Nonclinical Safety Evaluation

in Drug Development

Dr. Charlene Chen

Charlene@mail.dcb.org.tw
02-2695-6933 x 3116

Phases of Drug Development

Lead IND NDA

Target
Identification Preclinical Clinical Post
Identification
& Development Development Approval
& Validation
Optimization

Discovery Development
Stage Stage
 New Drug Discovery Process

Phases of Drug Development

Acute and Sub-chronic Toxicity Chronic Toxicity Carcinogenicity

Non
Non-clinical
Non-clinical Phase
Phase II Phase
Phase IIII Phase
Phase III
III Regulatory
Regulatory
Approved
Approved
Efficacy Safety Efficacy Confirmatory
Efficacy NDA
Metabolism PK-PD Safety in
100-300 Safety
Safety in 20-80 Health Patients Long-term
Animals Volunteers 1000-3000
Patients
2 – 5 yrs Up to 1 yr Up to 2 yrs 3 – 5 yrs Up to 2.5 yrs

10 Evaluated 5 Enter 3 Enter 2 Enter 1 Enter

 The Discovery Testing Scheme

Reasons for Attrition (1991 to 2000)

Nature Review/ Drug Discovery 3:711-715

Different Area of Toxicology

Mechanistic Toxicology

Risk
Regulatory Assessment Descriptive
Toxicology Toxicology

Guideline and Regulation

 OECD Guidelines
Duration Guideline No. Title
Acute 401 Acute Oral Toxicity
402 Acute Dermal Toxicity
403 Acute Inhalation Toxicity
420 Acute Oral Toxicity - Fixed Dose Method
423 Acute Oral Toxicity - Acute Toxic Class Method
425 Acute Oral Toxicity - Up-and-Down Procedure
Subacute 407 Repeat Dose 28-day Oral Toxicity Study in Rodents
410 Repeat Dose Dermal Toxicity - 21/28-Day Study
412 Repeat Dose Inhalation Toxicity - 28/14-Day
Subchronic 408 Subchronic Oral Toxicity - Rodent: 90-Day
409 Subchronic Oral Toxicity - Non-Rodent: 90-Day
411 Subchronic Dermal Toxicity: 90-Day
413 Subchronic Inhalation Toxicity: 90-Day
Chronic 451 Carcinogenicity Studies
452 Chronic Toxicity
453 Combined Chronic Toxicity/Carcinogenicity Studies

Pharmaceuticals
Guideline : ICH
S1: Carcinogenicity S1A: Need for Carcinogenicity S1B: Testing for S1C: Dose Selection (2008)
Studies (1996) Carcinogenicity (1998)
S2: Genotoxicity S2A: Specific Aspects of S2B: Standard Battery of S2 (R1): Genotox testing and
Regulatory Tests (1996) Tests (1997) Data Interpretation (2008)
S3: Kinetics S3A: Toxicokinetics (1995) S3B: Pharmacokinetics
(1995)

S4: Toxicity S4: Repeat Dose Toxicity in S4: Repeat Dose Toxicity in Non-Rodents (1999)
Rodents (1999)
S5: Reprotoxicology S5A: Toxicity to Reproduction S5B: Male Fertility (1996)
(1994)
S6: Biotech Safety S6: Safety Studies for S6 (R1): Safety Studies for Biotechnology Derived
Biotechnology Derived Products Products (2009)
(1997)
S7: Pharmacology S7A: Safety Pharmacology (2001) S7B: QT Prolongation (2005)
S8: Immunotoxicology S8: Immunotoxicity Studies (2006)
S9: Anticancer Safety S9: Non-clinical Evaluation for Anticancer Pharmaceuticals (2010)

• http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances
 ICH GUIDELINE
Duration of Repeated Dose Toxicity Studies to Support Phase I and II
Trials in EU and Phase I , II and III Trials in the US and Japan

Duration of Clinical Minimum Duration of Repeated

Trials Dose Toxicity Studies
Rodents Non-rodents
Single Dose 2-4 Weeks 2 Weeks
Up to 2 Weeks 2-4 Weeks 2 Weeks
Up to 1 Months 1 Months 1 Months
Up to 3 Months 3 Months 3 Months
Up to 6 Months 6 Months 6 Months
> 6 Months 6 Months Chronic
中華民國新醫藥品登記所需的毒性試驗

