2-Propenamide, N-[5-[[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-2-pyrimidinyl]amino]-4-methoxy-2-(4-morpholinyl)phenyl]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-18 to 2016-09-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: FDA Guidance for Industry, Single Dose Acute Toxicity Testing for Pharmaceuticals

Version / remarks:

CDER, August 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Nonclinical Safety Studies for the conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

Version / remarks:

CDER, January 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Food and Drug Administration Center for Drug Evaluation and Research (CDER)/International Conference on Harmonisation (ICH) Harmonised Tripartite Guidelines ICH-M3(R2)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide

EC Number:

841-500-6

Cas Number:

1903008-80-9

Molecular formula:

C30H34N8O3

IUPAC Name:

N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: 5138-117
- Retest date: 2016-07-07
- Appearance: Light yellow solid
- Purity: 98.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator, set to maintain 2 to 8°C and protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability has been evaluated within the bracketed concentrations of 0.1 and 200 mg/mL in a previous study (Covance Study No. 8333734). Formulations at 0.1 and 200 mg/mL were stable for up to 24 hours at room temperature (set to maintain 15 to 30°C), and up to 15 days at refrigerated (set to maintain 2 to 8°C) and frozen (set to maintain -10 to -30°C) conditions. Duplicate sets of samples (1.00 mL each) were taken from the top, middle, and bottom strata of the 20- and 200-mg/mL formulations prepared for administration on Day 1 of the dosing phase. One set of duplicate samples (for each stratum) was analyzed for test article content. The second set was retained as backup. Samples were analyzed on the day of preparation and stored at room temperature (15 to 30°C) and protected from light. Mean homogeneity values for the top, middle, and bottom of samples from dose formulation on Day 1, targeted 20-, 60-, and 200-mg/mL, had relative standard deviations ranging from 0.3% to 1%, and were acceptable for use on the study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stability of the YH25448 in the vehicle control article (Covance Study No. 8333734) was established within the bracketed concentrations of 0.1 mg/mL and 200 mg/mL.


FORM AS APPLIED IN THE TEST: liquid

Test animals

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female Crl:CD(SD) rats were received from Charles River Laboratories, Raleigh, North Carolina.
- Females (if applicable) nulliparous and non-pregnant: not indicated
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 160 to 185 grams for males and 149 to 176 grams for females
- Fasting period before study: not indicated
- Housing: Male and female rats were group-housed (two to three animals/sex/cage) in polycarbonate cages with hardwood chip bedding. Animals were individually housed in stainless steel or polycarbonate cages for study-related procedures.Animals were given various cage enrichment devices and dietary enrichment (that did not require analysis).
- Diet: ad libitum, Animals were offered Certified Rodent Diet #2014C (Envigo RMS, Inc.)
- Water: ad libitum
- Acclimation period: 7 days prior to initiation of dosing
- Method of randomisation in assigning animals to test and control groups: Animals were eliminated from consideration for study selection based on data collected during acclimation (predose phase). Animals were assigned to the study using a computerized procedure designed to achieve body weight balance with respect to group assignment. Prior to group assignment, animals may have been excluded from the selection pool/sex to produce minimal variation. After group assignment, the mean body weight for each group/sex was not statistically different at the 5.0% probability level, as indicated by analysis of variance F probability.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 10 or greater air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2016-01-25 To: 2016-02-08

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) methylcellulose (1500 cps) and 50 mM citric acid prepared in reverse osmosis water, pH adjusted to 3.0 - 3.2.

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0, 20, 60, 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: not indicated
- Lot/batch no.: Methylcellulose: SLBJ8516V (Retest date: 01 April 2017), Anhydrous Citric Acid, USP: 2DL0394 (Retest date: 28 October 2017)
- Purity: not indicated

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

DOSAGE PREPARATION:
- Test article formulations were prepared once by Covance according to the mixing procedure and apportioned for daily use. Dose concentrations were based on the test
article as supplied. Dose formulations were stored at room temperature (15 to 30°C), and protected from light.
- Dose formulations were allowed to equilibrate to approximately room temperature and stirred using a magnetic stir plate and stir bar for at least 30 minutes prior to and throughout dosing.

