Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Apr 2018 to 22 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
OECD Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodents, Paris, October 2008.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
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.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity/Composition: 99.73%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
5.7.1. Receipt
On 11 Apr 2018, Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld,
Germany. The animals were 9 weeks old at initiation of dosing and weighed between 152
and 250 g.
A health inspection was performed before the initiation of dosing.
5.7.2. Justification for Test System and Number of Animals
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 to be used in this study is 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.
Sex:
male/female
Details on test animals and environmental conditions:
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

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

5.7.4. Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

5.7.5. Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

5.7.6. Husbandry
5.7.6.1. 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.

5.7.6.2. 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°C with an actual daily mean relative humidity of 50 to 59%. A 12-hour light/12-hour dark cycle was maintained (nightlight during the night period). Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

5.7.6.3. 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.

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

5.7.6.5. 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.

5.7.6.6. 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:
5.8.1. Administration of Test Materials
The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 7 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:
propylene glycol
Remarks:
Specific gravity 1.036
Details on oral exposure:

5.8.2. Justification of Route and Dose Levels
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 the results of an acute oral toxicity study with oral exposure of PF-06961030 in rats (Test Facility Study No. 20141832), and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is 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 daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 females, 3 males per dose.
Control animals:
yes, concurrent no treatment
Details on study design:
5.3. Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item.
The Sponsor has appropriate documentation on file concerning the method of synthesis, fabrication or derivation of the test item, and this information is available to the appropriate regulatory agencies should it be requested.

5.4. Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiration date.

5.5. Test Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

5.6. Dose Formulation and Analysis
5.6.1. Preparation of Test Item
A trial preparation representative of the dosing concentrations was carried out prior to the start of the study to assess the suitability of the formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete.
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding vehicle to the test item.
No adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item. A factor of 1.036 was used to correct for the specific gravity of the vehicle.

Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were also stirred continuously during dosing. Any residual volumes were discarded.

5.6.2. Sample Collection and Analysis
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.

Examinations

Observations and examinations performed and frequency:
5.9.2.1. 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.

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

5.9.3. 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.

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

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

5.10.1.1. 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 (for animals surviving to planned necropsy and moribund animals).
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.

5.10.1.2. Hematology
Blood samples were analyzed for the parameters specified in the following table.
Text Table 4: Hematology Parameters
White blood cells (WBC) Red Blood Cell Distribution Width (RDW)
Neutrophils (absolute and relative) Haemoglobin
Lymphocytes (absolute and relative) Haematocrit
Monocytes (absolute and relative) Mean corpuscular volume (MCV)
Eosinophils (absolute and relative) Mean corpuscular haemoglobin (MCH)
Basophils (absolute and relative)
Red blood cells
Reticulocyte (absolute and relative) Mean corpuscular haemoglobin concentration (MCHC)
Platelets
A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. These smears were not examined

5.10.1.3. Coagulation
Blood samples were processed for plasma, and plasma was analyzed for the parameters listed in the following table.
Text Table 5: Coagulation Parameters
Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT)

5.10.1.4. Clinical Chemistry
Blood samples were processed for plasma or serum, and were analyzed for the parameters specified in the following table.
Text Table 6: Clinical Chemistry Parameters
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:
5.11.1. Unscheduled Deaths
A necropsy was conducted for two animals of Group 4 that died on study and specified tissues were saved.

5.11.4. Organ Weights
The organs identified in the following table were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanised in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratio/percentage (using the terminal body weight) ratios were calculated.
Text Table 8: Organs Weighed at Necropsy
Brain
Epididymis a
Gland, adrenal a
Heart
Kidney
Liver
Ovary a
Spleen
Testisa
Thymus
Uterus
a Paired organ weight.

5.11.5. 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.
Text Table 9: Tissue Collection and Preservation
Animal identification
Brain [cerebellum, mid-brain, cortex]
Cervix
Epididymisa
Gland, adrenal
Gross lesions/masses
Heart
Kidney
Liver
Ovary
Spleen
Stomach
Testisa
Thymus
Uterus
Vagina
a Preserved in Modified Davidson’s fixative prepared at Charles River Den Bosch.
Remaining animals of Group 4 were euthanized as per Test Facility SOPs. The body weights were recorded. These animals underwent necropsy, and specified tissues were retained.

5.11.2. Scheduled Euthanasia
Animals surviving until scheduled euthanasia 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.

5.11.3. 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.

Other examinations:
5.11.6. Histology
Tissues identified in Text Table 14 (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).

