(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

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:

11th May 2020 to 8th July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: 7 day repeat dose

Version / remarks:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) was used as a base for the study

Deviations:

yes

Remarks:

Adjusted to 7 day exposure

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 7 Day Oral repeat

Version / remarks:

EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008 was used as the based fpr the study

Deviations:

yes

Remarks:

Adjusted for 7 day day exposure

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

EC Number:

840-203-9

Cas Number:

2295746-51-7

Molecular formula:

C21H35N3O3

IUPAC Name:

(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 9 weeks old
- Weight at study initiation: Males weighed between 273 and 316 g and females weighed between 167 and 196 g.

- Housing: Polycarbonate cages ( Makrolon type IV, height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS -J.Rettenmaier &Söhne GmbH+ CO. KG, Rosenberg, Germany)equipped with water bottles. 3 animals of the same sex and same dosing group together. The room in which the animals were kept was documented in the study records. Cages were arranged on the racks according to a Latin-square model.
- Diet: SMR/M-Z from SSNIFF®Spezialdiäten GmbH, Soest, Germany ad libitum
- Water: Municipal tap water ad libitum
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days before the commencement of dosing.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature: Actual mean: 21°C
- Humidity: Actual mean: 45 to 56%
- Air changes: Ten or more air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
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 as a solution and dosed within 4 hours after preparation of the formulation. Adjustment was made for specific gravity of the vehicle. A factor of 1.036 was used to correct for the specific gravity of the test vehicle. No correction was made for the purity/composition of the test item. Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 30, 60 & 50 mg/ml respectively.
- Amount of vehicle: 5 ml/kg
- Lot/batch no.: K51750378936

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

Once daily

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.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Three animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Justification of Dose Levels
The dose levels were selected based on results of an acute oral toxicity study with oral exposure of PF-07202371-S7 in rats (Test Facility Study No. 20232344), and in an attempt to produce graded responses to the test item. At 2000 mg/kg, all animals were found dead or sacrificed in moribund condition on Days 1 or 2 post-treatment. At 300 mg/kg, only hunched posture in all animals between Days 1 and 3 and erected fur in three animals on Day 1 were noted. 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.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1 at 0 to 0.5 hours post-dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 1 and 8.

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 1, 4 and 7 (prior to dosing).

FOOD CONSUMPTION AND COMPOUND INTAKE
Time schedule: Over Days 1-4 and 4-7.
Quantitatively measured per cage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood sampling for clinical laboratory investigations was performed as part of the necropsy procedure immediately prior to sacrifice.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes. Overnight (maximum of 24 hours) before their scheduled necropsy.
- How many animals: All surviving animals
Parameters:
White Blood Cell Count(WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells(LUC) (absolute),
Red Blood Cell Count Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets.

Sacrifice and pathology:

Complete necropsy examination: 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.

Statistics:

Constructed Variables
Body Weight Gains: Calculated between at least each scheduled interval.

Food Consumption: Calculated between at least each scheduled interval.

Organ Weight Relative to Body Weight: Calculated against the Terminal body weight.

Descriptive Statistical Analysis

Means, standard deviations (or % coefficient of variation or standard error, when deemed
appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by
dataset.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hunched posture and erected fur was seen in all female animals at 750 mg/kg/day between
Day 3 and 7. Furthermore, one female at 750 mg/kg/day showed shallow and abnormal
breathing sounds between Days 3 and 6. Abnormal breathing sounds were also seen in one
female at 150 mg/kg/day on Day 7.
Salivation was seen among 5/6 animals at 150 mg/kg/day and in all animals at 300 and
750 mg/kg/day between Days 2 and 7, which was considered not toxicologically relevant,
taking into account the nature and minor to moderate severity of the effect and its time of
occurrence (i.e. after dosing). This sign was considered to be a physiological response related
to the taste of the test item rather than a sign of systemic toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effects on body weight and body weight gain were seen in males at 150 and 300 mg/kg/day and females at 300 mg/kg/day.
Males dosed at 750 mg/kg/day showed an overall body weight loss, which was -7.6% on Day 7 compared to Day 1. Females at 750 mg/kg/day had an overall body weight loss of - 10.5% from Day 1 to Day 4, followed by a slight increase in body weight on Day 7 (1.8% compared to Day 4).
At 150 mg/kg/day, the females showed only a small increase in body weight gain throughout
the study (1% on Day 7 compared to Day 1).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of males of at 150 mg/kg/day was similar to the control animals over the
study period.
A decrease in food consumption was seen in males dosed at 300 mg/kg/day over Days 4-7
(0.90x of controls) and a reduced food consumption was observed in males at 750 mg/kg/day
throughout the dosing period (0.56x and 0.37x on Day 1-4 and 4-7 respectively).
In females food consumption was reduced at 150 mg/kg/day over Days 1-4 and 4-7 (0.83x
and 0.81x of control, respectively). In females at 300 mg/kg/day food consumption was only
slightly lower over Days 1-4 and 4-7 (0.94x and 0.93x of control, respectively. For females
dosed at 750 mg/kg/day a reduced food consumption was seen over Day 1-4 and 4-7 (0.50x
and 0.36x respectively).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted hematology parameters in males up to
300 mg/kg/day.
An increase in neutrophil count was seen in females at 750 mg/kg/day. In males and females
at 750 mg/kg/day an increase in red blood cell count and a decrease in reticulocyte count was
observed. Furthermore, hemoglobin and hematocrit concentration was increased in males and
females at 750 mg/kg/day, while mean corpuscular volume was decreased in females at
750 mg/kg/day.
Lastly, an increase in platelets count was observed in males and females at 750 mg/kg/day.

