Methyl (1R,3S,4R,5R)-3-amino-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-D-galactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α -L-galactopyranosyl)oxy]cyclohexanecarboxylate hydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-29 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

The study integrity was not affected by the deviations

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The study integrity was not affected by the deviations

Principles of method if other than guideline:

List of protocol deviations:
1. From all animals, only 6 levels of the brain were processed since the olfactory bulbs were not discernable during trimming.
Evaluation: Sufficient information is available regarding the brain

2. On Days 1 and 4, the maximum time between preparing the formulation and dosing exceeded the 4 hours. The maximum deviation was approximately 30 minutes.

Evaluation: The deviation is slight and incidental and was considered not to have affected the study integrity
The protocol was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee (DEC 14-59) as required by the Dutch Act on Animal Experimentation (February 1997). The study procedures described in the protocol were based around the guidelines mentioned above.

GLP compliance:

yes (incl. QA statement)

Remarks:

dated: 17 February 2016

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (1R,3S,4R,5R)-3-amino-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-D-galactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α -L-galactopyranosyl)oxy]cyclohexanecarboxylate hydrochloride

EC Number:

945-311-0

Molecular formula:

C78H85NO18 HCl

IUPAC Name:

Methyl (1R,3S,4R,5R)-3-amino-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-D-galactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α -L-galactopyranosyl)oxy]cyclohexanecarboxylate hydrochloride

Details on test material:

- State of aggregation: white solid powder

Specific details on test material used for the study:

pH (1% in water, indicative range): 6.7-5.3 (determined by the test laboratory)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: Wi(Han), outbred, SPF-Quality

Details on species / strain selection:

Selected because they are recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Deutschland, Sulzfeld, Germany
Age at start of treatment: approx. 9 weeks
Randomisation: by computer generated randm algorithm according to body weight, with all animals within +/- 20% of the sex mean
Acclimatisation period: at least 5 days before the start of treatment under laboratory conditions
Health inspection: upon receipt of the animals

Animal husbandry
Environmental controls for the animal room were set to maintain 18 - 24C, a relative humidity of 40-70%, at least 10 air changes/hour and a 12h light / 12h dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Group housing of 3 animals per sex in Makrolon cages (MIV type, height 18cm) with sterilised sawdust as bedding material and paper as cage enrichment.

Animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaeten, Soest, Germany). They also had free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

A plastic feed tube was used.

Vehicle:

propylene glycol

Remarks:

based on trial formulations performed at WIL Research Europe and on information from the sponsor

Details on oral exposure:

Formulations were prepared daily within 4.5 hours prior to dosing and were homogenised to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

once daily,
approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Mortality / viability were observed at least twice daily.

At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed signs were recorded. Signs were gradd for severity and the maximum grade was predefined at 3 or 4. Grades were codes as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

Body weights were measured on Days 1, 4 and 7.

Food consumption was observed over Days 1-4 and 4-7.

Subjective appraisal of water consumption was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Sacrifice and pathology:

All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane. Blood samples for clinical laboratory investigations were drawn from the retro-orbital sinus of all rats/sex/group immediately prior to sacrifice. The animals were subsequently exsanguinated and subjected to a full post mortem examination. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

Samples of following tissues were collected from all animals at necropsy and fixed at 10% buffered formalin: adrenal glands, brain (cerebellum, mid-brain, cortex, 7 levels), epididymides, heart, kidneys.liver, ovaries, spleen, stomach, testes, thymus, uterus, all gross lesions

Organ weights were recorded from the animals on the scheduled day of necropsy.

A pathologist examined all tissues collected at the scheduled sacrifice from all group 1 (vehicle control) and 4 (1000 mg/kg bw/day) animals and all gross lesions. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings. Histopathology was subjected to a peer review.

All organ and tissue samples, as defined under histopathology above, were processed, embedded in paraffin wax and cut at a thickness of 2-4 micrometres and stained with haematoxylin and eosin.

Other examinations:

Blood samples were collected as part of the necropsy procedure under anaesthesia using isoflurane. Immediately thereafter, the animals were examined post mortem, between 7:00 and 10:30 am. Animals were deprived of food overnight (for a maximum of 24 hours) but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (0.45 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml). An additional blood sample (0.25 ml) was collected into untreated tubes for determination of bile acids.

Statistics:

A descriptive statistical analysis was performed (mean, SD and/or median)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain of male animals remained at the same range as controls over the study period.
Body weight loss or reduced body weight gain, observed among females and only one high dose male, is slight in nature and occurred in absence of a dose relation. Therefore this finding was considered ot be of no toxicological significance.

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

Description (incidence and severity):

Subjective appraisal was maintained during the study, but no wuantitative investigation was introduced as no effect was suspected.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematology parameters of treated rats.

Minor variations, such as higher mean corpuscular haemoglobin concentration (MCHC) in males and lower red blood cell count and haemoglobin and haematocrit levels in females, were considered not to represent a change of biological significance as they were very slight in nature and occurred in the absence of a clear treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

Slight variation in some parameters were considered to have arisen as a result of variation in individual data and slightly high or low control values and in the absence of a treatment-related distribution considered ot be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related higher absolute and relative ovaries weights were noted in the 1000 mg/kg group females (42 and 38% increase compared to mean control values, respectively). These values were considered to be caused by slightly low control value. Since all individuals were within the range considered normal for rats of this age and strain and in the absence of macro- or microscopic correlates, the higher ovaries' weight was considered not toxicologically relevant.

There were no other test item-related organ weight changes.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Wistar rats were treated with PF-06460259-01 for 7 consecutive days by daily oral gavage at dose levels up to 1000 mg/kg.
No treatment related changes were noted in clinical appearance, body weight, food consumption, clinical laboratory investigations, organ weights, macroscopic examinatin and microscopic examination.
From the results presented in this report it was concluded that PF-06460259-01 has no toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecutive days up to 1000 mg/kg.

Executive summary:

Title

7 -Day repeated dose study with PF-06460259 -01 by daily gavage in the rat.

Guidelines

The study was based on the following guidelines:

• EC No 440 /2008, B.7 Repeated Dose (28 days) Toxicity (rat), 2008
• OECD 407, Repeated dose 28 -day Oral Toxicity Study in Rodents, 2008

Rationale for dose levels

Dose levels for this 7 -day oral gavage study were selected to be 0, 150, 300 and 1000 mg/kg, based on the results of the acute oral toxicity study and on information from the sponsor.

Study outline

The test substance was administered daily for 7 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 3 males and 3 females.

Evaluated parameters

The following parameters were evaluated: clinical signs daily; body weight on Days 1, 4 and 7 and food consumption over Days 1 -4 and 4 -7; clinical pathology and macroscopy at terminationl organ weights and histopathology on a selection of tissues.

Results

No treatment related changes were noted in clinical appearance, body weight, food consumption, clinical laboratory investigations, organ weights, macroscopic examination and microscopic examination.

Conclusion

From the results presented in this report it was concluded that PF-06460259 -01 has no toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecituve days up to 1000 mg/kg.