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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Studies

Bacterial gene mutation study

A bacterial gene mutation study was carried out for BMS589152-01. The bacterial strains were exposed to concentrations of 50, 150, 500, 1500 and 5000 ug/plate both with and without metabolic activation. A positive increase in the mean number of revertants per plate was observed with 3 strains TA98, TA100 and TA1535 strain in the presence of S-9 mix. No positive increases were observed in any of the remaining tester strain/activation conditions combinations even up to the limit of 5000ug/plate.

In Vitro Gene Mutation

A mouse lymphoma assay test in L5178Y cells was carried out for BMS589152-01, according to Directive 88/302/EEC, Part B, OECD Guideline No.476. Three tests were carried out with different exposure concentrations which were determined based on a range finding experiment. (4h) without S9: 0.49, 0.98, 1.95, 3.91, 7.81, 15.63 µg/mL, with S9: 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL and 24 hour without S9 0.49, 0.98, 1.95, 3.91, 7.81, 15.63 µg/mL. both with and without metabolic activation. The results of the assay indicate that under the conditions of this study, the test substance did not increase the BMS-589152-01 induced no increases in mutant frequency at the TK locus in L5178Y cells and consequently was considered not mutagenic in this assay.

In vitro Cytogenicity Chromosomal Aberration Study in Mammalian Cells

A in vitro chromosomal aberration test was carried out to identify substances that cause structural chromosomal aberrations in cultured mammalian cells. Cells were exposed for 3 hours in the presence and absence of metabolic activation at concentrations of 125, 500 and 2000 ug/ml (without S9), 250, 500 and 2000 ug/ml (with S9) and 500, 1000 and 2000 ug/ml doses selected for metaphase analysis.

There was no evidence of increased presence of chromatid-type and chromosome-type aberrations and the test substance can be concluded as non- genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

thymidine kinase, TK +/-, locus

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.

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan. 24 to March 4th, 2005

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9

Test concentrations with justification for top dose:

Concentration range in the first test (with metabolic activation): 15.63 to 2000 µg/ml
Concentration range in the first test (without metabolic activation): 15.63 to 2000 µg/ml

Concentration range in the second test (without metabolic activation): 15.63 to 2000 µg/ml

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S-9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S-9

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours

Expression time:
48 hours

Selection time:
10-14 days for mutant frequency. Selective agent TFT

Fixation time:
Exposure period without metabolic activation:

First test:3 hours
Second test: 24 hours

Evaluation criteria:

Negative and positive are within ranges. Test substance positive if following are meet: 1) statistically significant increase in frequency of metaphases with aberrant chromosomes. 2). increase exceed the negative control range 3). increases are reproducable 4). not associated with large pH change or osmolality or extreme toxicity 5) evidence of dose relationship

Statistics:

Number of aberrant metaphase cells in each treatment group was compared with the solvent control values using a one tailed Fisher exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

test 1 without S-9: doses selected for metaphase analyis were 125, 1000 and 2000 ug/ml
test 1 with S-9: doses selected for metaphase analyis were 250, 1000 and 2000 ug/ml
test 2: doses selected for metaphase analyis were 500, 1000 and 2000 ug/ml

Remarks on result:

other: other: test 1

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

it was concluded that BMS 589152-02 did not demonstrate mutagenic potential in this in vitro gene mutation assay

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept. 30th to Nov. 14th, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

four histidine-requiring strains and one tryptophan-requiring strain

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post mitochondrial fraction (rat-liver S-9)

Test concentrations with justification for top dose:

Range finding experiment and main experiment carried out at concentrations from 0 to 5000 ug/plate with 10 concentrations tested. For the main study doses of 50, 150, 500, 1500 and 5000 ug/plate.

Vehicle / solvent:

Test solutions were prepared by dissolving BMS 589152-01 in sterile anhydrous grade dimethyl sulphoxide (DMSO).

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

use strain TA98 without S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

use strain TA100, TA 1535 without S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

use strain TA 1537 without S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

use strain TA98,TA 1537 and TA 100 with S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

use strain WP2uvrA and TA 1535 without S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

other: AF-2

Remarks:

use strain WP2uvrA without S-9

Details on test system and experimental conditions:

Experiment 1 involved the five strains with and without metabolic activation using seven concentrations. Normal plating treatment procedures followed. As a clear positive was observed in experiment 1, the same parameters were used for experiment 2 except 5 concentrations used.

Negative and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates were all acceptable.

