1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo-1-piperidinyl)phenyl]-, ethyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of the test substance was assessed on histidine dependent auxotrophic mutants of Salmonella typhimurium strains TA1535, TA1537, TA98 AND TA 100, and on a tryptophan dependent mutant of Escherichia coli WP2uvrA/Pkm101 in an Ames assay ( OECD guideline 471).There was no evidence of toxicity or mutagenic activity seen at any concentration of the test material in either repeat mutation test. Under ther test conditions, BMS 589154-01 showed no evidence of mutagenic activity in this bacterial system.

In a chromosome aberration study, cultured Chinese Hamster Ovary cells ( CHO-WBL) were exposed to different concentrations of BMS-589154-01 (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles.

BMS-589154-01 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. No statistically significant increase in the percent cells with numerical aberrations (polyploidy and/or endoreduplication) was observed in any test article treated group. Based on the results, BMS-589154-01 was not clastogenic in CHO-WBL cells when evaluated under conditions and at the maximum concentrations recommended by international guidelines for in vitro cytogenetic studies.

When tested in a mammalian gene mutation assy in accordance with OECD Guideline 490, the test item, BMS-589154-01 did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 April 2016 - 13 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) guideline S2(R1)

Version / remarks:

09 November 2011

Deviations:

no

GLP compliance:

yes

Remarks:

In compliance with the US FDA CFR Title 21, Part 58, with exception: no concentration analysis of dose formulations.

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The stock CHO-WBL cells were obtained from the Genetic Toxicology Laboratory at Pfizer, Inc. (Groton, CT)
- Number of passages if applicable: Cells were used at passage 22 for the range-finding assay, passage 4 for the aberration assay, passage 16 for the repeat aberration assay, and passage 3 for the 2nd repeat aberration assay.
- Modal number of chromosomes: modal chromosome number of 21
- Normal (negative control) cell cycle time: 12- to 14-hour cycling time.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cultures for the range-finding assay and Trial 1 were prepared by seeding with 1.0-1.5 × 10^6 CHO cells per 75 cm2 flask in McCoy’s complete media (McCoy’s medium supplemented with a final concentration of 10% (v/v) heat-inactivated fetal bovine serum, 2mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin). Cultures for Trials 2 and 3 were prepared by seeding with 0.9 – 1.2 × 10^6 CHO cells per 75 cm2 flask in McCoy’s complete media. All cultures were incubated at 36°C to 38°C in vented flasks in a humidified atmosphere of 4%
to 6% CO2 for approximately 24 hours prior to the initiation of treatment.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: The stock cells were found to be free of mycoplasma contamination
- Periodically checked for karyotype stability: Their karyotype was shown to have a modal chromosome number of 21 and a 12- to 14-hour cycling time.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3hr exposure; 20 hr fixation: 0.955, 1.91, 3.82, 7.64, 15.3, 30.6, 61.1, 122, 245 and 489 μg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 0.955, 1.91, 3.82, 7.64, 15.3, 30.6, 61.1, 122, 245 and 489 μg/mL
First cytogenetic test:
Without S9-mix, 3 hr exposure time, 20 hr fixation time: 16, 32, 64, 128, 250 and 489 μg/mL
With S9-mix, 3 hr exposure, 20 hr fixation time: 16, 32, 64, 128, 250 and 489 μg/mL
Without S9-mix, 20 hr exposure time, 20 hr fixation time: 16, 32, 64, 82, 103, 128, 160, 200, 250, 313 and 489 μg/mL
The following concentrations were selected for chromosome aberration analysis:
Without S9-mix, 3 hr exposure; 20 hr fixation: 64, 128 and 250 μg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 16, 160 and 200 μg/mL
With S9-mix, 3 hr exposure; 20 hr fixation: 128, 250 and 489 μg/mL

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choice of vehicle: A stock solution of the substance was prepared in DMSO at a concentration of 48.9 mg/mL on the day of each assay. The lower concentrations were prepared by serial dilution with DMSO to attain the appropriate concentration for testing.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): range-finding assay and Trial 1 were prepared by seeding with 1.0-1.5 × 10^6 CHO cells per 75 cm2 flask; for Trials 2 and 3 were prepared by seeding with 0.9 – 1.2 × 10^6 CHO cells per 75 cm2 flask