Remaining differences hematology parameters were considered not test item-related based on
the absence of a dose response, general overlap of individual values with the range of control
values, and/or were of a magnitude of change commonly observed in rats under similar study
conditions.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters in males
and females at 150 mg/kg/day.
At 750 mg/kg/day, an increase in aspartate aminotransferase activity was observed in males at
750 mg/kg/day, mainly caused by one male (No. 10). A dose-dependent increase of total
bilirubin and bile acid concentration was observed in males at 300 and 750 mg/kg/day. In
females, bilirubin concentration was also increased at 300 and 750 mg/kg/day).
In females dosed at 750 mg/kg/day a decrease in chloride concentration and an increase in
calcium concentration was observed.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with PF-07202371-S7 were noted in
the liver and thymus of the 750 mg/kg/day group males and females.

Minimal cytoplasmic alteration was observed in the liver of 2/3 750 mg/kg/day treated males
and 1/3 750 mg/kg/day treated females. The cytoplasmic alteration was characterized by
rearrangement of the cellular content: lighter stained content was present in the central part of
the cells, whereas darker stained content was present at the peripheral area of the cells. These
morphological changes were not accompanied by degenerative or inflammatory changes.
Mild to marked lymphoid apoptosis was observed in the thymus of 3/3 750 mg/kg/day treated
males and 3/3 750 mg/kg/day treated females. The lymphoid apoptosis in one male at 300
mg/kg/day and in one female at 150 mg/kg/day were considered not relevant because of the
low degree in the male and the lack of a dose-effect relationship in the females.
There were no other test item-related histologic changes. The remainder of 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.

Details on results:

The clinical signs of hunched posture and erected fur seen in females dosed at 750 mg/kg/day,
were considered to be test item-related and adverse at the incidence observed. Furthermore,
one female at 750 mg/kg/day and one females at 150 mg/kg/day showed shallow and
abnormal breathing sounds. As this finding was only observed directly after dosing it might
be caused by the dosing procedure, and therefore it is likely not caused by the test item.
Body weight loss was observed in males and females at 750 mg/kg/day, which was
accompanied by a clear decrease in food consumption. At the severity observed, these effects
were considered to be adverse. At 150 mg/kg/day, the females showed only a small increase
in body weight gain throughout the study, which was also accompanied by a lower food
consumption. As this was still an increase in body weight gain and as no effects on body
weight were seen at 300 mg/kg/day, this finding was considered to be not toxicologically
relevant. In males and females at 300 mg/kg/day, food consumption was also slightly lower,
but lacked any effects on body weight and was therefore considered to be not toxicologically
relevant.
Several hematology parameters were affected at 750 mg/kg/day consisting of an increase in
neutrophil count in females, red blood cell count, haemoglobin and hematocrit in males and
females and a decrease in reticulocyte count in males and females and mean corpuscular
volume in females. Furthermore, an increase in platelets count was observed in males and
females at 750 mg/kg/day. In absence of any histopathological correlation, these findings
were considered to be not adverse.
At clinical chemistry, an increase in aspartate aminotransferase activity was observed in
males at 750 mg/kg/day, which was mainly caused by one male. Furthermore, a dosedependent increase of total bilirubin concentration in males and females and bile acid
concentration in males was observed at 300 and 750 mg/kg/day. In females dosed at
750 mg/kg/day a decrease in chloride concentration and an increase in calcium concentration
was observed. In absence of any histopathological correlation, these findings were considered
to be not adverse.
At histopathological examination, minimal cytoplasmic alteration was observed in the liver
males and females at 750 mg/kg/day. The cytoplasmic alteration was characterized by
rearrangement of the cellular content: lighter stained content was present in the central part of
the cells, whereas darker stained content was present at the peripheral area of the cells. These
morphological changes were not accompanied by degenerative or inflammatory changes and
was therefore considered to be not adverse.
In the thymus, mild to marked lymphoid apoptosis was observed in males and females at 750
mg/kg/day, which correlates with lower thymus weight. It cannot be excluded that the
decreased thymus weight in combination with the lymphoid apoptosis is an adverse effect.
No test item-related or toxicologically significant changes were noted in any of the remaining
parameters investigated in this study (coagulation and gross examination).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

