ANIMAL TOXICITY TEST FOR ACUTE,
SUBACUTE & CHRONIC TOXICITY.
DEPT OF VPT, COVAS.
TOXICITY STUDIES - INTRODUCTION
• Toxicology classically has been defined as the study of poisons & concerned with the
adverse effects of xenobiotics.
• Casarett 1996 defined it as a science that defines the limits of safety of chemical
agents for human & animal populations.
• 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.
HISTORY OF TOXICITY STUDIES
• 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”
• Mathieu Orfila, determined the relationship between poisons and their
biological He is referred to as the father of modern toxicology.
SOURCES OF TOXIC SUBSTANCES
• Classified based on their
• chemical nature
• mode of action
• class (exposure class and use class)
• Exposure class: Food, air, water or soil.
• Use class: drugs as drug of abuse, therapeutic drugs, agriculture chemicals,
food additives, pesticides, plant toxins and cosmetics
The use of sheep brain for the production of rabies vaccine has been
phased out in 1992
Classical swine fever virus vaccine is produced in PK-15 Cells instead using
large number of rabits
PPR, FMD and sheep pox vaccines are also produced using cell culture vero,
BHK-21 and vero cell culture system respectively.
The use of laboratory animals e.g., rabbit and G. pig has been now
abandoned for the isolation or typing of mycobacetria with the availability
of improved synthetic media.
• Before conducting any toxicological testing in animals or collecting
tissue/cell lines from animals, the study should be approved by the
Institute Animal Ethics Committee (IAEC) or the protocol should
satisfy the guidelines of the local governing body.
NECESSITIES OF TOXICOLOGICAL STUDIES
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.
MAGNITUDE OF USE OF ANIMALS
It has been estimated that approximately 20 million animals are being
used for testing and are killed annually; about 15 million of them are
used to test for medication and five million for products.
China has become one of the biggest countries using lab animals, as is
evident in the higher numbers and quality of lab animals (e.g., specific
pathogen free, or SPF1; genetically modified) increasingly used in
scientific research—16 million a year, compared to 12 million in the 25
European Union countries in 2005 (FELASA 2007)—and the increased
publication of animal experiment results in international journals.
Japan was second only to the US, which used 17.3
million animals in 2005. The third largest use in
2005 was Great Britain, which used 1.9 million
People for the ethical treatment of animals (PETA)
reported that the National Centre for Laboratory
Animal Sciences (NCLAS) in Hyderabad, supplies
approximately 50,000 animals to laboratories every
year and to 175 institutions in India, including
pharmaceutical companies and educational
In India, among rodentia group of animals e.g. mice. rat, G.pig, rabbit,
mice is predominantly used in most of cases followed by other rodentia.
The number of monkeys used in research has now drastically reduced in
Use of some other unique animals like hamster, cotton rat, gerbil is not
There are inbred strains of mice which are used for research and
genetically modified laboratory animals are also imported into India by
some of institutes.
The information with regard to available laboratory animals resource in
different institutions/universities in India is not precisely known.
Multiple methods have been standardized (certified) by multiple
American Society for Testing and Materials (ASTM)
Organization for Economic Cooperation and Materials (OECD) –
National Toxicology Program (NTP)
All above standardized protocols available from US EPA,
Federal Register and researchers that developed the programs
REGULATORY MECHANISMS IN INDIA
• Institute Animal Ethics Committee (IAEC)
• Committee for the Purpose of Control and Supervision of Experiments in
• 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
RELEVANT TEST MODELS
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
IN-VIVO >< IN-VITRO
1.In Vivo Studies
In vivo safety pharmacology studies should be designed to define the
dose-response relationship of the adverse effect observed
The time course of the adverse effect should be investigated
e.g. onset and duration of response
2.In Vitro studies
In vitro studies should be designed to establish a concentration-effect
TYPES OF TOXICITY STUDIES
• Single dose studies/ Acute toxicity studies
• Repeated dose studies / sub-acute or Chronic studies
• Local toxicity studies
• Allergenicity / Hypersensitivity toxicology studies
• Genotoxicity studies
• Carcinogenicity / Oncogenicity studies
A. SYSTEMIC TOXICOLOGY STUDIES
• Maximum Non Lethal
• MTD and MLD
• Evaluate effects
• Target organ of toxicity
may be determined
a) SINGLE DOSE STUDIES/ ACUTE TOXICITY
,• Acute toxicity testing- study the effect of a single dose on a
particular animal species.
• Acute toxicity testing be carried out with two different animal
species (one rodent and one non-rodent).
• In acute toxicological testing, the investigational product is
administered at different dose levels, and the effect is observed for
14 days. All mortalities caused by the investigational product during
the experimental period are recorded and morphological,
biochemical, pathological, and histological changes in the dead
animals are investigated.
