isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24-12-2018 to 13-02-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The objective of this study was to determine the potential toxicity of PF-07097547-24, when given orally by gavage for 7 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.
The design of this study is based on the following study guidelines:
• OECD Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodents, Paris, October 2008.
• Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

EC Number:

950-484-0

Molecular formula:

C9H17NO2*C4H6O4

IUPAC Name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13335
Expiry date: 31 January 2020 (expiry date)
Physical Description: White solid
Purity/Composition: 97.1%
Storage Conditions: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Receipt
On 19 Dec 2018 Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. The animals were between 9 and 10 weeks old at initiation of dosing and weighed between 178 and 275 g.
A health inspection was performed before the initiation of dosing.

Animal Identification
At study assignment, each animal was identified using a chip that is implanted shortly after arrival at the Test Facility.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 9 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to groups by a stratified randomisation scheme designed to achieve similar group mean body weights. Males and females were randomised separately.

Husbandry
Housing
On arrival and following randomization, animals were group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. The room in which the animals were kept was documented in the study records.
Each cage was clearly labeled with a color-coded cage card indicating test facility study no., group, animal number(s), and sex.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21°C with an actual daily mean relative humidity of 27 to 53% (for exceptions, see deviations in Appendix 1). A 12 hour light/12 hour dark cycle was maintained (except during designated procedures). Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Administration of Test Materials
The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 7 consecutive days. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1.
The dosing formulations were stirred continuously during dose administration.
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Vehicle:

water

Details on oral exposure:

Justification of Route and Dose Levels
The oral route is selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

The dose levels were selected based on results of an acute oral toxicity study with oral exposure of PF-07097547-24 in rats (Test Facility Study No. 20171508), and in an attempt to produce graded responses to the test item. At 2000 mg/kg no mortality was observed and on the first day hunched posture and/or piloerection were observed. These signs were not observed from Day 2 onwards. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

In-life Procedures, Observations, and Measurements

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Cage Side Observations
Cage side observations were performed once daily, immediately after dosing up to 30 minutes after dosing, throughout the dosing period. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Detailed Clinical Observations
The animals were removed from the cage, and a detailed clinical observation was performed on Days 1 and 8.

Body Weights
Animals were weighed individually on Days 1, 4 and 7 (prior to dosing). A fasted weight was recorded on the day of necropsy.

Food Consumption
Food consumption was quantitatively measured on Day 1-4 and 4-7.

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

Laboratory Evaluations
Clinical Pathology
Sample Collection
Blood sampling for clinical pathology was performed as part of the necropsy procedure immediately prior to sacrifice when the animal was deeply anaesthetized.
Blood was collected from the retro-orbital sinus of fasted animals under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). After collection all samples were transferred the appropriate laboratory for analysis.

Hematology
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Blood samples were analyzed for the parameters specified in the following table:
White blood cells (WBC) Red Blood Cell Distribution Width (RDW)
Neutrophil (absolute) Haemoglobin
Lymphocyte (absolute) Haematocrit
Monocyte (absolute) Mean corpuscular volume (MCV)
Eosinophil (absolute) Mean corpuscular haemoglobin (MCH)
Basophil (absolute) Mean corpuscular haemoglobin concentration (MCHC)
Red blood cells Platelets
Reticulocyte (absolute)

A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. These smears were not examined.

Coagulation
Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Blood samples were processed for plasma, and plasma was analyzed for the parameters listed in the following table:
Coagulation Parameters: Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT)

4.10.1.4. Clinical Chemistry
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparin as anticoagulant. Serum samples at a target volume of 0.25 mL were collected in tubes without anticoagulant. Blood samples were processed for plasma or serum (bile acids) and were analyzed for the parameters specified in the following table:

Alanine aminotransferase (ALAT) Glucose
Aspartate aminotransferase (ASAT) Cholesterol
Alkaline Phosphatase (ALP) Triglycerides
Total protein Sodium
Albumin Potassium
Bile Acids Chloride
Total Bilirubin Calcium
Urea Inorganic Phosphate (Inorg. Phos)
Creatinine

Sacrifice and pathology:

Animals were weighed and euthanized using isoflurane, blood was sampled from the retro-orbital sinus (for clinical pathology) followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

All animals were necropsied, tissues coolected and organ weighed as per below. The tissues collected from groups 1 (control) and 4 (1000mg/kg bw/day) were assessed for histology and histopathology. Any gross lesions and the stomach collected from animals in groups 2 (150mg/kg bw/day) and group 3 (300 mg/kg bw/day) were assessed for histology and histopathology.

