ALTERNATIVES TO ANIMAL

EXPERIMENTS

BY
DR.ARUN.S
FIRST YEAR POST GRADUATE
DEPARTMENT OF PHARMACOLOGY
GMC, ANANTHAPURAMU
SPECIFIC LEARNING OBJECTIVES
AT THE END OF THE SEMINAR THE POST GRADUATE
STUDENTS WILL BE ABLE TO UNDERSTAND

1. The uses of animal testing

2. The need for alternative experiments
3. The Laws and regulations concerned
with animal experiments
4. 5 R’s to be followed
5. In Vitro models
6. In Chemico models
7. In Silico methods
USES OF ANIMAL TESTING
Animals are used in science for:
1. Undergraduates teaching to learn
physiological mechanism, anatomy and
effect of various drugs on human body.
2. Postgraduate teaching to show effects of
various drugs, to find out the nature of
unknown drug and for bioassay.
3. Research - to understand the working of
body and processes of disease and health.
4. Research to conduct screening for drugs,
bioassay and for preclinical testing of new
drug.
CONTD...
5. Animal models are used to test possibilities
that would be difficult or impossible to test using
the target species (Humans)
6. It is mandatory to do extensive toxicological
studies in animals before the candidate drug gets
approval for clinical trials in humans
7. There is no doubt that the best test species for
humans are humans. It is not possible to
extrapolate animal data directly to humans due to
interspecies variation in anatomy, physiology
and biochemistry.
NEED FOR ALTERNATIVES
In the laboratory an animal maybe
1. Poisoned
2. Deprived of food, water and sleep
3. Applied with skin and eye irritants
4. Subjected to psychological stress
5. Deliberately infected with disease
6. Brain damaged, Paralysed,
Surgically mutilated
7. Irradiated, burned, gassed
8. Force fed and electrocuted
CONTD..
Disadvantages of animal experiments:
1. Pain, distress and unethical
behaviour to animals
2. Requirement of skilled manpower
3. Time consuming protocols
4. High cost
LAWS AND REGULATIONS CONCERNED WITH
ANIMAL TESTING
YEAR LAW
1960 Prevention of Cruelty to Animals (PCA) Act 1960,
amended 1982
1964 Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA)
1972 Wild life protection act

1992 Indian National Science Academy (INSA)

“Guidelines for care and use of animals in scientific research”,
revised 2001
1998 “Breeding of and Experiments on Animals
(Control and Supervision) Rules, 1998”,
amended 2001, 2006
CONTD..
Year Law
2001 Indian Council of Medical Research (ICMR)
“Guidelines for use of Laboratory animals in Medical Colleges”

2009 MCI amendment - Recommends to use alternative store place animal

experiments

2012 Ministry of Health & Family Welfare bans use of animals in

educational institutes
2013 University Grants Commission (UGC)
“Guidelines for discontinuation of dissection and animal
experimentation in zoology / life sciences in a phased manner”
5 R’s TO BE FOLLOWED

Russel and burch in 1959 proposed that “if animals were to be used in
experiments, every effort should be made to replace them with non-
sentient alternatives”
They developed the 3R strategy which includes
1. Refinement- refine experimental methods to decrease unnecessary pain
and trauma to animals
2. Reduction- reduce the number of animals used in these experiments
3. Replacement- replace the animal experiments egcomputer simulation
models, In-vitro methods, cell culture techniques
REFINEMENT
Methods of Refinement:
1. Modified to reduce pain & distress in animal
2. Providing relief (pain & distress) by giving drugs like analgesics, anesthetics,
tranquilizers, sedatives.
3. By changing procedure
a) Small needle
b) Use non-invasive techniques like MRI, ultrasound
4. Use less sensitive species
5. Use smaller dose
6. Test can be ended at the earliest feasible time
7. Improve housing conditions
REDUCTION
Methods of Reduction:
1. Good planning of studies
a) Change in experimental design
b) Improve methods of data analysis
2. Sharing research animals
3. Re-designing Studies
c) To collect as much information as possible
d) Share information
4. Avoid duplicative testing
e) By improving communication and co-operation in the planning & execution of
testing
f) Sharing data – avoids unintentional repetitions
REPLACEMENT
Methods of Replacement:
1. Substitution of insentient material in place of
conscious higher animals.
2. Replace higher animals with lower animals
3. Replace live animals with dummies for teaching and
dissection purpose
4. Use computer simulation and in vitro methods
5. Use cell culture and tissue culture
Absolute replacement: Relative replacement:
No need to use animals Humane killing of animals to
(cell lines, tissue of human or provide cells or tissues for in vitro
invertebrate cells and tissues) studies