Evaluation criteria:

Acceptance criteria for validity of assay : the mean negative control counts fell within normal ranges, the positive control chemical induced clear increases in revertant numbers confirming discrimination between different strains and an active S-9 preparation, and no more than 5 % of the plates were lost through contamination or some other unforeseen event. Evaluation criteria: test article would be considered mutagenic if: the assay was valid, and an increase in frequency in revertant colonies in excess of 2 or 3 fold depending on the tester starin was noted verses the concurrent solvent controls.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

>5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(> 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(> 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(> 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(> 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(> 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: positive with and without metabolic activation

It was concluded that BMS 587172-01 induced mutation in three histidine-requiring strains of Salmonella typhimurium when tested under conditions of this study. These conditions included treatment concentrations up to 5000 ug/plate in the absence and in the presence of a rat liver metabolic activation system (S-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Vivo Comet Assay

It is concluded that BMS-589152-01 has not shown any evidence of causing an increase in DNA strand breaks or cytotoxicity in the liver or duodenum of male Crl:CD(SD) rats when administered orally by gavage in this in vivo test procedure

In Vivo Mouse Micronucleus Study

It is concluded that BMS-589152-01 did not show any evidence of causing an increase in the induction of micronucleated immature erythrocytes or bone marrow cell toxicity in CD-1 mice, when administered orally by gavage in this in vivo test procedure.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo comet assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 February 2017 to 09 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

BMS-589152-01
Lot number: 15600T0001
Appearance: Beige-yellowish
Purity/Assay: 100.1% HPLC

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

After arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and given a unique tail mark. Each group was kept with the sexes separated in cages. The animals were maintained in a controlled environment with the thermostat and relative humidity target ranges set at 20 to 24°C and 40 to 70%
respectively. The room was illuminated by artificial light for 12 hours per day.
All animals were allowed free access to pelleted Envigo Teklad 2014C diet and tap water ad libitum.
Food, chew blocks and tap water are routinely analysed for quality at source. All animals were given access to small soft white untreated wood (ASPEN) chew blocks and a red plastic shelter for environmental enrichment, were acclimatised for a minimum of 5 days.

Route of administration:

oral: gavage

Vehicle:

1% methylcellulose

Details on exposure:

All animals in the vehicle control group, BMS-589152-01 dose groups and positive control group were dosed orally by gavage using a dose volume of 10 mL/kg.

Duration of treatment / exposure:

The BMS-589152-01 was administered on two occasions approximately 24 hours apart.

Frequency of treatment:

2 times

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Preliminary study

Dose / conc.:

500 mg/kg bw/day

Remarks:

Final Comet assay

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Final Comet Assay

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Final Comet Assay

No. of animals per sex per dose:

2 males/2females in the preliminary study
6 males per dose in the final comet assay

Control animals:

yes

Positive control(s):

Ethyl methanesulfonate (EMS) batch number BCBM2272V, was used as the positive control compound. A solution was prepared using purified water at a concentration of 20 mg/mL just prior to administration.
Vehicle Control group also included

Tissues and cell types examined:

Cell suspensions from the liver and duodenum were obtained from animals in the vehicle control group and in each of the BMS-589152-01 groups 3 hours after administration of the second dose. Cell suspensions from animals in the positive control group were obtained approximately 3 hours after a single dose.

Details of tissue and slide preparation:

Sections of the liver and duodenum were placed into ice cold mincing solution; all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method.
Comet slides were prepared from all cell suspensions. Sections of the liver and duodenum were stored in 10% buffered formalin and stored within Cell and Molecular Sciences.

Slide Preparation
Glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer. For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.
Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored refrigerated overnight prior to electrophoresis.

Evaluation criteria:

The following criteria were applied for assessment of assay acceptability: The concurrent vehicle control is considered acceptable for addition to the laboratory
historical vehicle control database. Concurrent positive control should induce responses that are compatible with those generated
in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
A maximum tolerated dose or maximum feasible dose has been achieved. Adequate numbers of cells and doses have been analysed

Statistics:

For Groups 1, 2, 3 and 4, Bartlett’s test for variance homogeneity (Bartlett 1937) was applied.
If this test was not significant at the 1% level, then parametric analysis was applied. The F1 approximate test was applied to Groups 1 to 4. This test is designed to detect significant
departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test
statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual
error mean square, MSE. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/MSE which can be compared with standard tables of
the F distribution with 1 and error degrees of freedom. If the F1 test was not significant at the 1% level, Williams' test for a monotonic trend was applied to compare Groups 2, 3 and 4 to
the vehicle control group; otherwise Dunnett's test (Dunnett 1955, 1964) was performed instead. If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test wasstill significant, then non-parametric tests were applied. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied to Groups 1 to 4. This test is designed to be used when the
main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test. If the H1 test was not significant at the 1% level, Shirley's test for a
monotonic trend (Shirley 1977) was applied to compare Groups 2, 3 and 4 to the vehicle control group, otherwise Steel's test (Steel 1959) was performed instead.
Groups 1 and 5, Bartlett’s test for variance homogeneity was applied. If this test was not significant at the 1% level, then parametric analysis was applied.
Statistical significance was at the 5% level.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