新投與 新劑型 新使用 新單位

資料項目 新成份 途徑 新療效 新複方 控釋製 速放製 劑量 含量
1. 急性毒性試驗
● ● Ⅹ ● ▲ Ⅹ ▲ ▲
(Acute toxicity test)
2. 亞急性毒性試驗
● ● Ⅹ ● ▲ Ⅹ ▲ ▲
(Subacute toxicity test)
3. 慢性毒性試驗
● ● Ⅹ ● ▲ Ⅹ ▲ ▲
(Chronic test)
4. 生殖試驗
● ● Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ
(Reproductive test)
5. 依藥性試驗
▲ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ
(Drug dependence)
6. 抗原性試驗
▲ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ
(Antigenicity test)
7. 致突變性試驗
▲ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ
(Mutagenicity test)
8. 致癌性試驗
▲ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ
(Carcinogenicity test)
9. 局部刺激性
▲ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ Ⅹ
(Local irritation)

註 ●：表示須檢附該項目之資料 Ⅹ：表示不須檢附該項目之資 料 ▲：表示視個案而定

www.doh.gov.tw
中華民國藥品非臨床試驗規範

中華民國植物抽取新藥
申請進行臨床試驗所需的毒性試驗

申請項目 第一階段臨床試驗* 第二階段臨床試驗* 第三階段臨床試驗*

1. 急性毒性試驗 ▲ ▲ ●
(Acute Toxicity Study)

2. 亞急性毒性試驗 ▲ ▲ ●
(Subacute Toxicity Study)
毒
3. 基因毒性試驗 ╳ ╳ ●
性 (Genotoxicity Study)

試 4. 慢性毒性試驗
(Chronic Toxicity Study)
╳ ╳ ▲
驗 5. 致畸試驗
( Teratology Study)
╳ ╳ ●
項
6. 生殖試驗 ╳ ╳ ●
目 (Reproductive test)

7. 致癌性試驗 ╳ ╳ ●
(Carcinogenicity Study)

註 ●：表示須檢附該項目之資料
╳：表示不須檢附該項目之資料
▲：試狀況而定，若在藥物管理先進國核準以藥品上市或核準以藥品進行臨床試驗者
，可免除之。
*：若係在國外製造，且生產國或藥物管理先進國以藥品上市者，可免除其動物毒理試驗。
www.doh.gov.tw
 Regulatory Approaches for
a Botanical Product

Consumed for Botanical Applied to body

Food its taste, aroma, for cleansing,
or nutritive value
Product beautifying, or
altering appearance

Ingested to Ingested to affect

supplement structure or What is
diet function of body the intended
use?

Used to diagnose, Cosmetic

cure, mitigate,treat
or prevent disease
Is it intended
for use as a drug
Dietary under 21 U.S.C.
supplement No 321(g)? Yes
Drug

Herbal Medicine

„ Follow same regulation for chemical drugs

„ Mixture of multiple ingredients or plants
„ 衛生署中醫藥委員會：中藥新藥查驗登記須知
„ EMEA: Guidance on non-clinical testing of
herbal drug preparations with long-term
marketing experience
„ USFDA: Guidance for Industry: Botanical
drug products, 2004
 Genetic Toxicology

Genetic Toxicity Studies

Purpose: Access the likelihood that the drug compound is

mutagenic or carcinogenic.

AMES test: Detect genetic changes

conduct in bacteria
DNA damage: Micronucleus ( in vitro and in vivo )
CHO cell and mouse peripheral blood cell
Chromosomal damage: Chromosomal aberration
 Salmonella Reverse Gene Mutation
(Ames Test)
(SOP: DCB-DV-TE00201)

• Test System: Salmonella typhimurium

TA97 or TA1537, TA98, TA100, TA102, and TA1535
• Principles:
Histidine- Histidine+
• Metabolic Activation:
Aroclor 1254-induced rat liver homogenate
-- S9 mixture

Chromosome Aberration Assay

(SOP: DCB-DV-TE00217)

• Test system: CHO, V79 cells or Human peripheral

blood lymphocytes
• Max. conc.: >50% cytotoxicity or solubility limit , 5
mg/ml or 10 mM
• Conc. levels: ≥ 3 conc. ; positive and negative
controls with duplicate cultures
• TA treatment: 3h ± S9 and 20h (or 24h, 48 h) –S9,
cell harvest at 20h (or 24 h, 48 h)
• Analysis: code slides, score 200 metaphases
per conc.
 Micronucleus Assay
(Mouse Peripheral Blood)
(SOP: DCB-DV-TE00227)

