.alpha.-D-Glucopyranoside, methyl 1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, compd. with 2-butyne-1,4-diol (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June, 2015 - 27 October, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(2014)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Solvate of (2S,3R,4S,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(hydroxymethyl)-2-methoxytetrahydro-2H-pyran-3,4,5-triol with 2-butyne-1, 4-diol (1:1)

Cas Number:

960404-59-5

Molecular formula:

C26 H33 Cl O9

IUPAC Name:

Solvate of (2S,3R,4S,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(hydroxymethyl)-2-methoxytetrahydro-2H-pyran-3,4,5-triol with 2-butyne-1, 4-diol (1:1)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): BMS-587319-03
- CAS number: 960404-59-5
- Appearance: White powder
- Storage condition of test material: stored at room temperature (18°C to 24°C)

Test animals

Species:

mouse

Strain:

other: Crl:CD-1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: Range finding: 50 days; main test: 51 days.
- Weight at main study initiation: males: 25.1 - 30.9 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Upon arrival, all animals were housed 2 to 3 per cage by sex for approximately 3 days (± 1 day) following receipt. Thereafter, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: free access to basal diet, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water: free access to Reverse osmosis-treated (on-site) drinking water
- Acclimation period: 5 days (range finding), 13 days (main test)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): set to maintain 22 ± 3
- Humidity (%): set to maintain 50 ± 20
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: arachis oil (peanut oil)
- Justification for choice of vehicle: no data
- Concentration of test material in vehicle: 6.25, 12.5 and 25 mg/mL. The dosing formulations were adjusted for a purity of 97.7%.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article formulations were weight/volume (test article/vehicle) mixtures. For each phase of the study, the test article formulations were prepared on the day of dose administration as single formulations for each dosage level. The test article formulations were stirred continuously after preparation and during the dose administration procedures.

DOSING VOLUME:
The dosing volume for the test substance was 20 mL/kg body weight, and for the positive control 10 mL/kg body weight.

Duration of treatment / exposure:

Treatment:
Vehicle and test substance groups: approx. 18-24 hours
Positive control group: approx. 24 hours

Frequency of treatment:

Vehicle and test substance groups: once daily for 3 consecutive days
Positive control: single dose on one day (study day 2)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
125 mg/kg/bw/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg/bw/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg/bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CPS)
- Route of administration: Oral
- Doses / concentrations: 60 mg/kg/day

Examinations

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-The high (top) dose selected was expected to be the highest non-lethal dose based on the results of the dose range-finding phase. The mid- and low-dose levels were one-half and one-quarter of the selected high dose, respectively.

DETAILS OF SLIDE PREPARATION:
- Bone marrow was aspirated or flushed 2 to 3 times from each femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 4 appropriately labeled, clean microscope slides. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time.
The slides were stored and shipped at ambient temperature to WIL Research Skokie (8025 Lamon Avenue Skokie, IL 60077, USA) for analysis. Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution.

METHOD OF ANALYSIS:
Two separate evaluations were made for each slide: 1) a total of 500 erythrocytes (both polychromatic erythrocytes [PCEs] and normochromatic erythrocytes [NCEs]) per animal were counted and the PCE:total erythrocytes [TE] ratio was determined; and 2) the number of micronucleated PCEs from a total of 4000 PCEs was scored per animal.

Evaluation criteria:

The test article may be considered positive for induction of micronuclei if a doserelated increase in the percentage of PCEs with micronuclei is observed and a
statistically significant increase relative to the vehicle is seen at one or more dose levels. Statistical significance will not be the only factor in determining a positive response. Other criteria, such as historical control values, which take into account the biological relevance of the results, will be considered in the final evaluation.

The test article is considered to be negative for inducing the formation of micronuclei in PCEs if no statistically significant increase is evident at all dose
levels in the percentage of PCEs with micronuclei when compared to the concurrent vehicle.

Cases that do not clearly fit into the positive or negative criteria may be judged equivocal. In these cases, the Principal Investigator, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results. As a general rule the biological relevance of any result will be considered first.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the vehicle control group by sex.
Body weight, body weight change, and food consumption data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the vehicle control group. The positive control data were evaluated using the two-sample t-test (Sokal and Rohlf, 1981) and compared to the vehicle control group.

