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.
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
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
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