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Animal Toxicological Studies


• Man has always sort for ways to improve his health quality,

eradicate, and prevent disease conditions that always attack him.

• This has led to the discovery and development of many
pharmacological/chemical agents which are currently used for
diagnosis, treatment, and preventive purposes.

• These pharmacological/chemical agents have been developed
from synthetic, semi-synthetic, and natural (e.g., plant) materials.

• Although these agents have played a remarkable role in
improving man’s health quality, they are not all totally safe as
they possess the potential for causing harm. 2


• This has led to the inclusion of toxicity test for

pharmacological /chemical agents and food additives being
developed for use in man.

• This is with the purpose of ensuring that these agents are
relatively safe at a therapeutic dose (or intended dose for use)
before they could be used as medicines (and for other

• The safety of pharmacological/chemical agents and food
additives has become the main and longest-standing toxicology
issue of public interest.

• Risks associated with lack of safety of these agents are likely to
affect a large percentage of wpwwo.DuploMuix.lcaomtion. 3


• Exposure of the public to pharmacological/chemical agents

without proper knowledge about their toxicity has ended up in
several notable tragedies (e.g., the thalidomide disaster and
diethylene glycol tragedy). 4


• It was found that consumption of popular sedative thalidomide

by pregnant females resulted in birth of babies with sealed limbs.

• Thousands of children born with severe birth defects resulting
from pregnant women using thalidomide, an anti-nausea medicine

Phocomelia 5


• In 1968 an outbreak of Phenytoin toxicity was reported in

Australia which was because of change in formulation i.e.
switching over from calcium to lactose as an inert excipient in
the tablets.

• In them the blood phenytoin levels were above the therapeutic

• This change was probably causing the altered blood phenytoin

• Hence it was realized that studies must be done to investigate
bioavailability, pharmacokinetics and toxicity of different
formulations. 6


Drug-Discovery, Development


• IND – Investigational New Drug Application

• NDA – New Drug Application

7 7


What is Toxicology?

• Toxicology is the study of the adverse
effects of chemical, physical, or biological
agents on people, animals, and the

“Solely the dose determines

• Toxicologists are scientists trained to that a thing is not a poison.”

investigate, interpret and communicate the Paracelsus (German-Swiss
nature of those effects. physician), 1493-1541.

Water, in excess, is toxic. 8


Why study toxicology?

• Benefit –risk ratio can be calculated
• Prediction of therapeutic index
• Therapeutic index= Maximum tolerated dose

Minimum curative dose
• The larger the therapeutic index (TI), the safer the drug is.
• If the TI is small (the difference between the two
concentrations is very small), the drug must be dosed
carefully and the person receiving the drug should be monitored
closely for any signs of drug toxicity. 9


Why do we require non clinical studies IN ANIMALS before a new
molecule is ADMINISTERED to man??

• 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 10


General principles in toxicity studies

• 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 11


Toxicity Studies

In vivo Intact animals

– Isolated tissues
In vitro – Specific cells

– Organisms 12

Intact animals


In vitro toxicology

• In vitro toxicology

–The crossover point between drug discovery and drug development.

–Provide information on mechanism(s) of action of a drug

–Provides an early indication of the potential for some kinds of toxic

effects, allowing a decision to terminate a development program before

spending too much money.

• In vitro methods are widely used for:

–Screening and ranking chemicals

–Studying cell, tissue, or target specific effects

–Improve subsequent study design 13


In vitro toxicology

• Screening


–Protein binding

–CYP inhibition/induction

–Membrane permeability

–Metabolic stability

–Interspecies comparison 14


In Vitro Toxicology

• Cytotoxicity = toxicity to cells

•Many different types of cells can be used; cells from higher organisms

(e.g., liver cells, blood cells); bacteria; fungi; yeast

• Can be used to assess viability, structural effects, and/or function

–Structural – e.g., effects on membrane integrity

–Functional – e.g., effects on mitochondrial function

–Cell proliferation – decreases or increases 15


In Vitro Toxicology
Dermal or Ocular Toxicity

• Replace in vivo tests such as Dermal Corrosion, Skin Irritation,
Draize Eye Irritancy

