1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

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:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - - Environment Protection Agency

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese guideline: Eisei No. 127 - - Ministry of Health and Welfare

Test guideline 7

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

Test material information

Constituent 1

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

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.

Controls

Controls 1

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

Controls 2

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.