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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

EC Number:

611-033-0

Cas Number:

536759-91-8

Molecular formula:

C22H20N4O6

IUPAC Name:

ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

powder; stored at room temperature in the dark

Specific details on test material used for the study:

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Preliminary study

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

Final Comet assay

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Final Comet Assay

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Test results

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

Applicant's summary and conclusion

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.