Propan-2-yl-oxocyclobutane carboxylate

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:

07-12-2018 to 30-01-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

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-06238566, when
given orally by gavage for 7 days to Wistar 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 Guidline 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

1

Reference substance name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

EC Number:

834-970-9

Cas Number:

130111-95-4

Molecular formula:

C8H12O3

IUPAC Name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

Test material form:

liquid

Details on test material:

Batch (Lot) Number: GR13224
Expiry date: 31 January 2020 (expiry date)
Physical Description: Colourless to pale yellow liquid
Purity/Composition: 99.6%
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 05 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 280 g.
A health inspection was performed before the initiation of dosing.

Animal Identification
At study assignment, each animal was identified using a chip that was 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 7 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.
The disposition of all animals was documented in the study records.

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 20 to 21°C with
an actual daily mean relative humidity of 40 to 53%. 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.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations
or treatments were required.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was 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-06238566 in rats (Test Facility Study No. 20171526), and in an attempt to
produce graded responses to the test item. At 2000 mg/kg no mortality was observed and on
the first two days hunched posture, uncoordinated movements, piloerection and lethargy were
observed. These signs were not observed from Day 3 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.

Vehicle:

propylene glycol

Details on oral exposure:

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.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analysis of test item in vehicle for concentration, stability, homogeneity was not performed,
however, to limit the impact, the test item preparation was performed with approved
procedures and documented in detail. Formulations were visually inspected for homogeneity
prior to use and all formulations were used within 4 hours after adding vehicle to the test
item. This GLP exception was therefore considered as being minor with no impact on the
outcomes and the integrity and the achievement of the objective of the study.

Duration of treatment / exposure:

7 days

Frequency of treatment:

once daily

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 per sex per dose

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

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.

Sacrifice and 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 of hematology, coagulation and/or clinical chemistry

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
following parameters: Prothrombin Time (PT) and 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. 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

Unscheduled Deaths
No animals died during the course of the study.

Scheduled Euthanasia-conducted on all animals
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.

Necropsy-conducted on all animals
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-conducted on all animals
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.
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.

Animal identification
Brain [cerebellum, mid-brain, cortex] (8 levels according to Garman et al.)
Cervix
Epididymis
Gland, adrenal
Gross lesions/masses
Heart
Kidney
Liver
Ovary
Spleen
Stomach
Testis
Thymus
Uterus
Vagina

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:

CONSTRUCTED VARIABLES
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, treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted in females up to 300 mg/kg/day and in males up
to 1000 mg/kg/day.
Hunched posture, erected fur and/or abnormal breathing sounds were noted in 3/3 females at
1000 mg/kg/day on Day 2.
Severe salivation was observed in one control female (no. 15), which was not test item-
related.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

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):

Food consumption was similar to the control level over the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

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

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 differences noted, were considered not to be PF-06238566-related due to the
direction of the change, lack of dose-related pattern, and/or general overlap and variability in
individual values.

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:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
The histologic changes recorded were considered to be incidental findings and 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.

Histopathological findings: neoplastic:

not examined

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-06238566 by once daily oral gavage for 7 consecutive
days was well tolerated in rats at dose levels up to 1000 mg/kg/day. From the results
presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000
mg/kg/day was established for PF-06238566.

Executive summary:

The objective of this study was to determine the potential toxicity of PF-06238566, when

given orally by gavage for 7 days to Wistar rats. In addition, a No Observed Adverse Effect

Level (NOAEL) was evaluated.

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.

In females at 1000 mg/kg/day, non-adeverse test item-related hunched posture, erected fur

and/or abnormal breathing sounds were observed on a single day.  No other test item-related

changes were noted in any of the parameters investigated in this study.

In conclusion, administration of PF-06238566 by once daily oral gavage for 7 consecutive

days was well tolerated in rats at dose levels up to 1000 mg/kg/day.  From the results

presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000

mg/kg/day was established for PF-06238566.