Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January 2017 to 17 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407: "Repeated Dose 28-day Oral Toxicity Study in Rodents", Paris, 03 October 2008.
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA 870.3050
Version / remarks:
Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407: "Repeated Dose 28-day Oral Toxicity Study in Rodents", Paris, 03 October 2008.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity (by HPLC @ 225 nm) 100.0 % [a/a]

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
5.5. Test System
Test system Rat: Crl:WI(Han) (outbred, SPF-Quality).
Rationale Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Source Charles River Deutschland, Sulzfeld, Germany.
Total number of animals 30 males and 30 females (females were nulliparous and nonpregnant).
Age at start of treatment Approximately 6 weeks.
Identification Earmark and tattoo.
Randomization By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Acclimatization period At least 5 days before the start of treatment under laboratory conditions.
Health inspection Upon receipt of the animals.
Sex:
male/female
Details on test animals and environmental conditions:
5.7. Animal Husbandry
Room number MR1222 (MR 1221B for motor activity measurements; MR1220 for FOB test).

Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were evaluated and maintained in the raw data.

Accommodation
Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) 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). During motor activity measurements, animals will not have access to food for a maximum of 2 hours.

Water
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:
Oral gavage, using a plastic feeding tube.
Vehicle:
water
Details on oral exposure:
Dose volume 5 mL/kg body weight.
Actual dose volumes were calculated weekly according to the latest body weight.
Duration of treatment / exposure:
28 days. Animals were dosed up to the day prior to necropsy of Main Group animals
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
1 Main
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
2 Main
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
3 Main
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
4 Main
No. of animals per sex per dose:
5 females, 5 males
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Mortality / Viability
At least twice daily.

Clinical signs
At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior tostart of treatment and at weekly intervals during the treatment phase this was also performed outside the home cage in a standard arena (collected under Test Facility Study No. 515345 for logistic reasons and reported under Test Facility Study No. 510527). These clinical observations were at least conducted 1 hour (± 30 min) after dosing (based on results of the dose range finding study, Test Facility Study No. 516082). The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded 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. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.

Functional Observations
During Week 4 of treatment, functional observation tests were performed at 1 hour (± 30 min) after dosing (based on results of
the dose range finding study, Test Facility Study No. 516082) on all Recovery Group 1 and 4 animals and all Main Group 2
and 3 animals. The following tests were performed (abbreviations mentioned in the respective tables indicated between brackets):
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Since the abovementioned measurements did not reveal treatment-related effects, the functional observation tests were not performed at the end of the recovery phase.
Body weights Weekly.
Food consumption Weekly.
Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Sacrifice and pathology:
5.11.1. Necropsy
On the scheduled day of necropsy, animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Blood samples for clinical laboratory investigations were drawn from the retro-orbital sinus of all rats/sex/group (for details see par 5.10.) immediately prior to sacrifice. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Identification marks: not processed Ovaries
Adrenal glands (Pancreas)
(Aorta) Peyer's patches [jejunum, ileum] if detectable
Brain [cerebellum, mid-brain, cortex] (Pituitary gland)
Caecum (Preputial gland)
Cervix Prostate gland
(Clitoral gland) Rectum
Colon (Salivary glands - mandibular, sublingual)
Duodenum Sciatic nerve
Epididymides * Seminal vesicles including coagulating gland
Eyes (including optic nerve and harderian Skeletal muscle
gland) * (Skin)
(Female mammary gland area) Spinal cord -cervical, midthoracic, lumbar
Femur including joint Spleen
Heart Sternum with bone marrow
Ileum Stomach
Jejunum Testes *
Kidneys Thymus
(Larynx) Thyroid including parathyroid [if detectable]
(Lacrimal gland, exorbital) (Tongue)
Liver Trachea
Lung, infused with formalin Urinary bladder
Lymph nodes - mandibular, mesenteric Uterus
(Nasopharynx) Vagina
(Oesophagus) All gross lesions
* Fixed in modified Davidson's solution, prepared at Charles River Den Bosch using Formaldehyde 37-40%, Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
Other examinations:
5.11.2. Organ Weights
The following organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy:
Adrenal glands Spleen
Brain Testes
Epididymides Thymus
Heart Uterus (including cervix)
Kidneys Prostate
Liver Seminal vesicles including coagulating glands
Ovaries Thyroid including parathyroid

