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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2015 - September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD/EC guidelines and in compliance with GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(March 1996)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

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
- Physical state: White powder
- Storage condition of test material: At room temperature.

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-12 weeks.
- Weight at study initiation: males: 301-337 grams; females: 202-244 grams
- Fasting period before study: no
- Housing: Pre-cohabitation: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages. Cohabitation: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages. Post-cohabitation: Males were housed in their home cage (Macrolon plastic cages, with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages. Lactation: Pups were kept with the dam until termination in Macrolon plastic cages. During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.3
- Humidity (%): 41 - 73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 July 2015 To: 22 September 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Dose volume was 5 mL/kg bw/day. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe. Based on information provided by the sponsor.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (31 July 2015; Study Day 3), according to a validated method (Project 509278). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29-33 days, i.e. 2 weeks prior to cohabitation, during cohabitation and up to the day prior to scheduled necropsy.
Females were exposed for 40-55 days, i.e. during 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk and/or from exposure to maternal urine/feces.

Frequency of treatment:

Once daily for 7 days per week.

Doses / concentrations

Remarks:

Doses / Concentrations:
1.5, 15, 150 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight, with a maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters examined: according to guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes (overnight, with a maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters examined: according to guidelines

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected at the end of the cohabitation period (i.e. overnight prior to scheduled necropsy of males)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: During the sampling period animals were deprived of food but water was available.
- Parameters examined: according to guidelines

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period.
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (according to guidelines)
HISTOPATHOLOGY: Yes (according to guidelines)
organ weights: according to guidelines

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

statististically significant effects observed in high dose females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

statistically significant effects observed in high dose animals.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

statistically significant effects observed in high dose animals.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

effects were observed on liver, prostate gland, thymus and uterus.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

thyroid gland of both sexes, trachea, liver, spleen and adrenal glands of females

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period. No toxicologically relevant clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among all animals at 150 mg/kg bw/day was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence.

BODY WEIGHT AND WEIGHT GAIN:
No toxicologically relevant changes in body weight and body weight gain were noted. Males at 150 mg/kg showed a statistically significant reduced body weight gain during the mating period. Since the mean weight gain difference to control males over the treatment period was only 4%, food intake remained normal and general health condition was normal, this was considered not to represent a toxicologically meaningful difference.

FOOD CONSUMPTION:
No changes in food consumption before or after correction for body weight were noted that were considered to be related to treatment. Any variations noted in food intake did not show a dose-related trend and/or were not consistently seen as treatment-duration increased.

HAEMATOLOGY:
In high dose females statistically significant changes compared to controls included higher relative neutrophil counts, lower relative lymphocyte counts, higher relative reticulocyte count, higher red cell distribution width, and lower haemoglobin concentration.

CLINICAL CHEMISTRY:
In high dose animals statistically significant changes compared to controls included higher total protein (not statistically significant for females), higher cholesterol (not statistically significant for males), higher calcium, higher ALAT in females, lower chloride in females.

URINALYSIS:
In high dose animals statistically significant changes compared to controls included higher urinary volume, higher glucose, higher sodium excretion in males, higher urinary pH in females. The lower calcium concentration in females at 1.5, 15 and 150 mg/kg bw/day showed no dose-related trend. Also, when corrected for urinary volume, calcium excretion was not statistically different to controls at 1.5 and 150 mg/kg bw/day. Also, the lower potassium concentration in males at 150 mg/kg bw/day, was not statistically different to controls when corrected for urinary volume. These variations were therefore considered not toxicologically relevant.

NEUROBEHAVIOUR:
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

ORGAN WEIGHTS:
Statisticaly significant higher liver weights were observed in males at 150 mg/kg bw/day (relative to body weights) and females (absolute and relative to body weights); mean relative weight was approximately 17 and 37% higher than the control mean for males and females, respectively.
Statistically significant lower prostate gland weight (absolute and relative) was observed at 15 and 150 mg/kg bw/day (no clear dose-related trend); mean relative weight was approximately 23 and 19% lower than the control mean at 15 and 150 mg/kg bw/day, respectively.
Statistically significant lower thymus weight was observed in high dose females (absolute and relative); mean relative weight was approximately 29% lower than the control mean.
Statistically significant higher uterus weight was observed at 15 mg/kg bw/day (relative) and 150 mg/kg bw/day (absolute and relative); mean relative weight was approximately 41 and 40% higher than the control mean at 15 and 150 mg/kg bw/day, respectively.
The remaining organ weight differences observed, including those that reached statistical significance (increased relative weight of male kidneys), were considered related to the slightly lower terminal body weight and had no histopathological correlates. These variations were therefore considered unrelated to the administration of BMS-587319-03.

GROSS PATHOLOGY:
There were no test-item related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related microscopic findings were present in the thyroid gland of both sexes and trachea, liver, spleen and adrenal glands of females:
Thyroid glands: Hypertrophy of follicular cells was recorded at a slightly increased severity in males at 150 mg/kg bw/day and at an increased incidence and severity in females at 150 mg/kg bw/day. The incidences and severities recorded for the remaining dose groups are within background pathology for rats of this age and strain.
Trachea: Hypertrophy of the ciliated epithelium was recorded at a minimal or slight degree in 4/5 females at 150 mg/kg bw/day.
Liver: Hepatocellular hypertrophy was recorded at a minimal or slight degree in 2/5 females at 150 mg/kg bw/day.
Spleen: An increased incidence and/or severity of extramedullary hematopoiesis (up to slight at 1.5 mg/kg bw/day, up to moderate at 15 and 150 mg/kg bw/day) was present in females starting at 1.5 mg/kg bw/day.
Adrenal glands: An increased incidence and severity (up to slight degree) of hypertrophy of the zona fasciculata was recorded in females at 150 mg/kg bw/day.

Effect levels

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Accuracy of the preparations was between 100% and 103%. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Homogeneity coefficient of variation was 0.27 -0.75%. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Analysis of Group 2 formulations after storage yielded a relative difference of 0.6%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:

Based on test-substance related effects observed in high dose animals on haematological and clinical biochemical parameters, urinalysis, organ weight and microscopic findings, the NOAEL for BMS-587319-03 in this study is 15 mg/kg bw/day.

Executive summary:

BMS-587319-03 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 1.5, 15 and 150 mg/kg bw/day. Males were exposed for 2 weeks prior to cohabitation, during cohabitation, and up to termination (for 29-33 days). The females were exposed for 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and at least 4 days of lactation (for 40-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Treatment up to 150 mg/kg bw/day was well-tolerated; no mortality occurred, no treatment-related changes during functional observation tests were seen, and no toxicologically relevant clinical signs or changes in body weights and food intake were noted. Test-substance related effects were observed in high dose animals and included changes in haematological parameters (Hb, RDW, counts of neutrophils, lymphocytes, reticulocytes), clinical biochemical parameters (prot, chol, Ca, Cl, ALAT), urinalysis (volume, glu, Na, pH), organ weight of liver, prostate gland, uterus, thymus (f), and microscopic findings (hypertrophy in cells of thyroid, trachea, liver, adrenals, haematopoiesis spleen). Based on the effects obeserved in high dose animals, the NOAEL for parental (systemic) toxicity for BMS-587319 -03 in this study is 15 mg/kg bw/day.