5.11.7. Histopathology
All tissues as defined under Histology (section 5.11.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed at 50 mg/kg.
At 150 mg/kg, all animals had an erected fur on Days 4 and 5. Furthermore, laboured respiration was seen in one male (no. 8) at 150 mg/kg on Day 7.
No animals treated at 500 mg/kg survived to the end of the study period, therefore for this group clinical observations are mentioned under mortality.
Mild salivation and ploughing was seen in all animals after dosing at 150 mg/kg which was not considered to be toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity
Mortality:
mortality observed, treatment-related
Description (incidence):
One male (no.10) and one female (no.24) treated at 500 mg/kg/day were found dead on Day 4 and 6, respectively. Two males and two females treated at 500 mg/kg were euthanized for humane reasons on Days 5 (no.23), 6 (no. 22) and 7 (no. 11 and 12).
Clinical signs observed in all animals treated at 500 mg/kg were erected fur, salivation and hunched posture. Moreover, partly closed eyes (no. 11, 22 and 23), ploughing (no. 12, 22 and 24), weakness (no. 23), a pale tail (no. 23), thin (22), shallow breathing (no. 11) and discharge in the left eye (no. 11) were also observed in these animals. Furthermore, these animals also showed body weight loss, accompanied by decreased food intake.
Animals that were found dead showed a decrease in adipose tissue (male) and autolysis, abnormal content in cecum, white foci on the liver, a lack of adipose tissue and a thickened limiting ridge of the stomach (female). In the remaining animals which were killed in extremis, abnormal content in the gastrointestinal tract and a thickened limiting ridge of the stomach (no. 22 and 23) and a small spleen (no. 23) were observed.
The cause of death or clinical signs leading to euthanasia were not determined from the histopathology examination in any of the animals, however, the most significant microscopic changes were present in the gastrointestinal tract and lymphoid tissues. The main microscopic findings were decreased lymphoid cellularity in thymus and spleen and glandular mucosal
erosion, hyperplasia and necrosis in the stomach.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A decrease in mean body weight gain was seen in males (up to 23%) and females (up to 28%) treated at 50 mg/kg when compared to controls. In addition, a decrease in mean body weight gain was seen for males (up to 65%) and females (up to 72%) was seen in animals treated at 150 mg/kg.
No animals treated at 500 mg/kg survived to the end of the study period, therefor for this group body weight results are mentioned under mortality.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No effects on food consumption were seen in males at 50 mg/kg.
No animals treated at 500 mg/kg survived to the end of the study period, therefor for this group food consumption results are mentioned under mortality.
Males treated at 150 mg/kg and females treated at 50 and 150 mg/kg displayed a reduction in food consumption during the treatment period when compared to controls.
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):
A decrease in white blood cell count was seen in males treated at 50 and 150 mg/kg which also resulted in a decrease in absolute and/or relative neutrophils, lymphocytes and monocytes. Moreover, absolute and relative reticulocyte count were reduced in males at 150 mg/kg. Lastly, platelet levels were increased in both males and females treated at 150 mg/kg. No haematology data was recorded for animals treated at 500 mg/kg, due to early mortality.

No effect on coagulation parameters were observed in any of the other treatment groups.
No coagulation data was recorded of the animals treated at 500 mg/kg, due to early mortality.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males treated at 150 mg/kg a decrease in urea, cholesterol and inorganic phosphate concentrations and an increase in total bilirubin and bile acid concentrations (2 out of 3 animals) were seen. In addition, bile acid concentration was increased in one female treated at 150 mg/kg.
No clinical chemistry data was recorded of the animals treated at 500 mg/kg due to early mortality.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower thymus weights (absolute value and relative to body weights compared to controls) were noted in the males and females at 150 mg/kg/day. This was correlated with minimal decreased lymphoid cellularity microscopically in one male and was considered test itemrelated.

There were no other test item-related organ weight changes.
Description (incidence and severity):
There were no test item-related gross observations in the animals surviving to terminal euthanasia (50 and 150 mg/kg/day).
The few 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):
Test item-related microscopic findings after treatment with PF-06961030 were noted in the thymus and kidney of males at 150 mg/kg/day and are summarized in Text Table 4.

Text Table 4.:Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals (Day 8)
Males
Dose level (mg/kg/day) 0 50 150
Thymus a 3 3 3
Decreased cellularity; lymphoid
Minimal - - 1
Kidneya 3 3 3
Accumulation; hyaline droplets
Minimal - 2 -
Mild - 1 3
a = Number of tissues examined from each group.

Minimal, decreased lymphoid cellularity was noted in the thymus of 1/3 males at 150 mg/kg/day and correlated with lower thymus weights.
In the kidneys of males at 50 and 150 mg/kg/day, minimal to mild accumulation of hyaline droplets was noted.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. 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:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, administration of PF-06961030 by once daily oral gavage was well tolerated in rats at levels of 50 mg/kg/day. Early mortality was observed at 500 mg/kg and adverse clinical signs were observed at 150 mg/kg. Based on these results, the no-observed-adverseeffect level (NOAEL) was considered to be 50 mg/kg/day.