5.11.3. Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

5.11.4. Histopathology
The delegated phase was performed by the Principal Investigator for Histopathology (APPENDIX 5).
The following slides were examined by a pathologist:
• all tissues collected at the scheduled sacrifice from all Main Group 1 and 4 animals,
• 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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Yellow staining of the fur (urine) was noted for all animals treated at 250 mg/kg in the last week of the Main study and during the first week of the Recovery period. This was considered test item related but not adverse in the absence of any corroborative findings. Salivation noted for all high dose animals was not considered 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:
no mortality observed
Description (incidence):
No mortality occurred during the study period
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was shown by males treated at 250 mg/kg and to a lesser extent at 75 mg/kg. During the treatment free phase this was improving however body weights remained lower at the end of the treatment free phase. Body weights and body weight gain of females remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A decrease in food consumption was observed in both sexes treated at 250 mg/kg, especially during the first week of the study. During the recovery phase food consumption of the control and high dose group were similar.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters.
Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in clinical biochemistry parameters distinguished treated from control animals:
• Increased alanine aminotransferase (ALAT) at 250 mg/kg in both sexes at end of treatment and for males at the end of recovery.
• Increased alkaline phosphatase (ALP) at 250 mg/kg in females at the end of treatment and at end of recovery.
• Reduced total protein at 250 mg/kg in both sexes at the end of treatment and at end of recovery.
• Reduced albumin at 250 mg/kg in both sexes and 75 mg/kg for females at the end of treatment and for females at the end of recovery.
• Reduced cholesterol at 75 and 250 mg/kg in males at the end of treatment and end of recovery.
• Increased potassium at 250 mg/kg in both sexes at the end of treatment and end of recovery.
Any other (statistically significant) changes in clinical biochemistry parameters were considered to be unrelated to treatment as the changes were slight in nature or occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Motor activity was reduced in both sexes at 250 mg/kg. In females this effect is also seen at 75 mg/kg. All groups showed a normal motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Forepaw grip strength was increased in females treated at 250 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights.
All organ weight differences observed, including those that reached statistical significance, were considered incidental or related to the decrease in body weight and unrelated to the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
7.4.1. Macroscopic Examination
There were no test item-related gross observations.
All 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 recorded microscopic findings 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:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 250 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:
Wistar rats were treated with PF-06478031-01 for 28 consecutive days by daily oral gavage at dose levels of 0, 25, 75 and 250 mg/kg followed by a 14-day treatment-free recovery period.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No mortality occurred during the study period and no treatment-related changes were noted in haematology, macroscopic examination, organ weights and microscopic examination.

Clinical signs revealed yellow staining of the fur (urine) in all animals treated at 250 mg/kg.

This was considered test item related but not adverse in the absence of any corroborative findings.

Reduced body weight gain show by males during treatment, was recovering during the treatment free period. At a dose level of 250 mg/kg this was accompanied by a decrease in food consumption. In the absence of corroborative findings these changes were considered
test item related but not adverse at these dose levels.

Functional observation tests revealed increased forepaw grip strength in females treated at 250 mg/kg. Further lower motor activity was seen in both sexes at 250 mg/kg and in females at 75 mg/kg. These findings were considered test item related but not to represent an adverse neurobehavioral effect since the changes were not supported by clinical observations or other functional observation tests, were slight in nature, and had no supportive morphological correlates in examined neuronal tissues.\

Slight but not reversible changes in clinical biochemistry parameters as alanine aminotransferase, alkaline phosphatase, cholesterol, albumin, total protein and potassium could indicate an effect on the kidneys or liver but this was not supported by the morphology
of these tissues. Moreover the well-being of the animals was not affected therefore these changes were considered not adverse.

From the results presented in this report, the No Observed Adverse Effect Level (NOAEL) for PF-06478031-01 was established as being at least 250 mg/kg, since no adverse effects were observed at this dose level. Higher doses were not tested since it was expected that a dose level of 500 mg/kg or higher would not be tolerated (see dose range finding study APPENDIX 6).