• The LD50 was used as an indicator of acute toxicity previously. The
determination of the LD50 involves large numbers of animals, and the
mortality ratio is high. (24 h testing)
• Graphical method
• Arithmetical method (karbers’s) method. when number of animal is small)
Because of these limitations, modified methods
1. The fixed dose procedure
2. The acute toxic category method
3. The up-and-down method
The test report should include the following information:
• Test substance:
• Physical nature,
• Physicochemical properties;
• Identification data;
• Source of substance;
• Batch number
Vehicle (if appropriate):
• Justification for choice of vehicle (if other than water).
• Species/strain used and justification for choice made;
• Number, age, and sex of animals at start of test;
• Source, housing conditions, diet, etc.;
• Individual weights of animals at the start of the test.
Organization for Economic Cooperation and Development (OECD)Test
Guidelines (TGs 402, 403, 420, 423, and 425) describe acute systemic testing.
• Fixed Dose Procedure (OECDTG 420)
• AcuteToxic Class method (OECDTG 423)
• Up‐and‐Down Procedure (OECDTG 425)
• Acute DermalToxicity (OECDTG 402)
• Acute inhalation toxicity (OECDTG 403)
Six OECDTest Guidelines describe short‐term repeat‐dose toxicity
• Repeated Dose 28‐day OralToxicity Study in Rodents (TG407)
• Repeated Dose 90‐Day OralToxicity Study in Rodents (TG 408)
• Repeated Dose DermalToxicity: 21/28‐day Study (TG 410)
• Sub-chronic DermalToxicity: 90‐day Study (TG 411)
• Repeated Dose InhalationToxicity: 28‐day or 14‐day Study (TG 412)
• Sub-chronic InhalationToxicity: 90‐day Study (TG 413)
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:
b) REPEATED DOSE STUDIES/SUB-ACUTE OR
.• Rodents and non-rodents are used to study the sub-chronic toxicity of a substance.
• Dose: Expected therapeutic level (daily) or expected therapeutic level to increasing dose
• The test substance is administered orally for =/>90 days, and regular body weight
variations, biochemical and cardiovascular parameters changes, and behavioral changes
• At the end of the study, the experimental animals are sacrificed. Gross pathological
changes are observed, and all the tissues are subjected to histopathological analyses.
• There should be little individual variation between the animals, and the allowed weight
variation range is ±20%.
• Used to determine the maximum tolerable dose and nature of toxicity.
.Chronic toxicity studies are conducted with a minimum of one
rodent and one non-rodent species.
• The test compound is administered over more than 90 days, and the animals
are observed periodically.
• A chronic toxicology study provides inferences about the long-term effect of
a test substance in animals, and it may be extrapolated to the human safety
of the test substance.
• The report on chronic oral toxicity is essential for new drug entities. There
should be little individual variation between the animals, and the allowable
weight variation range is ±20%.
• High dose: Produce significant retardation of growth or some
pathological changes (10 times the expected maximum clinical
• The low dose is about twice the expected maximum clinical dose
• Third dose is medium dose fixed midway between the high and
.• During the study period, the animals are observed for normal
physiological functions, behavioral variations and alterations
in biochemical parameters at regular intervals (atleast every
• At the end of the study, tissues are collected from all parts of
the animal and subjected to histological analyses.
TYPES OF LOCAL TOXICITY STUDIES
Dermal toxicity studies
Vaginal toxicity studies
Rectal tolerance studies
Rats & Rabbit
Local signs (erythema, oedema)
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
Inhalation toxicity studies
Changes in cornea ,Iris & aqueous humor,
histological examination of eye
For intravenous/ intramuscular/ subcutaneous/
Sites of injection examined grossly and
One rodent and non rodent species
Acute , sub-acute and chronic studies performed
Observation of respiratory rate
Histological examination of respiratory passages,
Guinea Pig Maximization
Local lymph node assay
Determination of Maximum non
irritant or minimum irritant dose
Evaluation of Erythema and
Mice of one sex(either male or
Drug treatment given on ear skin
Auricular lymph node dissection
after 5 days
Increase in 3h-thymidine used for
E. GENOTOXICITY STUDIES
To detect early tumorigenic effects in cases of chronic
In vitro tests:
Test for gene mutation in Bacteria
Cytogenetic evaluation of chromosomal damage in mammalian
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
In vivo test for chromosomal damage using mammalian
Chromosome damage in rodent hematopoietic cells
E.g.; Micronucleus Assay
F. CARCINOGENICITY/ ONCOGENICITY
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
NOTE ON STEM CELLS
STEM CELLS NOW EMERGING AS AN ALTERNATIVE TO LABORATORY ANIMALS Drug company
interest in stem cell drug testing was demonstrated in July 2008, when GlaxoSmithKline
entered into a $25 million-plus agreement with the Harvard Stem Cell Institute.
By testing drugs on specific cells and tissues created from iPS cells, we can even predict a
patients individual response to a treatment realizing the vision of personalized medicine.
In the current issue of Stem Cells and Development (2007), Cezar and her colleagues revealed
a novel way to test drug toxicity: by monitoring the behaviour of embryonic stem cells
exposed to a drug-candidate compound.
Studying how potential drugs affect embryonic stem cells could provide a far more accurate
prediction of a drug's potential toxicity than conventional animal models can.
