The nonclinical safety study recommendations for the marketing approval of a pharmaceutical usually include single and repeated dose toxicity studies, reproduction toxicity studies, genotoxicity studies, local tolerance studies, and for drugs that have special cause for concern or are intended for a long duration of use, an assessment of carcinogenic potential. Other nonclinical studies include pharmacology studies for safety assessment (safety pharmacology) and pharmacokinetic (absorption, distribution, metabolism, and excretion (ADME)) studies. These types of studies and their relation to the conduct of human clinical trials are presented in this guidance.

1. Presented
by
Dr. Rahul G. Kadam
Ph.D Scholar
Roll NO. P1661

2.  Toxicology is a branch of science that deals with toxins and
poisons and their effects and treatment.
 Toxicological screening is very important for the development of
new drugs and for the extension of the therapeutic potential of
existing molecules.
 The US-FDA states that it is essential to screen new molecules for
pharmacological activity and toxicity potential in animals (21CFR
Part 314).
 Toxicity tests are mostly used to examine specific adverse events
or specific end points such as cancer, cardiotoxicity, and skin/eye
irritation.
 Toxicity testing also helps calculate the No Observed Adverse
Effect Level (NOAEL) dose and is helpful for clinical trails.

3.  Paracelsus (Father of Toxicology): determined
specific chemicals responsible for the toxicity of
plants and animals (dose-response relationship).
 "All substances are poisons; there is none which
is not a poison. The right dose differentiates a
poison and a remedy”
--Paracelsus
 Mathieu Orfila, determined the relationship
between poisons and their biological He is
referred to as the father of modern toxicology.
Paracelsus
(1493-1541)
Recent developments: after
1920
(introduced determine LD50

4. Benefit –risk ratio can be calculated
Prediction of therapeutic index
Therapeutic index= Maximum tolerated dose
Minimum curative dose
Smaller ratio, better safety of the drug

5.  Pharmacological effects are same in man as in animals
 Toxic effect in species will predict adverse effects in man
 Giving high doses in animals improves predictability to man
 Risk assessment can be made by comparison of toxic doses in
test species with predicted therapeutic dose in man

6. PHASES OF DRUG DEVELOPMENT
(ANIMAL MAN)
PHASE III PHASE IVPHASE I
PHASE IPHASE I
PRECLINICALPRECLINICAL PHASE II
Product Approval
(NDA/MAA)
Patient
studies
Entry to man
(IND / CTA)
NoneNone
Healthy subjects
Safety and
tolerability
Healthy subjects
Safety and
tolerability
Genetic toxicity
(in vivo)
Repeat dose
toxicity testing
+
Bioanalysis /
Toxicokinetics
Drug Metabolism
Reproductive
Toxicity Testing
(teratogenicity)
Genetic toxicity
(in vivo)
Repeat dose
toxicity testing
+
Bioanalysis /
Toxicokinetics
Drug Metabolism
Reproductive
Toxicity Testing
(teratogenicity)
Patients
Small scale
efficacy studies
Patients
Small scale
efficacy studies
Patients
Large scale
multicentre
studies
Patients
Large scale
multicentre
studies
Chronic (long term) toxicity testing
+
Bioanalysis / Toxicokinetics
Reproductive Toxicity Testing
(fertility and pre/post natal)
Carcinogenicity studies
Drug Metabolism
Chronic (long term) toxicity testing
+
Bioanalysis / Toxicokinetics
Reproductive Toxicity Testing
(fertility and pre/post natal)
Carcinogenicity studies
Drug Metabolism
Patients
Large scale
post-marketing
studies
Patients
Large scale
post-marketing
studies
As required
As required
Genetic toxicity
(in vitro)
Single / repeat
dose
toxicity studies
+
Bioanalysis /
Toxicokinetics
Safety
Pharmacology
Drug Metabolism
Lead
candidate
ClinicalNon-clinical
MOLECULE

7. Studies should comply with GLP
Performed by trained and qualified staff
Use of standardized and calibrated equipment
SOP’s followed in laboratory tasks
All documents should be preserved for minimum 5 years after
marketing of the drug

8.  OECD Guideline
 EPA Guideline
 FDA Guideline
 GAITONDE Guideline

10. TOXICOKINETIC STUDIES
Generation of Pharmacokinetic data to access systemic
exposure achieved in animals
Relation to dose level and the time course of toxicity study
To support choice of species & Treatment regimen
Design on clinical studies accordingly

11.  Pharmacodynamic responses
 Pharmacokinetic profile
 Species, sex, age of experimental animals
 Susceptibility, sensitivity and reproducibility of test
system
 In vitro: Isolated organs, tissues cell-cultures
 Mechanism of effect in vivo

12. Systemic toxicology studies
Single dose studies Repeated dose studies
Reproductive toxicology studies
Male fertility Female reproduction & Developmental
studies
Local toxicity studies
Hypersensitivity studies
Genotoxicity studies
Carcinogenicity studies

