PF-05201372

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th November 2011 to 26th March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-N-(5-Cyano-4-{(E)-[(dimethylamino)methylene]amino}-2-methoxyphenyl)-4-(piperidin-1-yl)but-2-enamide

Molecular formula:

C20 H27 N5 O2

IUPAC Name:

(2E)-N-(5-Cyano-4-{(E)-[(dimethylamino)methylene]amino}-2-methoxyphenyl)-4-(piperidin-1-yl)but-2-enamide

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han) (outbred, SPF-Quality).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females: nulliparous and non-pregnant
- Age at study initiation: Approximately 10 weeks.
- Weight at study initiation: all animals within ± 20% of the sex mean
- Housing: Group housing of 3 animals per sex in Makrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage- enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions

DETAILS OF FOOD AND WATER QUALITY: Results of analyses for nutrients and contaminants were examined and archived. Certificates of analysis (performed quarterly) were examined and archived.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.2ºC
- Humidity (%): 43 - 56%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 05 December 2011 To: 13 December 2011

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

water

Remarks:

Elix, Millipore S.A.S., Molsheim, France

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

Frequency of treatment:

Once daily for up to 7 consecutive days, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw (total dose)

No. of animals per sex per dose:

3 males and 3 females.

Control animals:

yes, concurrent no treatment

Details on study design:

Rationale for dose levels
Dose levels for this 7-day oral gavage study were selected to be 0, 50, 150 and 300 mg/kg, based on the results of the acute oral toxicity study (NOTOX project 497161) and in consultation with the sponsor.

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 to start of treatment and at weekly intervals this were also performed outside the home cage in a standard arena. Clinical observations were conducted between 1 and 2 hours after dosing (except for the last observations in a standard arena) which was considered to be the peak period of anticipated effects after dosing based on results of the dose range finding study. 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) were scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.
Body weights: On Days 1, 4 and 7..
Food consumption: Over Days 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:

Animals were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight prior to scheduled necropsy. Descriptions of all macroscopic abnormalities seen at post mortem were recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin. Ovaries, Adrenal glands, Spleen, Brain [cerebellum, mid-brain, cortex] Stomach, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver, All gross lesions

The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all 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, treatment-related

Description (incidence and severity):

Hunched posture and piloerection was observed for all males at 300 mg/kg on the last day of treatment only. No clinical signs were noted for control animals, all animals at 50 and 150 mg/kg and females at 300 mg/kg.

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):

Slight weight loss was observed among the animals and is more often seen in animals of this age.
However, slight body weight loss in males was observed for one male at 150 mg/kg and two males at 300 mg/kg only and appeared to be related to treatment.
The incidence of slight weight loss among females was unrelated to dose levels and therefore, this finding was considered to be of no toxicological significance in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females at 300 mg/kg showed lower food consumption before and after allowance for body weight when compared to control females.
Food consumption before or after allowance for body weight was similar between control animals and animals at 50 and 150 mg/kg and males at 300 mg/kg.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following haematology parameters appeared to differ from values in control animals:
- Lower relative reticulocyte counts in one male at 150 mg/kg and all males at 300 mg/kg.
- Lower mean corpuscular volume (MCV) in one male at 150 mg/kg and one male at 300 mg/kg.
Higher white blood cell counts among males at 300 mg/kg and lower platelet counts among males at 50, 150 and 300 mg/kg remained within the range considered normal for rats of this age and strain and/or occurred without a treatment related incidence. Therefore, these findings are considered to be of no toxicological significance.
No apparent changes occurred in haematology parameters of treated females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following clinical biochemistry parameters appeared to differ from values in control animals:
- Lower creatinine levels in one male at 300 mg/kg.
- Lower triglyceride levels in one male at 150 mg/kg and in two males at 300 mg/kg.
- Lower calcium levels in two males at 300 mg/kg.
Aspartate aminotransferase (ASAT) levels of females at 300 mg/kg appeared lower when compared to values of control values, but lower ASAT levels are no sign of toxicity. Higher alanine aminotransferase (ALAT) in all females at 150 mg/kg and one female at 300 mg/kg, higher cholesterol levels for one male and two females at 150 mg/kg and one female at 300 mg/kg and lower bile acid levels in one male at 50 and one male at 300 mg/kg remained within the range considered normal for rats of this age and strain and/or occurred without a treatment related incidence. Therefore, these findings are considered to be of no toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following organ weights and organ to body weight ratios appeared to differ from values in control animals:
- Higher liver weights in two females at 150 mg/kg and one female at 300 mg/kg and higher liver to body weight ratio in all females at 150 and 300 mg/kg.
- Lower thymus weight in two females at 300 mg/kg and lower thymus to body weight ratio in one of these females at 300 mg/kg.
- Higher kidneys weight and kidneys to body weight ratio of one male at 300 mg/kg.
- Higher adrenals weight and higher adrenals to body weight ratio in one male at 300 mg/kg.
- Lower spleen weight in one female at 150 mg/kg and two males and two females at 300 mg/kg and lower spleen to body weight ratio of one female at 150 mg/kg and one female at 300 mg/kg.
- Lower ovaries weight and ovaries to body weight ratio in one female at 300 mg/kg. Higher liver weights and liver to body weight ratios among treated males and higher heart weights and higher heart to body weight ratios among treated males and females were considered to have arisen as a result of slightly low control values and considered to be of no toxicological significance. The control female that showed presence of fluid in the uterus also showed a high uterus weight which is related to the stage in the oestrus cycle.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have
arisen as a result of treatment.
Incidental findings included a yellowish soft nodule on the tail of the right epididymis of one control male and presence of fluid in the uterus of one control female. These findings are occasionally seen among rats used in these types of studies and are considered changes of no toxicological significance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the absence of an apparent correlation between the findings and the absence of morphologic evidence of toxicity, it was concluded that PF-05201372 has no toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecutive days up to 300 mg/kg.

Executive summary:

The study was based around the following guidelines:
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
Rationale for dose levels
Dose levels for this 7-day oral gavage study were selected to be 0, 50, 150 and 300 mg/kg, based on the results of the acute oral toxicity study (NOTOX project 497161) and in consultation with the sponsor.
Study outline
The test substance, formulated in water, was administered daily for 7 days by oral gavage to SPF bred Wistar rats. One control group and three treated groups were tested, each consisting of 3 males and 3 females.
Evaluated parameters
The following parameters were evaluated: clinical signs daily; body weight on Days 1, 4 and 7 and food consumption over Days 1-4 and 4-7; clinical laboratory investigations and macroscopy at termination; organ weights and histopathology on a selection of tissues.
Results
Males at 300 mg/kg showed hunched posture and piloerection on the last day of treatment and reduced body weight gain or slight body weight loss, but normal food consumption. Females at 300 mg/kg showed lower food consumption, but normal body weight gain. Several heamatology and clinical biochemistry parameters and organ weights in animals at 150 and/or 300 mg/kg appeared to differ from control values. However, values of these parameters were only slightly above or below the range considered normal and occurred in the absence of any treatment-related macroscopic or microscopic findings.
Conclusion
Based on the absence of an apparent correlation between the findings and the absence of morphologic evidence of toxicity, it was concluded that PF-05201372 has no toxic potential when administered to rats by daily oral gavage for a period of up to 7 consecutive days up to 300 mg/kg.