• Many tests now available in kit form

• Example: EpiDerm
–Normal human epidermal keratinocytes
–Cultured on a permeable polycarbonate membrane
–Stratified, highly differentiated, model of human epidermis
–Metabolically and mitotically active cells organized into

differentiated layers 16


In Vitro Toxicology

• These are, by necessity, functional tests

• Examples

–Cytokine release. Assess ability of a chemical to induce release

of cytokines/chemokines. Can use cells from various sources,

including peripheral blood cells. Many variations of this assay.

–Drug-induced histamine release. 17


Advantages of in-vitro toxicity studies

• The main advantages of in vitro tests are as follows:

(1) Reduction of animal sacrifice during toxicity screening of various

(2) More controlled exposure conditions (hormonal and cofactor
make-up of the exposure media),

(3) Increased precision of the response (less biological variation than
with in vivo systems)

(4) High bioanalytical throughput for rapid screening investigations.

(5) Less regulatory constraints and ethical considerations 18


In-vivo toxicological models 19


In Vivo Toxicology – Purpose

Results from preclinical toxicology studies should, at a minimum:

–Establish a safe starting dose for clinical studies

–Provide information on a drug-treatment regimen that would

produce the least toxicity

–Assess target organ toxicity and its reversibility

–Provide insight into biomarkers for clinical monitoring 20


Standard methods

Multiple methods have been standardized (certified) by multiple

• American Society for Testing and Materials (ASTM)

• Organization for Economic Cooperation and Materials (OECD) –

(Europe based)

• National Toxicology Program (NTP) 21


Regulatory mechanism in India
• Institute Animal Ethics Committee (IAEC)

• Committee for the Purpose of Control and Supervision of
Experiments in Animals (CPCSEA)

• Drugs & Cosmetics Act, 1940, Appendix-I

• Department of Animal Husbandry, Dairying & Fisheries, Ministry
of Agriculture, New Delhi-2001

• Department of Biotechnology.

• The Prevention of Cruelty to Animals Act, 1960 as amended up to
30th July 1982 and Animal Welfare Board 22


Relevant Test Models

• 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 23


Types of toxicology studies

1. Systemic toxicology studies

Single dose studies Repeated dose studies

2. Reproductive toxicology studies

Male fertility Female reproduction & Developmental studies

3. Local toxicity studies

4. Hypersensitivity studies

5. Genotoxicity studies

6. Carcinogenicity studies 24


1. Systemic toxicology studies

a. Acute toxicity / Single dose studies

Preliminary Definitive

• Maximum Non Lethal dose • MTD and MLD determined
(MNLD) determined • Evaluate effects

• • MTD and MLD
• No-observed-adverse-effect • Target organ of toxicity may

level (NOAEL) determined be determined

(MTD) maximal tolerance dose
(MLD) minimal lethal dose 25


Preliminary studies

• 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 26


Definitive studies


• 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 27


Repeat Dose Studies/Sub-Acute Toxicity Studies

• Two mammalian species(one should be non-rodent)

• Long duration studies (30-180 days), usually 14 days

• Dose is dependent on dose-escalating studies

• Drug administered by clinical route

• Parameters monitored and recorded are:

– Behavioural



-Microscopic observations 28


Chronic Toxicity Studies
• To evaluate the cumulative toxicity of chemicals.
• To assess carcinogenic potential.

• Rodents – 6 to 24 months; non-rodents – 12 months or longer
• or up to 15 to 20% of species’ lifespan.

Test System/Animal System
• 2 species required. Rodents, non rodents.

• Mortality
• Pathology and histopathology
• Weight change
• Clinical pathology of all animals (mortalities and survivors) 29 30