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EC number: 611-033-0 | CAS number: 536759-91-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- 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
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- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Mouse Lymphoma Assay
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was conducted between 29 July 2016 and 23 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2R1 guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
- Version / remarks:
- except for the recommended dose level where the higher top dose level recommended in the OECD Test Guideline 490 was followed.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Japanese Guideline: Kanpoan No. 287 - - Environment Protection Agency
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Japanese guideline: Eisei No. 127 - - Ministry of Health and Welfare
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Japanese guidline: Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: mouse lymphoma assay
Test material
- Reference substance name:
- ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate
- EC Number:
- 611-033-0
- Cas Number:
- 536759-91-8
- Molecular formula:
- C22H20N4O6
- IUPAC Name:
- ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate
- Test material form:
- solid: particulate/powder
- Details on test material:
- powder; stored at room temperature in the dark
Constituent 1
- Specific details on test material used for the study:
- Physical State / Appearance: Amber colored viscous liquid
Batch: AAG8999N
Purity: 100.1%
Retest Date: 27 December 2017
Storage Conditions: Room temperature, protected from light
Correction factor to apply based on purity: None
Physical state / Appearance: Off-white to yellow to brown powder
Method
- Target gene:
- thymidine kinase, TK +/-, locus
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours
- Methods for maintenance in cell culture if applicable: The stocks of cells are stored in liquid nitrogen at approximately -196°C.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- Preliminary toxicity test:
The molecular weight of the test item was 436.42 therefore the maximum proposed dose level in the solubility test was set at 2000 µg/mL, the maximum recommended dose level. The dose range used in the preliminary toxicity test was 7.81 to 2000 µg/mL for all three of the exposure groups (4- hour exposure with and without S9, 24-hour exposure without S9).
Main test:
In the preliminary test there was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups when compared to the concurrent vehicle control groups. However, the reductions in %RSG values were only observed at concentrations beyond the onset of precipitate that occurred at 15.63 µg/mL in the 4 hour and 24 hour exposure groups in the absence of metabolic activation, and at 125 µg/mL in the 4 hour exposure group in the presence of metabolic activation. The maximum concentration in the subsequent Mutagenicity Test was therefore limited by the onset of precipitate.
The dose range of test item used in the main test was selected based on the results of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:
Group:
4-hour without S9: 0.49, 0.98, 1.95, 3.91, 7.81, 15.63 µg/mL
4-hour with S9 (2%): 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL
24-hour without S9: 0.49, 0.98, 1.95, 3.91, 7.81, 15.63 µg/mL - Vehicle / solvent:
- Following solubility checks performed in-house, the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO) prior to serial dilutions being prepared.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- absence of S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- presence of S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium, in suspension
- Cell density at seeding (if applicable):
preliminary toxicty tests 5 x 10^5 cells/ml for 4-hour exposure (with and without metabolic activation) and 1.5 x 10^5 cells/ml for 24-hour exposure (without metabolic activation). All groups were serially diluted to 2 x 10^5 cells/ml after the exposure period
Main test: 1 x 10^6 cells/ml for 4-hour exposures and 0.3 x 10^6 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 cells.ml after the exposure period. Before plating, cells were diluted to 1 x 10^4 cells/ml (or 10 cells/ml for viability assessment)
DURATION
- Preincubation period: Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment.
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days
SELECTION AGENT (mutation assays): 4 µg/mL 5 trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- Data evaluation:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system. - Statistics:
- Calculation of Percentage Relative Suspension Growth (%RSG):
The cell counts (x 10^5 cells/mL) obtained immediately post exposure (0 hour) and over the 2 day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100
Calculation of Day 2 Viability (%V):
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells / total wells plated
%V = (-ln P(0) x 100) / number of cells per well
Calculation of Relative Total Growth (RTG):
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = %V / mean solvent control %V
Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%
Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
Results and discussion
Test results
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Toxicity:
There was no evidence of any marked toxicity following exposure to BMS-589152-01 in any of the three exposure groups. There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had also not occurred. Acceptable levels of toxicity were seen with the positive control substances.
Controls:
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
Mutagenicity:
The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any concentration (including precipitating concentrations as recommended by the OECD 490 Guidelines), in any of the three exposure groups.
Precipitation:
Precipitate was observed at and above 15.63 µg/mL in the 4 hour and 24 hour exposure groups in the absence of metabolic activation, and at and above 125 µg/mL in the 4 hour exposure group in the presence of metabolic activation. Therefore, as sufficient precipitating concentration levels were observed (as recommended by the OECD 490 Guidelines), the 31.25 and 62.5 µg/mL concentrations in the 4 hour and 24 hour exposure groups in the absence of metabolic activation, and the 250 µg/mL concentration in the 4 hour exposure group in the presence of metabolic activation, were considered to be surplus to requirements and were not plated out for viability or 5-TFT resistance.