DURATION
- Preincubation period: 24 hours
- Exposure duration: 3 hr (with and without S9-mix) and 20 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hr

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): DiffQuik solution

NUMBER OF REPLICATIONS: duplicates in one experiment

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At harvest, the culture medium was removed and the cells were washed with phosphate buffered saline. The exposed monolayer of cells was trypsinized and an aliquot was removed for assessment of cell number via Coulter counter. Cells were then concentrated by centrifugation and resuspended in hypotonic solution (0.075 M KCl) and washed at least once with fixative (using an approximately 3:1 methanol:glacial acetic acid mix) before being stored in the refrigerator. These fixed cells were washed once more with fresh fixative prior to slide preparation. The final concentrated cell suspension was dropped on clean, cold wet glass slides, dried prior to staining with DiffQuik solution at room temperature, and coverslipped. At least two slides were prepared for each culture.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Structural chromosomal aberrations were analyzed from a total of 300 metaphase cells (150 from each culture) or ≥ 50 aberrant cells (25 aberrant cells from each culture) from each test article concentration or control.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity, as assessed using relative population doubling from Coulter counts, was measured in all cultures. Cells treated with test article or positive controls were compared with vehicle control cultures.
- Any supplementary information relevant to cytotoxicity: In addition, all cultures were examined visually for signs of cytotoxicity (i.e., visible cell abnormalities), changes in cell morphology, pH change and precipitates at the time of dosing, at the end of the 3-hour treatment (at wash), and prior to harvest.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

As a guideline, the test article would be considered positive for inducing chromosomal aberrations if a significant increase (p ≤ 0.05) in the mean percent of cells with chromosomal aberrations is observed at one or more dose levels. If a significant increase is seen at one or more dose levels, a dose-response should be observed. A response would be considered statistically significant for dose-response trend in the Cochran-Armitage test if p ≤ 0.05. At least one concentration should be associated with an increase that is outside the historical control range of previous vehicle control treated cultures.
The test article would be considered negative for inducing chromosomal aberrations if no statistically significant increase (p ≤ 0.05) is observed in the mean percent of cells with chromosomal aberrations at any of the test concentrations and there is no concentration-related increase when evaluated in the Cochran-Armitage test. All results should be comparable to the historical control range of previous vehicle control treated cultures.
Cases which do not clearly fit the positive or negative criteria may be judged equivocal (e.g., a response is statistically significant but not dose dependent). In these cases, the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results (e.g., reproducibility of response).

Statistics:

After completion of microscopic analyses, data were decoded and a One-tailed Fisher’s Exact test was performed on the total number of cells with structural aberrations and the total number of cells with more than one chromosome aberration comparing the treated cells to the results obtained from the relevant control group. The detection of dose-response trends was carried out using the Cochran-Armitage test. The same statistical analysis was performed for cells exhibiting polyploidy and/or endoreduplication.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No.
- Precipitation: Precipitates were observed at the end of test article treatment at ≥ 200 μg/mL in the 20-hour treatment without metabolic activation, ≥ 250 μg/mL in the 3-hour treatment without metabolic activation, and at 489 μg/mL in the 3-hour treatments with metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Population doubling in each of the range-finding treatments was below 1.5. However, the low growth of the cultures does not impact the study overall as solubility limits were able to be established from these trials.
Precipitates were observed at ≥ 245 μg/mL in each treatment at the end of test article treatment. Changes in cell morphology and the pH (as indicated by color change of the media) were not observed in any treatment at the end of test article treatment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The validity of this assay was demonstrated since positive controls (mitomycin C [MMC] and cyclophosphamide [CP]) induced statistically significant increases in the frequencies of structural aberrations and the vehicle control values were within the historical vehicle control range of 0% to 4% for the 3-hour exposure without metabolic activation, 0% to 5% for the 3-hour exposure with metabolic activation and 0% to 4% for the 20-hour exposure without metabolic activation.