• Test system: BALB/c or ICR mice

• Dose levels: 3 doses ; positive and vehicle
controls, 5 mice/group
• Drug treatment: gavage or i.p. injection; single or
multiple administration
• Blood collection: 48 and 72h for single dose;
36-48 h after the final dose for the
multiple dose study
• Analysis: score 2000 reticulocytes per animal
(SOP: DCB-DV-TE00258)

Animal Studies
 Test System
¾ IACUC-approved protocol

¾ Health screening prior to study start

Rodents: SPF
Dogs: standard vaccinations

¾ Quarantine: veterinary approval to release prior to

study initiation

¾ Acclimation
Rodents: usually 7 days
Dogs: at least 14 days

Acute Toxicity
One or more doses administered over a period of up to
24 hours

Goal: Determine toxic dose levels and observe

clinical indications of toxicity.

Purpose: Help to determine doses for repeated dose

studies in animals and Phase I studies in
human

Contents: Clinical observation (1, 2, 3, 4 hours after dosing

then daily)
Body weight
Gross necropsy
 Acute Toxicity
■ Weighing General: 4 groups (3 dose ＋ 1 control)
■ Dosing Limit Test: 2 groups (1 dose ＋ 1 control)
10 /group (5♂/ 5♀)

Clinical Observation
6 /group (3♂/ 3♀)
0 1 2 3 4 5 6 7 8

SD 1 :hours after dosing

Gross Necropsy
Extended single dose study:
SD2 and SD14 Histopath. Weighing
Weighing

Study Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Clinical Observation

Repeated Dose Studies

Depending on the duration of the studies, maybe referred
as subacute, suchronic, or chronic.

Specific duration should anticipate the length of the clinical trial

that will be conducted on the new drug

Contents: Clinical observation (daily)

Body weight (weekly)
Food consumption (weekly)
Ophthalmologic and EKG Examination
Gross necropsy
Clinical pathology (urine, whole blood, serum chemistry)
Organ weight
Histopathology
 Recovery
Repeated Dose Studies Dosing Period Period
Yes
Body Weight No
Pre-study Dosing Yes
Clinical Observation Yes
Body Weight Food Consumption
3 Dose +1 Control Clinical Observation Subacute (week) 1 2 3 4 1 2 3 4
Food Consumption
10+5R♂ Ophthalmologic Examination
10+5R♀ ECG & Clinical Pathology (CP)
4+2R♂
4+2R♀ Subchronic (week) 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4

20+10R♂
20+10R♀ CP, Ophthalmology & ECG

Chronic (week) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 1 2 3 4

R 5

1
C 3
4 5
Histopathology
1 3
Histopathology*
4

Ophthalmology & ECG 2

2 Gross Necropsy Ophthalmology & ECG Gross Necropsy
Clinical Pathology Clinical Pathology*
Body Weight
Body Weight

Clinical Observations
Respiratory Salivation
Difficult or labored breathing
Motor activities Piloerection
Changes in frequency and nature
of movement
Convulsion Analgesia
↓ reaction to induced pain
Reflexes Muscle tone
Hyptonia, Hypertonia
Ocular signs GI signs
Dropping, Emesis, Diuresis
Cardiovascular signs Skin
Vasoconstriction, Vasodilation, Edema, Erythema
↓ or ↑ heart rate
 Animal Weighing
■ Balance (g)
mice – 0.1
rats – 1
rabbits – 10
dogs – 100

■ Quality Control of Balance

- Check: twice daily
- Calibration: once a year (CNLA)

■ Historic Data
- Establish historical data
- Quality control reference

Urinalysis (10 items)

(SOP: DCB-DV-TE00451)

pH Glucose
Specific gravity Ketones
Nitrites Urobilinogen
Occult blood Bilirubin
Protein Leukocytes

Bayer Clinitek 100 Urine Chemistry Analyzer

(SOP: DCB-DV-EQ00066)

Gravity will be examined by Clinical Specific Refractometer

(SOP: DCB-DV-EQ00070)
 Hematology (12 items)
(SOP: DCB-DV-TE00425, DCB-DV-TE00413 )

Erythrocytes (Red blood cells) count

Hemoglobin Hematocrit
Mean corpuscular volume (calculated)
Mean corpuscular hemoglobin (calculated)
Mean corpuscular hemoglobin concentration (calculated)
Leukocytes (White blood cells) count
Leukocyte differential count
(Lymphocytes, Monocytes, Neutrophils, Eosinophils, Basophils, etc.)
Reticulocytes Platelet count