Any other information on results incl. tables
Preliminary cytotoxicity test:
There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups when compared to the concurrent vehicle control groups. However, the reductions in %RSG values were only observed at concentrations beyond the onset of precipitate that occurred at 15.63 µg/mL in the 4 hour and 24 hour exposure groups in the absence of metabolic activation, and at 125 µg/mL in the 4 hour exposure group in the presence of metabolic activation. The maximum concentration in the subsequent Mutagenicity Test was therefore limited by the onset of precipitate.
The concentration range of BMS-589152-01 used in the preliminary toxicity test was 7.81 to 2000 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:
Dose (mg/mL) |
% RSG (-S9) 4 Hour Exposure |
% RSG (+S9) 4 Hour Exposure |
% RSG (-S9) 24 Hour Exposure |
0 |
100 |
100 |
100 |
7.81 |
105 |
91 |
94 |
15.63 |
122 |
92 |
72 |
31.25 |
108 |
88 |
58 |
62.5 |
86 |
92 |
54 |
125 |
93 |
92 |
7 |
250 |
18 |
34 |
3 |
500 |
0 |
27 |
0 |
1000 |
0 |
5 |
0 |
2000 |
0 |
6 |
0 |
A summary of the results from the main mutagenicity test are shown in the table below
Concentration (µg/mL) |
4-Hours-S9 |
Concentration (µg/mL) |
4-Hours+S9 |
||||||||||||
|
%RSG |
RTG |
MF§ |
|
%RSG |
RTG |
MF§ |
||||||||
0 |
|
100 |
1.00 |
135.92 |
|
0 |
|
100 |
1.00 |
127.79 |
|
||||
0.49 |
|
97 |
0.95 |
156.87 |
|
1.95 |
Ø |
86 |
|
|
|
||||
0.98 |
|
91 |
1.02 |
130.18 |
|
3.91 |
|
90 |
0.87 |
140.00 |
|
||||
1.95 |
|
103 |
1.09 |
129.41 |
|
7.81 |
|
90 |
0.79 |
148.41 |
|
||||
3.91 |
|
95 |
1.03 |
120.77 |
|
15.63 |
|
82 |
0.89 |
115.14 |
|
||||
7.81 |
|
94 |
1.08 |
125.17 |
|
31.25 |
|
84 |
0.97 |
122.76 |
|
||||
15.63 |
|
99 |
1.12 |
122.28 |
|
62.5 |
|
88 |
0.97 |
124.84 |
|
||||
31.25 |
Ø |
96 |
|
|
|
125 |
|
86 |
0.81 |
149.39 |
|
||||
62.5 |
Ø |
91 |
|
|
|
250 |
Ø |
78 |
|
|
|
||||
MF threshold for a positive response = 261.92 |
MF threshold for a positive response = 253.79 |
||||||||||||||
Positive control |
|
|
Positive control |
|
|
||||||||||
EMS |
|
|
|
|
|
CP |
|
|
|
|
|
||||
400 |
|
82 |
0.60 |
1173.27 |
|
1.5 |
|
79 |
0.72 |
612.14 |
|
||||
|
|
|
|
|
|
|
|
|
|
|
|
Concentration (µg/mL) |
24-Hours-S9 |
||||
|
%RSG |
RTG |
MF§ |
||
0 |
|
100 |
1.00 |
146.50 |
|
0.49 |
|
107 |
1.12 |
137.72 |
|
0.98 |
|
105 |
1.03 |
146.65 |
|
1.95 |
|
96 |
1.01 |
139.07 |
|
3.91 |
|
97 |
1.04 |
140.42 |
|
7.81 |
|
89 |
1.01 |
134.35 |
|
15.63 |
|
85 |
1.02 |
136.96 |
|
31.25 |
Ø |
71 |
|
|
|
62.5 |
Ø |
66 |
|
|
|
MF threshold for a positive response = 272.50 |
|||||
Positive control |
|
|
|||
EMS |
|
|
|
|
|
150 |
|
50 |
0.38 |
1842.77 |
|
The tables below give a summary analysis for each exposure group:
4 -hour exposure ( - S9)
Concentration (µg/mL) |
|
SG |
%RSG |
%V |
RTG |
MF§ |
0 |
|
10.38 |
100 |
89.59 |
1.00 |
135.92 |
0.49 |
|
10.24 |
97 |
87.30 |
0.95 |
156.87 |
0.98 |
|
10.55 |
91 |
99.97 |
1.02 |
130.18 |
1.95 |
|
11.45 |
103 |
95.38 |
1.09 |
129.41 |
3.91 |
|
10.01 |
95 |
98.08 |
1.03 |
120.77 |
7.81 |
|
10.29 |
94 |
103.97 |
1.08 |
125.17 |
15.63 |
|
11.25 |
99 |
100.94 |
1.12 |
122.28 |
31.25 |
Ø |
11.13 |
96 |
|
|
|
62.5 |
Ø |
10.33 |
91 |
|
|
|
Positive control EMS |
||||||
Concentration (µg/mL) |
SG |
%RSG |
%V |
RTG |
MF§ |
|
400 |
|
8.88 |
82 |
65.80 |
0.60 |
1173.27 |