3-Hour Treatment without Metabolic Activation
Percent Cells with Aberrations (%)
Vehicle PositiveControl a
Mean 0.53 48.35
Standard Deviation 0.88 14.88
95% Confidence Interval Range 0.00-2.25 19.19-77.51
Range 0.00-4.00 27.30-83.30

20-Hour Treatment without Metabolic Activation
Percent Cells with Aberrations (%)
Vehicle Positive Control a
Mean 0.58 36.21
Standard Deviation 0.91 14.89
95% Confidence Interval Range 0.00-2.36 7.04-65.39
Range 0.00-4.00 4.00-65.20

3-Hour Treatment with Metabolic Activation
Percent Cells with Aberrations (%)
Vehicle Positive Control b
Mean 1.05 42.60
Standard Deviation 1.24 20.68
95% Confidence Interval Range 0.00-3.48 2.06-83.14
Range 0.00-5.00 15.00-93.80
a. Mitomycin C (MMC)
b. Cyclophosphamide (CP)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:
In the 3-hour treatment without metabolic activation, the concentrations selected for chromosome aberration analysis were as follows (with percent cytotoxicity by RPD): 64.0 μg/mL (6%), 128 μg/mL (0%) and 250 μg/mL (3%; lowest precipitating dose).
In the 20-hour treatment without metabolic activation, the concentrations selected for chromosome aberration analysis were as follows (with percent cytotoxicity by RPD): 16.0 μg/mL (1%), 160 μg/mL (37%) and 200 μg/mL (47%; lowest precipitating dose).
In the 3-hour treatment with metabolic activation, the concentrations selected for chromosome aberration analysis were as follows (with percent cytotoxicity by RPD): 128 μg/mL (3%), 250 μg/mL (6%) and 489 μg/mL (18%; lowest precipitating dose).

- Other observations when applicable: no statistically significant increase in the percent cells with numerical aberrations (polyploidy and/or endoreduplication) was observed in any test article treated group.

Conclusions:

A chromosome aberration study with BMS-589154-01 was performed according to OECD 473 guideline and GLP principles, in cultured Chinese Hamster Ovary (CHO-WBL) Cells in one experiment. It is concluded that BMS-589154-01 is not clastogenic in cultured mammalian cells.

Executive summary:

In a chromosome aberration study, cultured Chinese Hamster Ovary cells ( CHO-WBL) were exposed to different concentrations of BMS-589154-01 (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the cytogenetic assay, BMS-589154-01 was tested up to and including the precipitating concentration of 250 µg/mL for a 3 h exposure time with a 20 h fixation time in the absence of S9 -mix, and up to and including the precipitating and cytotoxic concentration of 489 µg/mL for a 3 h exposure time with a 20 h fixation time in the presence of S9 -mix.

In the absence of S9 -mix, BMS-589154-01 was tested up to and including the precipitating and cytotoxic concentration of 200 µg/mL for a 20 h continuous exposure time. BMS-589154-01 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. No statistically significant increase in the percent cells with numerical aberrations (polyploidy and/or endoreduplication) was observed in any test article treated group.

Based on the results, BMS-589154-01 was not clastogenic in CHO-WBL cells when evaluated under conditions and at the maximum concentrations recommended by international guidelines for in vitro cytogenetic studies.

Reference 2

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:

29 October to 18 November, 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)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Remarks:

OECD ENV/MC/CHEM(98)17

Type of assay:

bacterial reverse mutation assay

Target gene:

The mutagenic potential of the test substance was assessed on histidine dependent auxotrophic mutants of Salmonella typhimurium strains TA1535, TA1537, TA98 AND TA 100, and on a tryptophan dependent mutant of Escherichia coli WP2uvrA/Pkm101 (cm891)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aclor 1254-induced rat liver

Test concentrations with justification for top dose:

Range-finding concentrations: 1, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Second, pre-incubation test concentrations: 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

solvent; dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

vehicle controls used in parallel with the test material)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

In the presence of S9 mix

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

In the range finding first test:
0.1ml of the test solution, positive control or negative control were placed in glass vesssels. 0.5ml of S9 mix or 0.5ml of 0.1M pH7.4 phosphate buffer were added, followed by 0.1ml of a 10 hr bacterial culture and 2ml agar containing histidine (0.5 mM), biotin (0.5 mM) and tryptophan (0.5 mM).
The mixture was shaken and overlaid onto previously prepared petri dishes containing 25ml minimal agar. Three petri dishes per concentration were incubated at 37°C for ca. 72 hours.The number of revertant colonies were then counted.

