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

Genetic toxicity: in vitro

Administrative data

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

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:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

BMS-589154-01

IUPAC Name:

BMS-589154-01

Details on test material:

Identification: BMS-589154-01
Description: Off white powder
Batch: 1M49303NA
Purity: Not supplied
Structure: Please see Attachment 1
Expiry / Retest Date: Not supplied
Storage Conditions: Room temperature in the dark

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.