GEF =126, therefore MF threshold for a positive response = 261.92
4 -hour exposure ( + S9)
Concentration (µg/mL) |
|
SG |
%RSG |
%V |
RTG |
MF§ |
0 |
|
12.81 |
100 |
98.55 |
1.00 |
127.79 |
1.95 |
Ø |
11.21 |
86 |
|
|
|
3.91 |
|
12.31 |
90 |
95.38 |
0.87 |
140.00 |
7.81 |
|
11.86 |
90 |
86.56 |
0.79 |
148.41 |
15.63 |
|
11.24 |
82 |
107.20 |
0.89 |
115.14 |
31.25 |
|
10.71 |
84 |
114.35 |
0.97 |
122.76 |
62.5 |
|
11.70 |
88 |
108.32 |
0.97 |
124.84 |
125 |
|
12.53 |
86 |
92.81 |
0.81 |
149.39 |
250 |
Ø |
10.69 |
78 |
|
|
|
Positive control CP |
||||||
Concentration (µg/mL) |
SG |
%RSG |
%V |
RTG |
MF§ |
|
1.5 |
|
9.55 |
79 |
90.38 |
0.72 |
612.14 |
GEF =126, therefore MF threshold for a positive response = 253.79
24 -hour exposure ( - S9)
Concentration (µg/mL) |
|
SG |
%RSG |
%V |
RTG |
MF§ |
0 |
|
70.34 |
100 |
95.82 |
1.00 |
146.50 |
0.49 |
|
74.55 |
107 |
101.93 |
1.12 |
137.72 |
0.98 |
|
70.85 |
105 |
98.08 |
1.03 |
146.65 |
1.95 |
|
67.46 |
96 |
100.94 |
1.01 |
139.07 |
3.91 |
|
69.99 |
97 |
99.97 |
1.04 |
140.42 |
7.81 |
|
66.88 |
89 |
101.93 |
1.01 |
134.35 |
15.63 |
|
65.37 |
85 |
105.02 |
1.02 |
136.96 |
31.25 |
Ø |
56.11 |
71 |
|
|
|
62.5 |
Ø |
53.94 |
66 |
|
|
|
Positive control EMS |
||||||
Concentration (µg/mL) |
SG |
%RSG |
%V |
RTG |
MF§ |
|
150 |
|
41.35 |
50 |
62.06 |
0.38 |
1842.77 |
The following tables give a summary of mutation frequencies in each exposure group
4 -hour exposure ( - S9)
Concentration (µg/mL) |
|
Small colonies |
Large colonies |
Proportion small colony mutants |
||||||
|
Viable |
Mutants |
|
Mutants |
|
|
||||
|
Yv |
Nv |
Ym |
Nm |
MF§ |
Ym |
Nm |
MF§ |
|
|
0 |
|
128 |
768 |
702 |
768 |
50.1 |
668 |
768 |
77.9 |
0.40 |
0.49 |
|
67 |
384 |
349 |
384 |
54.7 |
327 |
384 |
92.0 |
0.38 |
0.98 |
|
52 |
384 |
353 |
384 |
42.1 |
327 |
384 |
80.4 |
0.35 |
1.95 |
|
57 |
384 |
356 |
384 |
39.7 |
328 |
384 |
82.6 |
0.33 |
3.91 |
|
54 |
384 |
349 |
384 |
48.7 |
338 |
384 |
65.0 |
0.43 |
7.81 |
|
48 |
384 |
352 |
384 |
41.8 |
328 |
384 |
75.8 |
0.36 |
15.63 |
|
51 |
384 |
353 |
384 |
41.7 |
331 |
384 |
73.6 |
0.37 |
400 EMS |
|
103 |
384 |
245 |
384 |
341.5 |
221 |
384 |
419.8 |
0.46 |
4 -hour exposure ( + S9)
Concentration (µg/mL) |
|
Small colonies |
Large colonies |
Proportion small colony mutants |
||||||
|
Viable |
Mutants |
|
Mutants |
|
|
||||
|
Yv |
Nv |
Ym |
Nm |
MF§ |
Ym |
Nm |
MF§ |
|
|
0 |
|
107 |
768 |
695 |
768 |
50.7 |
670 |
768 |
69.3 |
0.43 |
3.91 |
|
57 |
384 |
345 |
384 |
56.1 |
333 |
384 |
74.7 |
0.43 |
7.81 |
|
68 |
384 |
347 |
384 |
58.5 |
334 |
384 |
80.6 |
0.43 |
15.63 |
|
45 |
384 |
354 |
384 |
37.9 |
330 |
384 |
70.7 |
0.36 |
31.25 |
|
39 |
384 |
352 |
384 |
38.0 |
322 |
384 |
77.0 |
0.34 |
62.5 |
|
44 |
384 |
351 |
384 |
41.5 |
326 |
384 |
75.6 |
0.36 |
125 |
|
60 |
384 |
353 |
384 |
45.3 |
322 |
384 |
94.9 |
0.33 |
1.5 CP |
|
63 |
384 |
221 |
384 |
305.7 |
290 |
384 |
155.3 |
0.63 |
24 -hour exposure ( - S9)
Concentration (µg/mL) |
|
Small colonies |
Large colonies |
Proportion small colony mutants |
||||||
|
Viable |
Mutants |
|
Mutants |
|
|
||||
|
Yv |
Nv |
Ym |
Nm |
MF§ |
Ym |
Nm |
MF§ |
|
|
0 |
|
113 |
768 |
715 |
768 |
37.3 |
633 |
768 |
100.9 |
0.28 |
0.49 |
|
50 |
384 |
349 |
384 |
46.9 |
325 |
384 |
81.8 |
0.37 |
0.98 |
|
54 |
384 |
352 |
384 |
44.4 |
320 |
384 |
92.9 |
0.33 |
1.95 |
|
51 |
384 |
353 |
384 |
41.7 |
321 |
384 |
88.8 |
