3.73 | 20.56 | 9.03 | 20.76 | 8.85 | 99 | |
Mean | 38.49 | 6.07 | 13.92 | 2.31 | 24.57 | 3.77 | 2494 | 3.85 | 98 |
SD = Standard deviation The Standard deviation shown for a each animal is tha deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting Of three animals.
Table 10.4.2 Mean nucleus, cytoplasmic area, and net grains at 16 hr
Test group | Animal No. | Mean Nuclear Grain Count | Mean Cytoplasmic grain count§ | Mean Net Grain Counts | Mean Nuclear Grains of Cells in Repair | % Cells in Repair | ||||
Mean | S.D. | Mean | S.D. | Mean | S.D. | Mean | S.D. | |||
Vehicle control 30% DMSO / 70% PEG 400 | 17 | 10.49 | 3.99 | 14.02 | 5.22 | -3.53 | 4.75 | 6.5 | 1 | 4 |
18 | 14.13 | 7.31 | 20.1 | 8.92 | -5.97 | 8.24 | 8.88 | 3.68 | 8 | |
20 | 14.73 | 7.2 | 20.71 | 7.68 | -5.98 | 5.95 | 8.33 | 2.08 | 3 | |
Mean | 13.12 | 2.29 | 18.28 | 3.7 | -5.16 | 1.41 | 7.9 | 1.24 | 5 | |
500 mg/kg b.w. Arquad 2C | 21 | 6.88 | 3.59 | 10.76 | 4.11 | -3.88 | 4.89 | 6 | 1 | 3 |
23 | 10.72 | 5.85 | 16 | 5.97 | -5.28 | 5.9 | 11.67 | 5.77 | 3 | |
24 | 10.08 | 5.11 | 15 | 5.51 | -4.92 | 5.27 | 6.5 | 2.38 | 4 | |
Mean | 9.23 | 2.06 | 13.92 | 2.78 | -4.69 | 0.73 | 8.06 | 3.14 | 3 | |
1000 mg/kg b.w. Arquad 2C | 25 | 12.55 | 6.53 | 17.41 | 6.69 | -4.86 | 7.41 | 7.45 | 2.46 | 11 |
26 | 12.41 | 6 | 15.5 | 6.79 | -3.09 | 6.47 | 7.11 | 2.32 | 9 | |
28 | 11.63 | 7.47 | 15.13 | 8.26 | -3.49 | 6.28 | 6.5 | 2.19 | 11 | |
Mean | 12.2 | 0.49 | 16.01 | 1.22 | -3.81 | 0.93 | 7.02 | 0.48 | 10 | |
Positive control 2-AAF 100 mg/kg b.w. | 30 | 20.57 | 6.17 | 12.45 | 488 | 8.12 | 5.66 | 1042 | 4.52 | 74 |
31 | 37.69 | 15.57 | 25.94 | 9.56 | 11.75 | 11.32 | 16.23 | 8.07 | 77 | |
32 | 31.1 | 16.16 | 20.71 | 11.07 | 10.39 | 10.32 | 14.43 | 9.08 | 72 | |
Mean | 29.79 | 8.64 | 19.7 | 6.8 | 10.09 | 1.83 | 13.69 | 2.98 | 74 |
SD = Standard deviation The Standard deviation shown for a each animal is tha deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting Of three animals.
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Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
A study was conducted to evaluate the mutagenic potential of the test substance, C12-18 DAQ (76.4% active in hydroalcoholic solution) in the Ames test, according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. In the study S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains were used and S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats was used as metabolic activating system. A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100 was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix. Experiment 1: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. Experiment 2: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test (Scheres, 1990).
No mammalian clastogenicity or mutagenicity study could be located on C12 -18 DAQ. Therefore, read across studies available with structurally similar substance DDAC is presented. Both the test and read across substances are di-alkyl dimethyl ammonium chloride compounds. DDAC is structurally the same but only differs in a slightly lower average alkyl chain length. Slightly shorter alkyl chains do not change possible chemical reactivity or genotoxicity potential.
