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EC number: 841-500-6 | CAS number: 1903008-80-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2016-03-17 to 2016-03-31
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- other: International Conference on Harmonisation Harmonised Tripartite Guideline, Safety Guidelines, Genotoxicity Studies S2(R1) Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
- Version / remarks:
- 09 November 2011
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Ministry of Food and Drug Safety Notification No. 2015-82 Test Guidelines for Safety Evaluation of Drugs Annex 4 Genetic Toxicity Study-Bacterial Reverse Mutation Test
- Version / remarks:
- 11 November 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide
- EC Number:
- 841-500-6
- Cas Number:
- 1903008-80-9
- Molecular formula:
- C30H34N8O3
- IUPAC Name:
- N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- KIT Code No.: K-5523
- Batch/Lot No.: 5219-10
- Purity: 99.6% (by HPLC)
- Purity test date : 2016-01-07
- Expiry date: 2016-07-07 (retest date)
- Appearance: Light yellow solid
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated, protected from light and tightly closed
-Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test item in the solvent/vehicle: not indicated
OTHER SPECIFICS: As per the sponsor’s request, the correction for purity was not conducted.
Method
- Target gene:
- Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- not relevant
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: Experiment 1: Sprague-Dawley rat (male) liver tissue; Experiment 2: human (male) liver tissue (pooled)
- Method of preparation of S9 mix: The final concentration for the composition of cofactor mix per 1.0 mL of metabolic activation system was as follows:
- MgCl2 8 μmol,
- KCl 33 μmol,
- Glucose 6, phosphate (G-6-P) 5 μmol,
- Nicotinamide adenine dinucleotide phosphate reduced (NADPH) 4 μmol,
- Nicotinamide adenine dinucleotide reduced (NADH) 4 μmol,
- Sodium phosphate buffer (pH 7.4) 100 μmol
- Concentration or volume of S9 mix and S9 in the final culture medium: The metabolic activation system containing 5% v/v of S9 fraction consisted of the following: S9 fraction 0.5 mL, Cofactor mix 9.5 mL.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 fraction was checked by the manufacturer and the quality control statement, for each batch and type of S9 fraction used, was included in the raw data
- Use of metabolic activation system: The metabolic activation system was prepared immediately prior to use and placed in an ice bucket during experiment. The remaining metabolic activation system was discarded after the treatment. Each plate was treated with 0.5 mL of the prepared metabolic activation system, which was held in an ice batch for use. Same volume of 0.2 M sodium phosphate buffer solution (pH 7.4) was used for treatment in the absence of metabolic activation system. The activity of metabolic activation system was confirmed by the induction of revertant colonies in the plates treated with 2-AA and BP in S. typhimurium TA1535 and TA98, respectively. - Test concentrations with justification for top dose:
- DRF: 50, 100, 500, 750, 1000 and 2000 μg/plate
Experiment 1: 0, 2.5, 8, 23, 70 and 200 µg/plate with and without metabolic activation (rat liver S9 fraction)
Experiment 2: 0, 2.5, 8, 23, 70 and 200 µg/plate with and without metabolic activation (human liver S9 fraction)
See section Any other information on materials and methods incl. tables for the justification - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as vehicle according to the information provided by sponsor and was used to dilute for dose formulation.
- Justification for percentage of solvent in the final culture medium: not indicated
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Without S9; S. typhimurium TA100 and TA1535 (0.5 μg/plate per strain)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Without S9; S. typhimurium TA98 (2 μg/plate)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without S9; S. typhimurium TA1537 (50 μg/plate)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without S9; E. coliWP2uvrA (1 μg/plate)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- With S9; S. typhimurium TA1535 (2 μg/plate) and E. coliWP2uvrA (4 μg/plate) in exp 1. S. typhimurium TA1535 (0.5 and 1 μg/plate), TA98 (0.1 and 0.5 μg/plate), TA1537 (0.5 and 1 μg/plate) and E. coliWP2uvrA (4 and 6 μg/plate) in exp 2.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9; S. typhimurium TA100, TA98 and TA1537 (2 μg/plate per strain)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- With S9; S. typhimurium TA100 (0.5 and 1 μg/plate) in the second experiment
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
- Preincubation period, if applicable: approximately 10 hours in a shaking incubator (120 to 200 rpm) at 37±1°C
- Treatment of test and control items: Three plates for each concentration were used in the absence and presence of metabolic activation system, respectively. Autoclaved top agar (2.0 mL) was dispensed into each 12×75 mm tube maintained at approximately 45°C in a heating block. Then 0.1 mL of test item solution, 0.5 mL of metabolic activation system (or 0.2 M sodium phosphate buffer, pH 7.4), and 0.1 mL of bacterial culture were added to each tube. This mixture (2.7 mL) was stirred for 2 to 3 seconds using a vortex mixer and overlaid onto the surface of a minimal glucose agar plate.