- Rationale for the selection of the starting dose: In previous 7-day studies in the rat conducted by the sponsor, dose levels of 62.5, 125, and 250 mg/kg/day were tolerated. Soft feces and perinasal red discharge were noted at doses of 62.5 and 125 mg/kg/day. Additional observations at a dose of 125 mg/kg/day included soiled anogenital area and perianal discharge. At a dose of 250 mg/kg/day, observations included mucous feces, hunched posture, hypoactivity, partial eyelid closure. Decreased body weight gain (47%) and food consumption (19%) were noted at a dose of 125 mg/kg/day. Similar effects were noted at a dose of 250 mg/kg/day (16% decrease in body weight and 58% decrease in food consumption). Clinical pathology changes were dose-related in terms of parameters affected, and included increased white blood cell, neutrophil, and red blood cell count, decreased mean cell volume, reticulocytes, and lymphocytes, decreased albumin, and alkaline phosphatase, and increased total cholesterol, blood urea nitrogen, and/or creatinine. Organ weight changes included increased thymus at a dose of 125 mg/kg/day, and decreased heart, kidneys, spleen and thymus, and increased adrenal at a dose of 250 mg/kg/day. Macroscopic findings included pale kidneys (62.5 and 125 mg/kg/day), dark spleen (125 mg/kg/day), and small kidneys and spleen, white spot in liver and enlarged adrenals (250 mg/kg/day). In addition, in a preliminary single dose study in the rat conducted by the sponsor, 1000 and 2000 mg/kg was tolerated. Therefore, dose levels of 200, 600, and 2000 mg/kg have been selected as the dose levels in this single dose rat study.

Doses:

0, 200, 600, 2000 mg/kg bw

No. of animals per sex per dose:

5 males and 5 females per dose, 4 groups in total

Control animals:

yes

Remarks:

5 males and 5 females

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/viability: twice daily
Clinical signs: Day 3 of the dosing phase through the dosing phase (except on the days of detailed observations). Detailed observations were conducted for each animal once during the predose phase, prior to dosing on Day 1, and on Days 8 and 14 of the dosing phase. Detailed observations were also collected on the days of scheduled sacrifice (all surviving animals).
Body weight: Body weights for animals were recorded once during the predose phase, before dosing on Day 1, and on Days 3, 8, 11, and 14 of the dosing phase.
- Necropsy of survivors performed: On Day 8 of the dosing phase, all males and females given 2000 mg/kg (Group 4) were sacrificed at an unscheduled interval. On Day 15 of the dosing phase, all surviving animals were fasted overnight, anesthetized with sodium pentobarbital, exsanguinated, and necropsied. Terminal body weights were recorded. The necropsy included an examination of the external features of the carcass; external body orifices; abdominal, thoracic, and cranial cavities; organs; and tissues.
- Other examinations performed:
Post dose observations: On the day of dosing, cageside observations were conducted for each animal approximately 1, 4, and 24 hours postdose. Postdose observation start times for each toxicity group were based on the dosing completion time for each animal.
Food consumption: Quantitative food consumption for animals was recorded on Days 1 to 3, Days 3 to 8, Days 8 to 11, and Days 11 to 14 during the dosing phase for each cage of animals.
Histology: the following tissues (unless noted as missing) from each animal were saved for possible future evaluation: adrenal, muscle (biceps femoris), optic nerve, aorta, ovary, brain, pancreas, cecum, pituitary gland, cervix, prostate, colon, rectum, duodenum, salivary gland (mandibular), epididymis, seminal vesicle, esophagus, skin/ subcutis eye, spinal cord (cervical, thoracic, and lumbar), femur with bone marrow (articular surface of the distal end), spleen, gross lesions, sternum with bone marrow, Harderian gland, stomach, heart, testis, ileum, thymus, jejunum, thyroid (2 lobes), with parathyroid, kidney, tongue, liver, trachea, lungs with large bronchi, urinary bladder, lymph node (mandibular), uterus, lymph node (mesenteric), vagina, mammary gland (females).