A study was conducted to determine the clastogenic potential of the read across substance, DDAC (51.3% active) in cultured human lymphocytes, according to OECD Guideline 473 and EU method B.10, in compliance with GLP. After a preliminary toxicity test, read across substance was tested in two independent experiments, with and without a metabolic activation system, the S9- mix. In the absence of S9 -mix read across substance was tested up to 5.6 µg/mL for a 24 and 48 h fixation time in the first experiment. In the second experiment read across substance was tested up to 7.5 µg/mL for a 24 h fixation time. In the presence of S9 -mix read across substance was tested up to 24 µg/mL for 24 h fixation time and up to 18 µg/mL for a 48 h fixation time in the first experiment. In the second experiment it was tested up to 24 µg/mL for a 24 h fixation time. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since, the types of aberrations observed were only breaks, the increase was not dose related. Experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, therefore the increase was not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix read across substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly (Van der waart, 1996). Based on the results of the read across study, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation. Based on results of the read across study, similar absence of clastogenic potential is expected for the test substance.
A study was conducted to determine the potential of the read across substance, DDAC (40.37% active) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells, according to OECD guideline 476 and EU method B17, in compliance with GLP. After a preliminary toxicity test, read across substance was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5E+6 (3-hour treatment: preliminary toxicity test and all experiments except for the second experiment without S9 mix) or 0.15E+6 (24-hour treatment: second experiment without S9 mix) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the read across or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. The read across substance was dissolved in dimethylsulfoxide (DMSO). All the dose-levels were expressed as active substance, taking into account the active material content of 40.37%. The selected dose-levels for read across substance treatment without S9 mix were as follows: (a) 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL, for the first experiment (b) 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL, for the second experiment. The selected dose-levels for read across substance treatment with S9 mix were as follows: (a) 0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL, for the first experiment (b) 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL, for the second experiment. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking. The dose-levels for the positive controls were as follows: (1) without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL (3-hour treatment) or 5 µg/mL (24-hour treatment) (2) with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL. Cytotoxicity was then determined using cloning efficiency (CE0) before expression of the mutant phenotype. Cell viability (using cloning efficiency CE2) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Other parameters such as cell concentration, relative survival (RS), relative suspension growth (RSG) and/or relative total growth (RTG) were also taken into consideration. In experiments without S9 mix. a slight to strong toxicity was induced, depending on the dose-levels and the treatment duration. No noteworthy increase in the mutation frequency was induced both experiments without S9 mix after the 3 and 24-hour treatments. In experiments with S9 mix, the read across substance was moderately to strongly toxic, depending on the dose-levels. No noteworthy increase in the mutation frequency was induced in both experiments with S9 mix. The cloning efficiencies CE0 and CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid (Haddouk, 2002). Based on results of the read across study, similar absence of clastogenic potential is expected for the test substance.
A in vivo study was conducted on Quaternary ammonium compounds, dicoco alkyldimethyl, chlorides (CAS: 61789-77-3), which was previously registered as Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides (CAS: 68391-05-9). The test item Arquad 2C was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. After an initial pre-experiment was conductd to determine tolerabel dose limits, dose levels of 500 mg/kg b.w. and 1000 mg/kg b.w. (4 h and 16 h preparation interval) were choosen for the main experiment. Experimental results show that viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups. No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 4 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. High variation in observed cells in repair for one animal in the 1000 mg/kg b.w. dose group resulted in the examination of a third slide to verify results (150 cells total at 50 cells per slide). The cells in repair data from the third slide confirmed a low incidence rate.
Justification for classification or non-classification
Based on data available for C12-18 DAQ and read across substances, genotoxic potential is not expected. Therefore no classification is required for gentoxicity according to EU CLP criteria (Regulation 1272/2008/EC).
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