Negative control (vehicle) group was treated with 0.1 mL of vehicle instead of test item solution.
Positive control group was treated with 0.1mL of specific positive control for each tester strain.
Sterility checks were performed on test item solution (maximum concentration only, 0.1 mL) and metabolic activation system (0.5 mL), without bacterial culture, using additional plates. After the overlay solidified, the plates were incubated upside down for approximately 48 hours at 37±1°C in the dark.
- Harvest time after the end of treatment (sampling/recovery times): Cultures were harvested when the tester strain culture titers were more than 1×10^9 cells per milliliter.
- Counting of revertant colony: Upon completion of the incubation, the plates were scored immediately. The numbers of revertant colonies were counted using a hand counter. Absence or diminution of background lawn, formation of microcolonies, and the presence of turbidity and /or precipitation resulting from test item for each plate were evaluated with the unaided eye and recorded.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: a concentration-dependent decrease in the revertant counts, or absence or diminution of background lawn or formation of microcolonies - Rationale for test conditions:
- In the DRF study, YH25448 did not cause a positive response in all strains in the absence and presence of metabolic activation system. However, antibacterial effect (toxicity), turbidity and/or precipitation were observed at some concentrations regardless of the metabolic activation system application. Therefore, to include the concentration with a visible precipitate on the basis of these results, 200 μg/plate was selected as the highest concentration in the definitive study. Treatment concentrations of the definitive study were 0, 2.5, 8, 23, 70 and 200 μg/plate.
- Evaluation criteria:
- The assay must be considered valid in accordance with the assay acceptance criteria (see additional information on material and methods)
Antibacterial effect (toxicity): A ≥50% reduction in the number of revertants per plate relative to the negative control (vehicle) values and exhibiting a concentration-dependent decrease in the revertant counts, or absence or diminution of background lawn or formation of microcolonies (Richard et al., 2002).
The assay was considered positive if there is a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one tester strain with or without the metabolic activation system, ≥ two times (for S. typhimurium TA98, TA100 tester strains, and E. coli WP2uvrA tester strain) or ≥ three times (for S. typhimurium TA1535 and TA1537 test strains) that of the mean concurrent negative control (vehicle) value (Richard et al., 2002).
An equivocal response was a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
The assay was considered negative: if none of the above criteria are met. - Statistics:
- No statistical analyses were performed. The ‘fold-increase’ relative to the negative control
(vehicle) value was calculated in order to compare the mean values for all treatment groups
with those obtained for negative control (vehicle) groups.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- rat S9-mix
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- observed at the highest dose level 200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- rat S9-mix
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- rat S9-mix
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- observed at the highest dose level 200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- rat S9-mix
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- rat S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- rat S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- observed at the highest dose level 200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- rat S9 mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- human S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- human S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- observed at the highest dose level 200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- human S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- human S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- observed at the highest dose level 200 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- human S9-mix
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- 200 μg/plate was the highest concentration tested
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The turbidity of test item was observed at 200 μg/plate concentration in all strains in the absence and presence of metabolic activation system when the test item solutions were mixed with top agar in the first and second experiments.
When the test item solutions were incubated for 48 hours in the first experiment, the precipitation of test item was observed at 200 μg/plate concentration in all strains except for S. typhimurium TA1537 strain in the
absence of metabolic activation system. The precipitation of test item was observed at 70 μg/plate and 200 μg/plate concentrations in S. typhimurium TA1537 strain in the absence of metabolic activation system.
In the second experiment, the precipitation of test item was observed at 200 μg/plate concentration in S. typhimurium TA1535, TA98 and E. coli WP2uvrA strains, and at 70 μg/plate and 200 μg/plate concentrations in S. typhimurium TA100 and TA1537 strains in the absence of metabolic activation system. The precipitation of test item was observed at 200 μg/plate concentration in S. typhimurium TA100 and E. coli WP2uvrA strains, and at 70 μg/plate and 200 μg/plate concentrations in S. typhimurium TA1535, TA98 and TA1537 strains in the presence of metabolic activation system.