NO effects seen at 500, 1000 and 2000mg/kg/day

Toxicity:

no effects

Remarks:

no effects see up to limit dose of 2000mg/kg

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that BMS-589152-01 has not shown any evidence of causing an increase in DNA strand breaks or cytotoxicity in the liver or duodenum of male Crl:CD(SD) rats when administered orally by gavage in this in vivo test procedure

Executive summary:

This study was designed to assess the potential of BMS-589152-01 to induce DNA strand breaks in the liver and duodenum of male Crl:CD (SD) rats. Animals were treated with BMS-589152-01 orally on two occasions, the second dose being administered approximately 24 hours after the first dose and 3 hours before sampling. All animals in the vehicle control, positive control and BMS-589152-01 dose groups were dosed orally by gavage using a dose volume of 10 mL/kg. A preliminary toxicity test has shown that a dose of 2000 mg/kg/day, (the standard limit dose for the comet test) administered on two consecutive occasions approximately 24 hours apart was tolerated. On the basis of this result, dose levels of 500, 1000 and 2000 mg/kg/day were selected for the comet test. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, in line with current guidelines the test was performed using male animals only. The vehicle control group received 1% methylcellulose and the positive control group received Ethyl methanesulfonate at 200 mg/kg. Cell suspensions from the liver and duodenum were obtained from animals in the vehicle control group and in each of the BMS-589152-01 groups 3 hours after administration of the second dose. Cell suspensions from animals in the positive control group were obtained approximately 3 hours after a single dose. Following electrophoresis three slides per animal per tissue were analysed for comets. Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy. 150 morphologically normal cells were analysed for the presence of comets per animal per tissue. DNA strand breaks were assessed by comparing the mean and median % tail intensities (% TI) from BMS-589152-01 treated animals with vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. ‘Hedgehog’ or ‘Ghost’ cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts. Results No statistically significant increases in the % TI were observed in the liver or duodenum of male Crl:CD(SD) rats administered BMS-589152-01 at any dose level, compared with vehicle control values. The positive control compound, Ethyl methanesulfonate, produced statistically significant increases in the % TI when compared with vehicle control values. No Hedgehog “Ghost” cells were observed in either the liver or duodenum of male Crl:CD(SD) rats administered BMS-589152-01 at any dose level.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Decemeber 2004 - January 2005

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: bone marrow micronucleus test

Specific details on test material used for the study:

Identity: BMS-589152-01
Appearance: Tan, yellow solid
Storage conditions: Room temperature, in the dark
Batch number: PRF 02-06-2-43-1
Expiry date: 7 December 2006
Purity: 99.5% by area, 102.5% by weight

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

All animals used on this study were CD-1 mice, ca 8 weeks old at dosing. Males weighed between 28 and 32 grams and females weighed between 22 and 26 grams on despatch from Charles River UK Limited, Margate, Kent, England. On arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and tail marked. Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the thermostat and relative humidity target ranges set at 22±3˚C and 55±15%, respectively. Temperature and humidity were within
range throughout the study. The room was illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Food and tap water are routinely analysed for quality at source. All animals were housed in cages containing wood shavings (Lignocel Type ¾) and were given Nestlets for environmental
enrichment. The animals were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.

Route of administration:

oral: gavage

Vehicle:

1% w/v methylcellulose

Details on exposure:

Suspensions of the test substance were freshly prepared on each day of dosing in
1% w/v methylcellulose, obtained from Aldrich, batch number 01419EB.
Stability and homogeneity of the test substance and of the test substance in the vehicle were not
determined in this test and remain the responsibility of the Sponsor. Chemical analysis of dosing
formulations for achieved concentration was not performed in this study.
Mitomycin C, obtained from Sigma Chemical Company, batch number 103K0496, was used as
the positive control compound. It was prepared as a solution in purified water at a concentration
of 0.6 mg/ml, just prior to administration.
All animals in all groups were dosed orally by gavage with the standard volume of 20 ml/kg
bodyweight. Animals in negative and test substance groups were dosed on two occasions, the
second dose being administered approximately 24 hours after the first dose. The positive control
group was dosed on one occasion, approximately 24 hours prior to termination.

Duration of treatment / exposure:

24hr

Frequency of treatment:

Single dose

Post exposure period:

animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Seven males from the negative control and each of the test substance groups were sacrificed 24 hours after administration of the second dose. In addition five male animals in the positive control group were sacrificed 24 hours after a single dose. Animals not used for analysis were killed and discarded.

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

7 animals per dose

Control animals:

yes

Positive control(s):

Mitomycin C

Tissues and cell types examined:

The animals were killed by exposure to rising levels of carbon dioxide followed by cervical dislocation. Both femurs were dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone.