Analysis of the slides:
The percentages of micronucleated cells in PCEs and in the ratio of PCEs to TEs for the test article-treated and vehicle control group were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p ≤ 0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the vehicle control group (Group 1). In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA. Statistical significance was assessed at a 95% confidence level (p≤0.05).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg/bw/day
- Clinical signs of toxicity in test animals:
One male in the 1000 mg/kg/bw/day group and all animals in the 2000 mg/kg/bw/day group were found dead and/or euthanized for humane considerations; all deaths were considered to be test article-related. One male in the 1000 mg/kg/bw/day group was found dead on study day 1. There were no test article-related clinical signs noted for this animal. In the 2000 mg/kg/bw/day group, 3 animals were euthanized for humane considerations and 2 animals were found dead on study day 0 at approximately 4 hours after dose administration, and one male was found dead on study day 1. Test
article-related clinical signs noted in the 2000 mg/kg/bw/day group included prostration, flattened body, pale and/or cool extremities, cool body, hypoactivity, decreased respiration rate, yellow material on various body surfaces (around the mouth, hindlimb[s], ventral neck and/or trunk, anogenital and/or urogenital areas, and/or base of the tail), partial closure of the eye(s), mucoid feces, and/or diarrhea. There were no macroscopic findings noted for these animals. All other animals survived to the scheduled euthanasia.
Test-article related clinical signs noted in the 1000 mg/kg/bw/day group animals that survived to the scheduled euthanasia included pale and/or cool extremities on study days 1 and 2, yellow material around the mouth and/or on the anogenital and/or urogenital areas, diarrhea on study day 0. No other test article-related clinical signs were observed.
Test article-related effects on body weights were noted in the 250 and 500 mg/kg/bw/day group females and the 1000 mg/kg/bw/day group males and females. Body weight losses were noted for 1 of 3 females in the 250 and 500 mg/kg/bw/day group and the two surviving males and 2 of the 3 females in the 1000 mg/kg/day group from study days 0 to 2. Based on the results of the range-finding phase, dosage levels of 125, 250, and 500 mg/kg/bw/day were selected for evaluation in the micronucleus assay (definitive phase).
Males were chosen for the definitive phase of this study because they exhibited a slightly greater decrease in body weight gains and an increased incidence of test article-related post-dose clinical observations during the range-finding phase.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The test substance did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) compared to the vehicle control group. No bone marrow cytotoxicity was noted in any male mice at any test substance dosage level.
- Ratio of PCE/NCE (for Micronucleus assay): At 0, 125, 250, and 500 mg/kg/bw/day, the mean %MN-PCE frequencies in the bone marrow of male mice were 0.05%, 0.24%, 0.21%, and 0.15%, respectively.
- Appropriateness of dose levels and route: The high (top) dose selected was the highest non-lethal dose based on the results of the dose range-finding phase. The selected route of administration for this study was orally by gavage because this is the recommended route of exposure according to testing guidelines.

Any other information on results incl. tables

- Clinical signs of toxicity in test animals:

Test article-related clinical observations noted in the 500 mg/kg/bw/day group included clear material around the mouth and/or on the ventral neck on study days 0 and 2, and yellow material on various body surfaces (hindlimb[s], ventral neck, anogenital area, and urogenital areas), and unkempt appearance on study days 1 and 2.

Test article-related clinical observations noted in the 250 mg/kg/bw/day group included clear material on the forelimb(s), ventral neck, and/or ventral trunk on study day 2. All other clinical findings in the test article-treated groups were noted with similar incidence in the vehicle control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory mice of this age and strain.

- Test article-related effects on body weights were noted in the 500 mg/kg/bw/day group. Statistically significant mean body weight losses were noted in the 500 mg/kg/bw/day group from study days 0 to 2 compared to the vehicle control group. As a result, mean body weights were 9.5% and 9.8% lower than the vehicle control group on study days 2 and 3, respectively. There were no other test article-related effects on body weight.

- Test article-related effects on food consumption were noted in the 500 mg/kg/bw/day group. Lower mean food consumption values were noted in the 500 mg/kg/bw/day from study days 0 to 3 and correlated with body weight losses noted for this group. There were no other test article-related effects on food consumption.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
An in vivo Micronucleus test with BMS-587319-03 was performed according to OECD 474 guideline and GLP principles, in male CD-1 mice. It is concluded that BMS-587319-03 is not clastogenic under the conditions of the Micronucleus test.

Executive summary:

In an in vivo Micronucleus test, male CD-1 mice were exposed once daily for 3 consecutive days to 125, 250 and 500 mg/kg/bw/day of BMS-587319-03 (suspended in arachis oil (peanut oil)) according to OECD 474 guideline and GLP principles.

The test substance did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) compared to the vehicle control group. No bone marrow cytotoxicity was noted in any male mice at any test substance dosage level. Reliable negative and positive controls were included.

Based on the results, it is concluded that BMS-587319-03 is not clastogenic under the conditions of the Micronucleus test.