In the second test:
Plates were incubated at 37°C for 30 minutes.

Evaluation criteria:

Evaluation criteria: test article would be considered mutagenic if it produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA 1537) the concurrent solvent/vehicle controls.
The positive responses described above were reproducible in an independent assay.

Statistics:

Mean and standard deviation of the plate counts for each treatment were determined

Species / strain:

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

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:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks on result:

other: all strains/cell types tested

The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.

Conclusions:

Interpretation of results
negative

Under ther test conditions, BMS 589154-01 showed no evidenceof mutagenic activity in this bacterial system.

Executive summary:

No evidence of toxicity or mutagenic activity was seen at any concentration of the test material in either mutation test.

Under ther test conditions, BMS 589154-01 showed no evidenceof mutagenic activity in this bacterial system.

Positive controls (with S9 mix where required) induced substantial increasses in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2017- 31st October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

according to guideline

Guideline:

other: ICH S2R1 (adopted June 2012)

Deviations:

yes

Remarks:

the higher top dose level recommended in the OECD Test Guideline 490 is followed

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from 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.

Metabolic activation:

with and without

Metabolic activation system:

Lot No. PB/BNF S9 20/08/17 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test:

Species: HSD:CD Sprague Dawley Liver S9 fraction Inducer: Phenobarbital/B-Naphtha 80/100mg/kg

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM),
glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

4 hr: 0, 12.5, 25, 50, 100, 200, 300, 400, 500 µg/mL
24hr: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 187.5, 250 µg/mL

Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 2000 µg/mL or 10 mM, whichever is the lowest
concentration.
ii) The presence of precipitate regardless of where test item-induced toxicity was
observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to
20% survival (the maximum level of toxicity required). This optimum upper level of
toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al.,
2002).

Vehicle / solvent:

Solvent (DMSO)
(Fisher batch 1684307 Expiry 19.09.22 Purity >99%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

see below

Rationale for test conditions:

see below

Evaluation criteria:

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.

Statistics:

see below

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see below

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 1.95 to 500 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (µg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
1.95	136	101	86
3.91	89	102	85
7.81	107	94	89
15.63	107	99	87
31.25	119	106	62
62.5	124	106	32
125	101	99	21
250	87	86	0p
500	0p	5p	0p

p = precipitate of test item at the end of exposure

In the all three exposure groups there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. A precipitate of the test item was observed at 500 µg/mL in both 4-hour exposure groups. A precipitate of the test item was observed at 250 µg/mL in the 24- hour -S9 exposure group. In the subsequent mutagenicity experiments the maximum dose level was limited by a combination of test item induced toxicity and precipitate.

Mutagenicity Test

A summary of the results from the test is presented in Table 1 (attached).

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 10. (attached) In the all three exposure groups, there was evidence of marked test item induced toxicity, as indicate by the %RSG and RTG values (Tables 3, 6 and 9). There was no evidence of marked reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred (Tables 3, 6 and 9). Near to optimum levels of toxicity were achieved in in the 4-hour-S9 and 24-hour –S9 exposures. Optimum levels of toxicity were not achieved in the 4-hour +S9 as the dose level where optimum levels of toxicity occurred (500 µg/mL) was excluded due to excessive precipitate. The dose levels of 500 and 250 µg/mL in the 4-hour –S9 and 24-hour –S9 respectively, were not plated out for 5-TFT resistance and % viability due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances (Tables 3, 6 and 9).

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 (Tables 3, 6, and 9).

The test item did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups including several dose levels beyond the acceptable level of toxicity. A precipitate of the test item was observed at and above 400 µg/mL in both 4-hour exposure groups. Also a precipitate of the test item was observed at 250 µg/mL in the 24-hour –S9 exposure. The numbers of small and large colonies and their analysis are presented in Tables 4, 7 and 10.

Conclusions:

The test item, BMS-589154-01 did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of
126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the negative results found in the in vitro battery of Ames test, mammalian chromosome aberration and mammalian gene mutation, there is no justification to test the substance in an in vivo genotoxicity model. Therefore, there is no concern for mutagenic potential and the hazard classification critieria for germ cell mutagenicity as set out in 1272/2008/EC (as amended) are not met.