13. Preliminary Definitive
• Maximum Non
Lethal dose
(MNLD) determined
• MTD and MLD
determined
• Evaluate effects
• Target organ of toxicity
may be determined
a) SINGLE DOSE STUDIES/ ACUTE TOXICITY

14. METHOD
 Single dose tested in 2 rodent species
 2 routes of administration
 Oral dosing of 2g/kg or 10 times of normal human dose
 Observation for 14 days after dosing
 MNLD established
 Symptoms , signs reported
 Microscopic and Macroscopic evaluation

15. METHOD
 Group of 20 animals of either sex dosed at MNLD
 5 animals of each sex are observed for 48 hr and conduct
autopsy for early pathological changes
 Remaining 5 of each sex are observed for 14 days
 MTD and MLD established
 Signs of intoxication or recovery, changes in body weight,
pathological changes
 Complete macroscopic and microscopic examination
 Target organs can be identified

16.  Two mammalian species(one should be non-rodent)
 Long duration studies (30-180 days)
 Dose is dependent on dose-escalating studies
 Drug administered by clinical route
 Parameters monitored and recorded are:
 Behavioral
 Physiological
 Biochemical
 Microscopic observations
b) REPEATED DOSE STUDIES/SUB-ACUTE
OR CHRONIC TOXICITY

17. a) MALE FERTILITY
METHOD
One rodent species(rat)
3 dose groups taken
(each with 6 adult males),
1 control
Drug treatment by clinical route for 28-72 days

18. Mated with females in 1:2 ratio
Females getting pregnant should be examined
After 13 days of gestation
All male animals sacrificed
•Weights of testis, epididymus recorded & examined for
their histology
•Sperms examined for motility & morphology

19. Segment I
19
Fertility and general reproductive
performance study
Segment II Teratogenicity
Segment III Peri and post-natal study
Fertility and early embryonic
development (rat)
Embryo- foetal development
(rat & rabbit)
Post natal development (rat) (post natal
survival of offspring), growth parameters,
vital senses, behavioral effects
b) FEMALE FETILITY
Drug administered to both males (28days) and females (14
days) before mating
Implantation
Embryogenesis

20.  Required when drug is administered by special route
(other than oral) in humans
 Study design:
 2 species along with control used
 Dose dependent on dose escalating studies
 3 dose levels

21. Dermal toxicity studies
Dermal photo-toxicity
studies
Vaginal toxicity studies
Rectal tolerance studies
Rats & Rabbit
Local signs (erythema, oedema),
histological examination
Guinea pig
Used in treatment of leucoderma
Examination of erythema &
oedema formation
Rabbit or Dog
Observation of swelling,
histopathology of vaginal wall
Rabbit or Dog
Signs of pain, blood or mucous,
histology examination of rectal
mucosa

22. Ocular toxicity studies
Parenteral drugs
Inhalation toxicity studies
Albino Rabbit
Changes in cornea ,Iris &
aqueous humor, histological
examination of eye
For intravenous/
intramuscular/ subcutaneous/
intra-dermal injection
Sites of injection examined
grossly and microscopically
One rodent and non rodent
species
Acute , sub-acute and chronic
studies performed
Observation of respiratory rate
Histological examination of
respiratory passages, lung tissue

23. Guinea Pig Maximization
test
Local lymph node assay
Determination of Maximum non
irritant or minimum irritant dose
Evaluation of Erythema and
oedema
Mice of one sex(either male or
female)
Drug treatment given on ear skin
Auricular lymph node dissection
after 5 days
Increase in 3h-thymidine used for
evaluation

24. To detect early tumorigenic effects in cases of chronic illness
In vitro tests:
Test for gene mutation in Bacteria
Cytogenetic evaluation of chromosomal damage in
mammalian cells
E.g.; Ames’s Salmonella Assay detects increased number
of aberrations in metaphase chromosomes
DNA strand breaks, DNA repair or recombination,
Measurements of DNA adducts
In vivo tests:
Chromosome damage in rodent hematopoietic cells
E.g.; Micronucleus Assay

25.  Life-time Bioassays
 Carcinogenicity studies are performed on:
 Drug used for >6 months or frequent intermittent use for
chronic diseases
 Chemical structure of drug indicates carcinogenic potential
 Therapeutic class of drugs which have produced positive
carcinogenicity

26. Group sizes of 50 animals/sex at each of 3 dose
levels
Control group is of double size
Record for onset of tumor development
Usually carried out for 24 months in rats and 18
months in mice (life span studies)
CONDUCT OF STUDY

27. EVALUATION OF RESULT
Incidence of cancers in control and test
 Trend towards increasing incidence with
increasing doses
Number of animals with single/multiple tumors
Macroscopic changes observed by autopsy
 Histopathology of organs and tissues