ANIMAL TOXICITY STUDIES

Dr.k.vishnuvardhan babu

Why study toxicology???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

why do we require non clinical

studies IN ANIMALS before

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

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

Types of toxicology studies

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

A. Systemic toxicology studies

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

Preliminary studies

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

Definitive studies

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

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

Chronic toxicity

Objectives To evaluate the cumulative toxicity of chemicals.

To assess carcinogenic potential.

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

Test System/Animal System2 species required. Rodents, non rodents.

Parameters

Mortality

Pathology and histopathology

Weight change

Clinical pathology of all animals (mortalities and survivors)

B. Reproductive toxicology studies

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

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

Segment I

13

Fertility and general reproductive performance study

Segment II Teratogenicity

Segment III Perinatal 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

C. Local toxicity studies

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

Types of local toxicity studies

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

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

D. Allergenicity/hypersensitivity

toxicology studies

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

E. Genotoxicity studiesTo 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 adductsIn vivo tests:

Chromosome damage in rodent hematopoietic cells

E.g.; Micronucleus Assay

f. Carcinogenicity/ oncogenicity studies

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

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

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

Thank you