Statistics:

See section Any other information on materials and methods incl. tables.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

other: Maximum tolerated dose (MTD)

Effect level:

600 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No LD50 determined

Mortality:

All animals given 2000 mg/kg were sacrificed early on Day 8 of the dosing phase due to test item-related moribund condition and clinical observations. Although a definitive cause of death could not be determined based on macroscopic evaluation, these animals had reduced food consumption, decreased body weights, poor clinical condition (thin, hunched appearance, and mucoid feces) and abnormal macroscopic findings in the stomach (firm, abnormal lumen contents, and large, non-glandular mucosa), which were attributed to administration of the test article. All other toxicity animals survived to their scheduled sacrifice.

Clinical signs:

bodyweight loss

other:

Body weight:

greater than 10% body weight loss

Remarks:

Males and females given 200, 600 or 2000 mg/kg gained less, had no weight gain, or lost weight during the first three days of the study compared to controls. While animals given 200, or 600 mg/kg recovered from this initial effect, animals given 2000 mg/kg continued to experience significant body weight loss leading to early termination of the group. Although overall body weight gain (Days 1 through 14) was significantly less for females given 600 mg/kg, this was attributed to the initial weight loss during the first three days of the study. Body weight gain at subsequent intervals was comparable with controls. No test item-related alterations in mean body weight or body weight gains were noted for animals given 200 mg/kg from Days 3 through 14. test item-related decreases in mean body weights on Day 8 (-39.3% in males and -31.7% in females), and mean body weight gains for Days 1 through 8 (-183.6% in males and -217.8% in females) were observed in animals given 2000 mg/kg compared with the control values. Body weight loss observed in animals given 2000 mg/kg ranged from -30 to -57 g in males and -26 to -40 g in females from Day 1 to 8 and was considered adverse which correlated with decreased mean food consumption. Due to the severity of body weight loss, these effects were considered to be adverse and the group was terminated early.

Gross pathology:

test item-related macroscopic findings were present in the stomach of all animals given 2000 mg/kg and included abnormal, firm luminal contents, and large, non-glandular mucosa.
All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), or were as expected for animals of this strain and age; therefore, they were not considered test article related.

Other findings:

Food consumption:
In general, dose-dependent test item-related decreases in mean food consumption was observed from Days 1 through 8; however, animals given 200 or 600 mg/kg recovered and had food consumption values that were comparable with controls from Days 8-14. Test article-related decreases in mean food consumption corresponding with effects on body weight were observed in animals given 2000 mg/kg during Days 3 through 8 (up to -91.3% in males and up to -76.4% in females compared with controls). Due to the severity of this decrease, this effect was considered adverse and the group was terminated early.

Any other information on results incl. tables

Number of dead animals in each test group with 10 animals each:
Animals (male and female) 2000 mg/kg: 10
Animals (male and female) 600 mg/kg: 0
Animals (male and female) 200 mg/kg: 0

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In conclusion, male and female rats were given vehicle control article or YH25448 at a dose level of 200, 600, or 2000 mg/kg via oral route as a single dose and dose levels up to 600 mg/kg were well tolerated. Animals given 2000 mg/kg were sacrificed early at an unscheduled interval on Day 8 due to test article-related lower mean body weights and mean food consumption, and were associated with large non-glandular mucosa and abnormal firm luminal contents in the stomach. Lower mean body weights and mean food consumption was observed from Days 1 through 3 in animals given 200 or 600 mg/kg; however, the animals recovered and gained body weights comparable with
controls from Days 3 through 14. Therefore, the dose level of 600 mg/kg is considered the maximum tolerated dose (MTD) for this study.
Based on these results the estimated LD50 is concluded to be between 600 and 2000 mg/kg bw and the test item should be classified as Acute Oral Toxicant Category 4 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.