Prothrombin time Activated partial thromboplastin time

Bayer ADVIA 120 Hematology Analyzer (SOP: DCB-DV-EQ00091)

Sysmex CA-1000 Automated Coagulation Analyzer (SOP: DCB-DV-EQ00071)

Serum Chemistry (22 items)

(SOP: DCB-DV-TE00442)

Alkaline phosphatase Albumin/Globulin ratio (calculated)

Alanine aminotransferase Sodium
Aspartate aminotransferase Potassium
ϒ-glutamyltransferase Chloride
Glucose Calcium
Total bilirubin Phosphorus
Direct bilirubin Cholesterol
Creatinine Triglycerides
Blood urea nitrogen Lactate dehydrogenase
Total protein Creatine phosphate kinase
Albumin Uric acid

Hitachi Model 7060 Automated Analyzer (SOP: DCB-DV-EQ00075)

 Histopathology Examination
(37 items)
Adrenals Pancreas
Aorta Penis (male only)
Bone with bone marrow (femur & Pharynx/Larynx
sternum) Pituitary gland
Brain (medulla/pons, cerebellum, Prostate (male only)
cerebrum) Salivary glands (submandibular & parotid
Esophagus Sciatic nerve
Eyes including optic nerve Skeletal muscle (thigh)
Gall bladder Skin
Gonads: Small intestine:
Ovaries and oviducts (female only) Duodenum
Testes with epididymis (male only) Ileum
Heart Jejunum
Kidneys Spinal cord (cervical, thoracic & lumbar)
Large intestine: Spleen
Cecum Stomach
Colon Thymus
Rectum Thyroid/Parathyroid
Liver Tongue
Lung (with main-stem bronchi) Tonsil
Lymph nodes (submandibular & Trachea
mesenteric) Urinary bladder
Masses/Target organs Uterus (both horns) and cervix (female only
Mammary glands (female only) Vagina (female only)

Reproduction Studies

Segment I : Provide the overview of reproductive toxicity

with treatment of both sexes throughout
gametogenesis
(60 days for males and 15 days for females)

Segment II: Teratology, screen the capability of an agent to

elicit toxicity during the period of organogenesis

Segment III: Assess agent-induced effects during

organogenesis in neonatal life
 Teratology Studies
Purpose: Evaluate the effects of the test article on maternal
pregnancy, embryo-fetal development and fetuses while
administered orally to pregnant rats from gestation day
6 to day 15.

Contents: Clinical observation (daily)

Body weight (at least twice weekly)
Food consumption (weekly)
Reproductive performance
Gross necropsy (external, visceral and skeletal of fetus)

Safety Pharmacology
Radiotelemetry
Safety pharmacology core battery (conscious animals)
(GLP compliant ICH S7A) Blood pressure
Heart rate
Core battery ECG/QT interval

Cardiovascular system Motor activity

Behavior changes
Central Nerve system
Coordination
Respiratory system Body temperature

Urine and blood parameter

Renal/Urinary system
Creatinine clearance
Gastrointestinal system Functional measure
Respiratory rate
Tidal volume
Respiratory function Gastric emptying & GI
propulsion in rodents
 Carcinogenesis
Purpose: Observe test animals for a major life span for the
development of neoplastic lesions during or after
exposure to test article

Duration: 18 months for mice, 24 months for rats

Contents: Clinical observation (daily)

Body weight (weekly for first 13 weeks, once every 4 weeks thereafter)
Food consumption (weekly for first 13 weeks, approximately 3 months
thereafter)
Differential blood count (12, 18 months and prior to sacrifice)
Gross necropsy
High standard Histopathology

Animal condition: Carcinogenesis study Related condition:

Routine monitoring
Additional monitor
Necropsy
Histopathology
Animal facility
Dosing training
Historical data establish Animal allocation
Study conducting

References

„ ICH: www.ich.org -> Safety Topics

„ USFDA: www.fda.gov/cder/PharmTox/guidance.html
„ USFDA: Botanical drug products, 2004
„ USFDA: www.cfsan.fda.gov/~redbook/red-toca.html
„ OECD: www.sourceoecd.org
„ EMEA: www.emea.eu.int
„ OPPTS Harmonized Test Guidelines (US EPA)
„ DOH: www.doh.gov.tw
„ ISO10993
„ USP