Necropsy
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Organ Weights
The organs identified in the following table were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) were calculated.

Organs Weighed at Necropsy:
Brain
Epididymis
Gland, adrenal
Heart
Kidney
Liver
Ovary
Spleen
Testis
Thymus
Uterus

Tissue Collection and Preservation
Representative samples of the tissues identified in the following table were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Tissue Collection and Preservation:
Animal identification
Brain [cerebellum, mid-brain, cortex] (8 levels)
Cervix
Epididymis a
Gland, adrenal
Gross lesions/masses
Heart
Kidney
Liver
Ovary
Spleen
Stomach
Testis a
Thymus
Uterus
Vagina
a Preserved in Modified Davidson’s fixative prepared at Charles River Den Bosch.

Histology
Tissues identified in the table above (except animal identification) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology
All tissues as defined under Histology were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
A peer review on the histopathology data was performed by a second pathologist.

Other examinations:

Body Weight Gains: calculated against the body weight on Day 1
Organ Weight Relative to Body Weight: calculated against the Terminal body weight

Statistics:

Descriptive statistics number, mean and standard deviation were reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during the observation period.
A scab in the dorsal cervical region was observed in one female at 1000 mg/kg/day (no. 22). This finding is commonly noted in rats of this age and strain, housed under laboratory conditions and is therefore not considered test item-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption were recorded.

Food efficiency:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters in treated females.
Higher triglyceride levels were observed in all test item-treated males, when compared to controls, without reaching statistical significance. In the absence of a dose-related trend and at the severity observed, this alteration was not considered to be toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Any minor organ weight differences observed were considered incidental, occurred in the absence of a dose-related trend, and/or remained within the range considered normal for rats of this age and strain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males only, epithelial hyperplasia and vacuolation at the limiting ridge of the stomach (squamous mucosa) was noted at 300 and 1000 mg/kg/day with a dose-dependent increase in incidence and severity (up to mild). In addition at 1000 mg/kg/day increased severity of mucosal neutrophilic infiltrates (diffuse, up to mild) was also noted. The few histologic changes noted in test item-treated females were comparable to the controls or minimal and without a dose response, and compatible with background changes in the rat.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Description (incidence and severity):

Coagulation parameters of treated rats were considered not to have been affected by treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 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

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of PF-07097547-24 by once daily oral gavage for 7 consecutive days was well tolerated in rats at dose levels up to 1000 mg/kg/day. Non-adverse target organ effects were observed in the stomach of males at levels of 300 and 1000 mg/kg/day (hyperplasia and vacuolation of the epithelium of the limiting ridge and increased severity of diffuse neutrophilic infiltrates of the stomach mucosa). Based on the results obtained in this study, the no-observed-adverse-effect level (NOAEL) for PF-07097547-24 was considered to be at least 1000 mg/kg/day.

Executive summary:

The objective of this study was to determine the potential toxicity of PF-07097547-24, when given orally by gavage for7 days toWistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows:

Text Table1
Experimental Design

Group No.	Test Item Id.	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	-	0 (Vehicle)	5	0	3	3
2	PF-07097547-24	150	5	30	3	3
3		300	5	60	3	3
4		1000	5	200	3	3

Id.= identification.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Test item-related microscopic findings were present in the stomach of males at 300 and 1000 mg/kg/day and consisted of hyperplasia and vacuolation of the epithelium of the limiting ridge starting at 300 mg/kg/day, and increased severity (up to mild) of diffuse neutrophilic infiltrates of the stomach mucosa at 1000 mg/kg/day. These findings were considered to be non-adverse based on the observed incidence and severity. 

No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, clinical pathology, macroscopic examination and organ weights).

In conclusion, administration of PF-07097547-24 by once daily oral gavage was well tolerated in rats at dose levels up to 1000 mg/kg/day. Non-adverse target organ effects were observed in the stomach of males at 300 and 1000 mg/kg/day. Based on these results, the no-observed-adverse-effect level (NOAEL) for PF-07097547-24 was considered to be at least 1000 mg/kg/day.