immune system

Organ:

thymus

Treatment related:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Administration of PF-07202371-S7 by once daily oral gavage for 7 days to rats was well tolerated in male and female rats at levels of 300 mg/kg/day. Adverse clinical signs (hunched posture and erected fur), body weight loss and lower food consumption was seen in males and/or females at 750 mg/kg/day. Test item-related morphologic alterations were observed in the liver (cytoplasmic alteration) and thymus (lymphoid apoptosis, with correlating lower thymus weight) at 750 mg/kg/day. The morphological change in the liver was considered not adverse while the change in the thymus might be adverse. Furthermore, several non-adverse hematology and clinical chemistry changes were observed in males and females at 300 and/or 750 mg/kg/day.
Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg for males and females.

Executive summary:

The objective of this study was to determine the potential toxicity of PF-07202371-S7, when given orally by gavage for 7 days to Wistar rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.
The study design was as follows:

Group No.	Test Item Id.	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	Control	0 (vehicle)	5	0	3	3
2	PF-07202371-S7	150	5	30	3	3
3		300	5	60	3	3
4		750	5	150	3	3

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.
At 150 mg/kg/day, no test item-related effects were seen.
At 300 mg/kg, only a non-adverse increase in total bilirubin concentration in males and females and bile acids in males were observed.
At 750 mg/kg/day, hunched posture and erected fur was seen in females, which were considered to be adverse. Body weight loss was observed in males and females, which was accompanied by a clear decrease in food consumption. At the severity observed, these effects were considered to be adverse. At hematology several parameters were affected consisting of an increase in neutrophil count
in females, red blood cell count, hemoglobin and hematocrit in males and females and a decrease in reticulocyte count in males and females and mean corpuscular volume in females. Furthermore, an increase in platelets count was observed in males and females. In absence of any histopathological correlation, these findings were considered to be not adverse.
At clinical chemistry, an increase in aspartate aminotransferase activity was observed in males, which was mainly caused by one male. Furthermore, an increase of total bilirubin concentration in males and females and bile acid concentration in males was observed. In females, a decrease in chloride concentration and an increase in calcium concentration was observed. In absence of any histopathological correlation, these findings were also considered to be not adverse.
At histopathological examination, minimal cytoplasmic alteration was observed in the liver males and females. The cytoplasmic alteration was characterized by rearrangement of the cellular content: lighter stained content was present in the central part of the cells, whereas darker stained content was present at the peripheral area of the cells. These morphological changes were not accompanied by degenerative or inflammatory changes and was therefore considered to be not adverse.
In the thymus, mild to marked lymphoid apoptosis was observed in males and females, which correlates with lower thymus weight. It cannot be excluded that the decreased thymus weight in combination with the lymphoid apoptosis is an adverse effect.
No test item-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (coagulation and gross examination).
In conclusion, administration of PF-07202371-S7 by once daily oral gavage for 7 days to rats was well tolerated in male and female rats at levels of 300 mg/kg/day.
Adverse clinical signs (hunched posture and erected fur), body weight loss and lower food consumption was seen in males and/or females at 750 mg/kg/day. Test item-related morphologic alterations were observed in the liver (cytoplasmic alteration) and thymus (lymphoid apoptosis, with correlating lower thymus weight) at 750 mg/kg/day. The morphological change in the liver was considered not adverse while the change in the thymus might be adverse. Furthermore, several non-adverse hematology and clinical chemistry changes were observed in males and females at 300 and/or 750 mg/kg/day.
Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg for males and females.