RANGE-FINDING/SCREENING STUDIES: Increase in the number of colonies compared to the negative control (vehicle) in S. typhimurium TA1535, TA98 and TA1537 strains in the absence of metabolic activation system, and in S. typhimurium TA98 and TA1537 strains in the presence of metabolic activation system. To confirm the results of these strains, the verification test of colony was conducted using sixteen randomly selected colonies per plate that were removed and streaked onto minimal glucose agar plates supplemented with biotin but without histidine or the test item. No growth was observed at 500 to 2000 μg/plate concentrations in S. typhimurium TA1535 and TA1537 strains confirming that the colonies were not revertants, but growth was observed at 500 μg/plate concentration and at 500 to 2000 μg/plate concentrations in S. typhimurium TA98 strain in the absence and presence of metabolic activation system, respectively. However, the criterion for considering test item as positive (≥ two times for S. typhimurium TA98 strain) was not met in S. typhimurium TA98 strain regardless of metabolic activation system application. Antibacterial effect (toxicity) defined as a diminution of background lawn, formation of microcolonies was observed at ≥500
μg/plate concentrations in all strains in the absence of metabolic activation system, and at ≥500 μg/plate concentrations in S. typhimurium TA98 and TA1537 strains and at ≥750 μg/plate concentrations in S.
typhimurium TA100, TA1535 and E. coli WP2uvrA strains in the presence of metabolic activation system. Antibacterial effect (toxicity) defined as the ≥50% reduction in the number of revertants relative to the negative control (vehicle) values were also observed at ≥500 μg/plate concentrations in S. typhimurium TA100 strain in the absence of metabolic activation system, and at 500, 750 and 2000 μg/plate concentrations in S. typhimurium TA100 and TA1535 strains in the presence of metabolic activation system. In addition, turbidity of test item was observed at ≥500 μg/plate concentrations, and precipitation of test item was observed at ≥100 μg/plate concentrations in all strains when the test item solutions were mixed with top agar. After 48 hours incubation, precipitation of test item was observed at ≥100 μg/plate concentrations in all strains regardless of the metabolic activation system application. In particular, precipitation of test item was observed at 50 μg/plate concentration in S. typhimurium TA100 strain in the absence of metabolic activation system.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: The activity of the metabolic activation system was verified using 2-AA and BP, both of which produced significant mutagenicity in the presence of metabolic activation system. In the absence of metabolic activation system, 2-AA and BP produced no mutagenicity in the S. typhimurium TA1535 or TA98 stains. The number of revertant colonies in the negative control (vehicle) group was within the range of historical control data. The positive control groups showed significant increases (≥ two times) in mutagen-induced revertant colonies compared to that of the concurrent negative control (vehicle) group. These data indicated that the present study was performed under acceptable experimental conditions and the results were valid.
- Signs of toxicity: In both experiment, antibacterial effect (toxicity) defined as a diminution of background lawn, formation of microcolonies and/or the ≥50% reduction in the mean number of revertants per plate relative to the mean negative control (vehicle) values was observed at the highest concentration (200 μg/plate) in some strains in the absence and presence of metabolic activation system.
- There was no increase in the number of revertant colonies compared to the negative control (vehicle) at any concentrations of YH25448 in the absence and presence of metabolic activation system in any strain tested in the first experiment. Therefore, to confirm the results of the first experiment, the second experiment was performed by changing the type of S9 fraction of metabolic activation system. The results of S. typhimurium TA1535 strain were rejected according to the criteria for assay acceptance in the first experiment, since the contamination was observed at all plates. So, the retest for S. typhimurium TA1535 strain was additionally carried out when performing the second experiment. The number of revertant colonies was not increased at any concentrations of YH25448 in the absence and presence of metabolic activation system in any strain tested in the second experiment.
- In case of S. typhimurium TA98 strain, there was a slight discrepancy between the results of DRF study and the definitive study. The primary difference between DRF and definitive studies is the preparation method of 10-fold stock solutions of each target concentration for treatment. Since the concentration of the highest stock solution in the definitive study was 10 times lower than in the DRF study, the stock solution was clearly dissolved by using the unaided eye. The results of the DRF study was obtained with only one plate per each concentration and did not meet the criteria for considering test item as positive (≥ two times
for S. typhimurium TA98 strain). In addition, the negative results were obtained from two independent experimental conditions of the definitive study. Therefore, it is considered that a biologically relevant increase in the number of revertant colonies was not observed following treatment with YH25448 at any concentrations in the absence and presence of metabolic activation system.
Applicant's summary and conclusion
- Conclusions:
- All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Study of YH25448 in the Salmonella typhimurium/Escherichia coli Plate Incorporation Assay indicated that, under the conditions of this study, YH25448 did not cause a positive response up to 200 μg/plate in the absence and presence of Aroclor 1254-induced rat liver S9 homogenate. The second experiment with human liver S9 did not show mutagenic potential in all tester strains in the absence and presence of metabolic activation system.
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