0.33 |
3.91 |
|
52 |
384 |
351 |
384 |
44.9 |
323 |
384 |
86.5 |
0.35 |
7.81 |
|
50 |
384 |
354 |
384 |
39.9 |
322 |
384 |
86.4 |
0.33 |
15.63 |
|
47 |
384 |
354 |
384 |
38.7 |
318 |
384 |
89.8 |
0.31 |
150 EMS |
|
111 |
384 |
211 |
384 |
482.5 |
212 |
384 |
478.6 |
0.50 |
KEY TO TABLES
$ = Cell counts (x105 cells/ml). Set up on previous day to 2 x 105 cells/ml unless otherwise stated in parenthesis.
%RSG = Relative Suspension Growth
RTG = Relative Total Growth
%V = Viability Day 2
§ or # = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis
A,B = Replicate cultures
CP = Cyclophosphamide
EMS = Ethylmethanesulphonate
MF§ = 5-TFT resistant mutants/106 viable cells 2 days after exposure
NP = Not plated, surplus to requirements
Ø = Not plated for viability or 5-TFT resistance
Nv = Number of wells scored, viability plates
Yv = Number of wells without colonies, viability plates
Ym = Number of wells without colonies, mutation plates
Nm = Number of wells scored, mutation plates
Applicant's summary and conclusion
- Conclusions:
- BMS-589152-01 induced no increases in mutant frequency at the TK locus in L5178Y cells that exceeded the GEF, and consequently was considered not mutagenic in this assay.
- Executive summary:
Introduction
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase (TK) locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, ICH S2R1 guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) except for the recommended dose level where the higher top dose level recommended in the OECD Test Guideline 490 was followed, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.
Methods
One main Mutagenicity Test was performed. In this main test, duplicate cultures of L5178Y TK+/-3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with eight concentrations of BMS-589152-01, in addition to vehicle (DMSO), and positive controls for 4 hours both in the absence and presence of metabolic activation (2% S9), and for 24 hours in the absence of metabolic activation.
The dose range of test item used in the main test was selected based on the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:
Mutagenicity Test
Group
Concentration of BMS-589152-01 (µg/mL) plated for mutant frequency
4 hour without S9
0.49, 0.98, 1.95, 3.91, 7.81, 15.63
4 hour with S9 (2%)
3.91, 7.81, 15.63, 31.25, 62.5, 125
24 hour without S9
0.49, 0.98, 1.95, 3.91, 7.81, 15.63
Results……..
The maximum concentration used in the Mutagenicity Test was limited by the presence of precipitate, which is indicative of test article maximum exposure. Precipitate was observed at >15.63 µg/mL in the absence of S9 metabolic fraction and at >125 µg/mL in the presence of S9 metabolic fraction. At the doses plated for mutant frequency, no marked toxicity was observed for any treatment condition.
Vehicle control mutant frequency values met assay acceptance criteria and positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. BMS-589152-01induced no toxicologically significant increases in mutant frequency at any of the concentrations evaluated, in any of the three exposure conditions.
Conclusion
BMS-589152-01 induced no increases in mutant frequency at the TK locus in L5178Y cells that exceeded the GEF, and consequently was considered not mutagenic in this assay.
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