Currently, the most successful development of stem cells as in vitro models for toxicology
testing is in human cardiac tissue.
• 407 ~ Repeated Dose Oral Toxicity ( Rodents : 28 days / 14 days study )
• 408 ~ Sub-chronic Oral Toxicity Test ( rodents : 90 days study )
• 409 ~ Sub-chronic Oral Toxicity Test ( Non-rodents : 90 days study )
• 410 ~ Repeated Dose Dermal Toxicity ( 21/28 days study )
• 411 ~ Sub-chronic Dermal Toxicity (90 days study )
DEFINITIONS RELATIVE TO DOSES
ED 50 : The median effective dose = the dose for which
half(50%) of the animals exhibit an effect (E) and half of
animals exhibit no effect
LD 50 : The median lethal dose = the effect may be defined as
specific toxic event (tremors) & sometimes defined as lethality
ED10 & ED90 are doses at which 10% & 90% of the animals
respectively demonstrate effect
• LIMIT DOSE : a dose which is considered high enough that if no mortality /
significant toxicity is seen in animals receiving this dose, no higher doses are
eg : Limit doses(EPA/OECD)
1.Acute Oral Toxicity 5000mg/kg
2.Acute Dermal Toxicity 2000mg/kg
3.21-days Dermal Toxicity 1000mg/kg
4.Chronic Studies of Pesticides 1000mg/kg
Note: the limit dose of a non-nutritive material added to diet is generally
considered to be 5% (50,000ppm)
SUB-CHRONIC & CHRONIC STUDIES
• ADI: Acceptable Daily Intake (established for food
additives/ residues & published by EPA)
• NOEL: No Observed Effect Level. Dose at which no effect is
• NOAEL: No Observed Adverse Effect Level. Dose at which
no adverse/toxic effect is seen
• MTD: Maximum Tolerated Dose. Is the highest dose that can be tolerated
without significant lethality from causes other than tumors.
(for EPA studies MTD for chronic studies with pesticides is a dose which
produces an approximate 10-15% decrement in body weight gain)
• HTD: Highest dose tested. Highest dose that can be expected to yield
results relevant to humans. This is a proposed new dose which would be
selected based on evaluation of results of sub-chronic studies.
VEHICLES USED FOR DOSING
• ORAL: Water, Methylcellulose or Carboxymethylcellulose(0.5-5%
aqueous suspension), Oil( corn, peanut, sesame)
• DERMAL: Physiological saline, Water, Ethanol, Acetone, Mineral
• PARENTERAL: Physiological saline(sterile), sterile water for
CLINICAL SIGNS OF TOXICITY
RESPIRATORY: blockage in the nostrils, changes in rate & depth of
breathing, changes in color of body surfaces
• Signs like dyspnea, abdominal breathing, gasping, Apnea, Cyanosis,
Tachypnea, nostril discharge = involvement of CNS respiratory
center, pulmonary edema, cholinergic inhibition, pulmonary cardiac
Changes in frequency & nature of
Signs like decrease/increase in
spontaneous motor activity,
Somnolence, Loss of righting reflex,
Anesthesia, Catalepsy, Ataxia, Unusual
locomotion, prostrations, Tremors &
fasiculations = CNS, sensory, autonomic
& neuromuscular system
Marked involuntary contractions or seizures of contraction of
Signs like Clonic convulsions, Tonic convulsions, Tonic-Clonic
convulsions, Asphyxial convulsion, Ophisthotonos = CNS, respiratory
failure, neuromusculsr & autonomic systems.
• STARTLE REFLEX
All signs indicates involvement of CNS, sensory, autonomic &
• Lacrimation, Miosis, Mydriasis, Exophthalmos, Ptosis,
Chromodacryorrhea ( red lacrimation ), relaxation of nictating
membrane, corneal opacity, iritis, conjunctivitis.
Mainly due to autonomic system involement
• Bradycardia, tachycardia, vasodilation, vasoconstriction,
• Due to autonomic, CNS, cardiac-pulmonary insufficiency,
myocardiac infarction, cold environment.
EFFECTS OF DECREASED BODY WEIGHTS ON RELATIVE
ORGAN WEIGHTS OF RATS
• Decreases = liver
• No change = heart, kidneys, prostrate, spleen, ovaries.
• Increases =adrenal gland, brain, epididymides, pituitary,
testes, thyroid, uterus.
1. General considerations for clinical studies
2. Specific considerations for clinical studies
a. Protocol design
b. The study population
c. Statistical analyses
3. Sequence of clinical studies
a. Early clinical studies
b. Further clinical studies
4. Submitting reports of clinical studies to CFSAN (Center for Food
Safety and Applied Nutrition)
PHASES OF DRUG DEVELOPMENT
PHASE III PHASE IV
PHASE IPRECLINICAL PHASE II
Patient studiesEntry to man
(IND / CTA)
Chronic (long term) toxicity testing
Bioanalysis / Toxicokinetics
Reproductive Toxicity Testing
(fertility and pre/post natal)
Single / repeat dose