ICH S2: Genetic Toxicity• Genetic toxicology studies include in vitro and in vivo

systems

• Evaluate the potential to induce mutations and chromosomal damage

– bacterial mutation

– cytogenetics

– mammalian gene mutation

Genetic Toxicology• “Standard Battery” for Genotoxicity

– A test for gene mutation in bacteria• Ames assay (bacterial reverse

mutation assay)• Identifies comparatively subtle

effects on chromosomes (point mutations, substitutions, frame shifts)

• Tester strains of E. coli and Salmonella typhimurium

– An in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells (Chromosomal aberrations assay)

– An in vivo test for chromosomal damage using rodent hematopoietic cells

ICH S7: Safety Pharmacology• In vitro and in vivo studies conducted to determine

whether this compound has any effects on:

• Brain –central nervous system

• Lungs –respiratory system

• Heart –cardiovascular system

Safety Pharmacology• “Core Battery”

– Cardiovascular• Generally use ionic current assay (hERG), followed by in vivo QT assessment• In vivo cardiovascular safety studies in anesthetized or conscious telemetrized dogs

or monkeys evaluate both electrophysiology and hemodynamics– Respiratory

• Can be included in some in vivo cardiovascular or general toxicology studies• Usually single dose study in rats

– Neurobehavioral• Functional Observational Battery or Irwin Assay• Usually single dose study in rats• Evaluates learning/memory, muscle grip strength, cranial nerve functions, and open

field motility• Additional studies may be triggered by findings from general tox studies

– Immunotoxicology (NK and CMI parameters, immunization studies, etc.)– Renal Toxicology– Gastrointestinal pharmacology (motility)

General Toxicology• Two species, at least one of which is not a rodent

– Generally rat as rodent– Either cynomolgus monkey or dog– Pigs, rhesus, ferrets, etc. sometimes used as second species

• Normally dose via the intended route of human exposure

• No single study design or group of studies fits all molecules– Program tailored to clinical plan, availability of API, PK/metabolism

data, “formulatability”, etc.– In U.S., duration of preclinical studies supporting human trials is

generally equal units of time

General Toxicology• Duration of dosing guided by indication and anticipated

duration of therapeutic use– Acute toxicity– Subchronic

• 2, 4, 13-weeks of daily dosing• Sequence dependent on requirements for clinical trial support

– Chronic (6-months in the rodent; 9-months in the non-rodent)• Why do toxicologists always want to dose to such high levels?

– Highest dose produces toxicity– Low dose ideally is not associated with toxicity (establish TI)– Middle dose(s) produce intermediate results (establish NOEL/LOEL)

Endpoints• Evaluation of toxicity is based upon

– Clinical observations• At least once daily• Ideally conducted at/near Tmax

– Body weight/food consumption• Daily body weights for short duration studies• Twice weekly for studies of 4-week or longer duration

– Ophthalmic examination• Pre-study• During last week

– Electrocardiography (non-rodent)• Pre-study• During last week

Endpoints (cont.)• Clinical pathology– Pre-study and during last week (non-rodent)– At/near termination (rodent)– Clinical chemistry– Hematology– Urinalysis– Coagulation

Endpoints (cont.)• Gross post mortem evaluation– External examinations to include any in-life

observations– Evaluation of body cavities• Thoracic• Abdominal• Pelvic• Cranial

Endpoints (cont.)• Organ weights– Absolute– Relative

• To brain weight• To terminal body weight

– Typical list: adrenal gland, brain, epididymis, heart, kidney, liver, lung, ovary, pituitary, salivary gland, spleen, testis, thymus, thyroid/parathyroid

Endpoints (cont.)• Histopathology– Microscopic examination of tissue sections– Formalin-fixed, paraffin-embedded, H&E-stained– Includes gross:micro correlation– Non-rodent: All tissues, all animals– Rodent

• Frequently compare high dose with control initially• “Read down” of target tissues in low, mid, and recovery groups