Details of tissue and slide preparation:

The bone marrow of both femurs from
each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum
by aspiration. The cells were sedimented by centrifugation, the supernatant was discarded and
the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension
was transferred to a glass microscope slide and a smear was prepared in the conventional manner
(Schmid 1976). Four smears were made from each animal. The prepared smears were fixed in
methanol (> 10 minutes). After air-drying at least one smear from each animal were rinsed in
purified water, stained in acridine orange solution (0.01 mg/ml using purified water) for 3
minutes, washed in purified water for 5 minutes and then rinsed in cold tap water for 2 minutes.
All stained slides were held in the dark at ca 4˚C for a minimum of 1 hour until required.
Immediately prior to scoring, slides were wet mounted with a glass coverslip using purified
water.
The stained smears were examined (under code) by fluorescent microscopy to determine the
incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. One smear
per animal was examined and the remaining smears were held temporarily in reserve in case of
technical problems with the first smear.
The proportion of immature erythrocytes for each animal among total erythrocytes was assessed
by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature
erythrocytes observed during assessment of this proportion was also kept as recommended by
Schmid (1976).

Evaluation criteria:

Acceptance criteria
The following criteria was applied for assessment of assay acceptability:
1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent with the laboratory historical negative control data.
3. Positive controls must show clear unequivocal positive responses.
4. The proportion of immature erythrocytes among total erythrocytes in treated groups is not less than 20% of the control value.

Criteria for assessing clastogenic/aneugenic potential
A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the vehicle control group (P<0.01); individual and/or group mean values should exceed the laboratory historical
control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated
immature erythrocytes for the group treated with the BMS-589152-01 are not significantly greater than incidences for the vehicle control group and where these values fall within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response. Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of immature erythrocytes (P<0.01)

Statistics:

The results for each treatment group were compared with the results for the vehicle control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided P-values are calculated
by permutation (StatXact, CYTEL Software Corporation, NC, USA). Comparison of several dose levels are made with the vehicle control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (StatXact, CYTEL Software Corporation,
NC, USA). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versionsof Wilcoxon’s sum of ranks test (Wilcoxon 1945) and Jonckheere’s test for trend (Jonckheere 1954).

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

up to highest dose tested of 2000mg/kg/day

Toxicity:

yes

Remarks:

small decrease in bodyweight and clinical signs of fast respiration only

Vehicle controls validity:

valid

Remarks:

1% w/v methylcellulose

Positive controls validity:

valid

BMS-589152-01 did not cause any statistically significant increases in the number of micronucleated immature erythrocytes compared to vehicle control values.

BMS-589152-01 did not cause any substantial increases in the incidence of micronucleated mature erythrocytes compared to vehicle control values.

BMS-589152-01 failed to cause any significant decreases in the proportion of immature erythrocytes.

Conclusions:

It is concluded that BMS-589152-01 did not show any evidence of causing an increase in the induction of micronucleated immature erythrocytes or bone marrow cell toxicity in CD-1 mice, when administered orally by gavage in this in vivo test procedure.

Executive summary:

This study was designed to assess the potential induction of micronuclei of BMS-589152-01 in bone marrow cells of treated CD-1 mice. Mice were treated with BMS-589152-01 orally on two occasions approximately 24 hours apart. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg (the standard limit dose for the micronucleus test) was tolerated. This level was therefore selected as an appropriate maximum for use in the micronucleus test. On the basis of results from the preliminary toxicity test, dose levels of 500, 1000 and 2000 mg/kg were selected for the micronucleus test. Following the preliminary toxicity test, no substantial differences in toxicity were observed between the sexes, therefore in line with current guidelines, the main test was performed using male animals only. All animals were dosed orally by gavage. The negative control group received the vehicle, 1% w/v methylcellulose and the positive control group received mitomycin C at 12 mg/kg. Bone marrow smears were obtained from seven male animals in the negative control and each of the test substance groups 24 hours after administration of the second dose. In addition bone marrow smears were obtained from five male animals in the positive control group 24 hours after a single dose. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept. In addition, blood samples were collected from three male mice 1 hour after the first administration of BMS-589152-01 at 2000 mg/kg. However, although resultant plasma samples were obtained during the in-life phase of this study, it was not possible to detect the presence of BMS-589152-01 and confirm systemic exposure to BMS-589152-01 as a validated analytical procedure for this test material was not achieved. No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with BMS-589152-01, compared to vehicle control values. The positive control compound, mitomycin C, produced significant increases in the frequency of micronucleated immature erythrocytes (P<0.01)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in-vitro and in-vivo studies conducted on the test substance it can be concluded that BMS 589152 -01 is non mutagenic