Tissues Examined• Adrenal Gland• Aorta• Bone Marrow• Bone Marrow Smear (collected; examined if necessary)• Bone (femur)• Bone (sternum)• Brain (cerebellum)• Brain (cerebrum)• Brain (brainstem)• Cecum• Cervix• Colon• Duodenum• Epididymis• Esophagus• Eye• Gall Bladder (not in rat)• Heart• Ileum• Jejunum• Kidney• Lacrimal Gland• Liver• Lung• Lymph Node (cervical) (not in rat)• Lymph Node (mandibular)• Lymph Node (mesenteric)

• Mammary Gland• Optic Nerve• Ovary• Pancreas• Parathyroid Gland• Pituitary Gland• Prostate• Rectum• Salivary Gland• Sciatic Nerve• Seminal Vesicle (not in dog)• Skeletal Muscle• Skin• Spinal Cord (cervical)• Spinal Cord (thoracic)• Spinal Cord (lumbar)• Spleen• Stomach• Testis• Thymus• Thyroid• Tongue• Trachea• Urinary Bladder• Uterus• Vagina

ICH S3: Toxicokinetics - Objectives• Verify exposure to the test material in dosed groups and lack

of exposure in control groups• TK parameters of interest to toxicologists

– AUC = Area under the plasma concentration vs time curve• Note units – AUC(0-t); AUC(0-24) ; AUC(0-inf)

– Cmax = Maximum plasma concentration– Tmax = Time to achieve maximum plasma concentration– T1/2 = Elimination half-life– F = Percent bioavailability

0.1

1

10

100

1000

10000

0 4 8 12 16 20 24 28

Time (hr)

Pla

sma

Co

nce

ntr

ati

on

(n

g/m

L)

Male Rats

Female Rats

Must evaluate exposure in animals

Interpretation of Toxicology Data• NOEL = No-Observable Effect Level• NOAEL = No-Observable Adverse Effect Level• LOEL = Lowest Observable Effect Level• MTD = Maximum tolerated or minimally toxic dose• Therapeutic exposure multiple = Exposure at NOAEL divided

by exposure at maximum human dose– Exposure usually determined by AUC ratios– Occasionally, Cmax or Cmin define both efficacy and toxicity; TEM’s

based on these parameters may also be justified• Cancer indications

– STD10: Severely toxic dose in 10% of rodents– HNSTD: Highest non-severely toxic dose in non-rodents

Interpretation of Toxicology Data

• Important factors for human risk assessment– Ability to identify NOAEL– Existence of biomarkers of injury– Reversibility of finding(s)– Likely relevance of finding(s) to human– Known or postulated mechanism of toxicity

How Much of an Exposure MultipleDo I Need?

• There is no answer to this question; however, feel better with 100X than with 5X!

• Answer is dependent upon– Therapeutic indication– Unmet medical need– Nature of toxicity observed in animals– Ability to monitor toxicity clinically– Reversibility of toxicity

• The therapeutic exposure “window” does not widen with increasing duration of dosing; only stays the same or narrows

• From early, short-duration studies, want TEM to be as wide as possible

Complete Toxicology Profile

Preliminary Evaluation

Genetic Toxicology BatteryAcute Toxicology

Subchronic Toxicology(14-day to 90-days)

Chronic Toxicology(6-month to 12-

month)

Oncogenic Studies(2-year)

Genetic ToxicologyKinetics

Acute and Multidose Toxin vitro Tox

Definitive toxicokinetics

Reproductive and Developmental Tox(timing affected by inclusion of WCBP)

Environmental Assessment

Toxicology of major human metabolites

Toxicology of degradation products

Safety Pharmacology

Dorato, M. A., Vodicnik, M. J. The Toxicologic Assessment of Pharmaceutical and Biotechnology Products. in Principles and Methods of Toxicology. Raven Press, 1994

Minimum For IND…• Genetic Toxicity “Standard Battery”• Safety Pharmacology “Core Battery”• General Toxicology with Toxicokinetics– Two species– Duration sufficient to cover duration of initial

clinical studies

• All studies conducted according to Good Laboratory Practices (GLP)