REHABILITATION
The concept of Rehabilitation has been recognised in India as the 4th R and evolved as
an official policy of the CPCSEA in 2004
The concept of 4th R “Rehabilitation” of laboratory animals is defined as “the aftercare
rendered to animals that have been
(i) bred for the purpose of experimentation
(ii) subject to any form of experimentation
(iii) retained in laboratory animal houses or breeding houses for the purpose of
experimentation, both for education and research,
with the sole intention of alleviating the pain/distress or suffering due to the physical,
physiological and psychological trauma that the animals have been exposed to and to
provide the animal a life distinctly different from laboratory housing and care, until the
point of natural death”
RESPONSIBILITY
1. The use of animals in biomedical research may be summed up in only few
words, “it is a privilege, not a right”.
2. We, in the research community have a responsibility to the animals we work
with.
3. It is our responsibility to treat these animals with respect and acknowledge
and address the basic issues of humane care and thoughtful stewardship of
their use.
4. We must be ever mindful of the rules and regulations that govern the use
of animals in all types of research including, behavioral, medical and
psychological studies
ALTERNATE METHODS
• IN VITRO
• IN CHEMICO
• IN SILICO
IN VITRO METHODS

Mainly two types:

1) Based on animal or human components - cell,
tissue and organ culture.
2) Based on organisms not considered animals -
micro-organisms and invertebrates
 IN VITRO METHODS - CONTD..
ADVANTAGES DISADVANTAGES
1. Small quantity of test substance is needed 1. In vivo dose-response not available
2. Experiment done under controlled conditions 2. No systemic effect could be studied
3. Result is obtained quickly 3. Organ specificity lacking
4. No major infrastructure is required 4. Chronic and long-term effects could not be studied
5. Transportation of material not easy
SOURCE OF TISSUE FOR IN VITRO
METHODS
1. Avian - chick embryos
2. Rodents - rats and mice (wild types and transgenic): embryonic, post-
natal and adult
3. Human:
a) Neural progenitor cells from aborted foetuses and stem cell lines.
b) Cord blood derived stem cells
ANIMAL OR HUMAN COMPONENTS
Cells, tissues & organs kept alive outside a living organism
Advantages:
- Less distress to animals
- Less number of animals are required
- Human tissues – no need to extrapolate results from animal data
Disadvantage:
- Unable to produce complete physiological response
- Components lose their ability to perform special function
- Can not determine the effect of route of administration
WHAT TO MEASURE?
• Rate of synthesis of certain substances e.g : In response to irritation, cell produces
prostaglandins, certain enzymes, proteins, antibodies
• Changes in membrane permeability
• Damage to some part of cell structure. e.g. Liver cells – retain most of special
functions when cultured
• Beating heart cells:
- to detect the effect of drugs
CONTD..
• Response to toxic substances
a) Use of sugar as an indication of metabolic activity
b) Production of proteins or other substances
c) Uptake of amino acids as an indication of protein synthesis
d) Morphological changes
e) Reduction in viability
• Rabbit kidney tubules – to detect substances that can cause acute renal
failure
• Rat vaginal tissue – to vaginal irritancy of contraceptives
IN VITRO MODELS TO ANIMAL
TESTING
Cell Based –in vitro models
The closer a cultured cell system matches cells in real human target tissues, the
more likely that results obtained using such systems will be predictive of drug
efficacy, safety and toxicity In vivo

Ideal Characteristic of in vitro model

The most predictive cell system would be a primary culture of fully differentiated
cells derived from a relevant human tissue
TYPES OF CELL CULTURES
Two types of cell culture
• Primary Culture
• Cell Line Culture (finite / continuous / established / secondary / subclone /
immortalized cell culture)
TISSUE CULTURE
Removal of cells, tissues, or organs from an animal or plant and their subsequent
placement into an environment conducive to growth
Environment:
Suitable plastic or glass culture vessel with liquid / semi solid medium + nutrients
for survival and growth
Types of Tissue Culture:
• Organ Culture
• Explant Culture
• Cell Culture
• Organotypic Culture
ADVANTAGES OF TISSUE CULTURE
LIMITATIONS OF TISSUE CULTURE
NON-ANIMAL ORGANISMS
Ranging from plants to single celled organism to invertebrates
Micro-organism
• Use of bacteria and fungi to measure genotoxic effects
• More easily & quickly cultivated
• Simple genetic makeup
• Protozoa have specialized functions. e.g.- cilia of protozoans responds
to smoke or phenols
INVERTEBRATES

Example:
1. Drosophila melanogaster – ‘fruit fly’ for mutagenic, teratogenic and
reproductive toxicity
2. Sea urchin – for basic reproductive research
IN VITRO METHODS USED
COMMONLY
1. In vitro Pyrogen test
2. Embryonic stem cell test
3. Local lymph node assay for skin sensitization
4. Skin patch test
5. Neutral red uptake assay
6. Carcinogenicity test
7. Acute toxicity test
8. Repeated dose toxicity test
9. Developmental neurotoxicity test
IN-VITRO PYROGEN TEST
1. Limulus amoebocyte lysate (LAL) test
2. monocyte activation test (MAT)

Limulus amoebocyte lysate (LAL) test

Principle:
• The principle of the LAL test is that the lipopolysaccharides (LPS)
cause extracellular coagulation of the blood (hemolymph) of the
horseshoe crab Limulus polyphemus (Levin and Bang, 1964).
• Does not detect pyrogens other than the bacterial endotoxins (Gram -
positive endotoxins), viruses and fungi.
CONTD..
Three techniques to perform this test:
• Gel clot technique - based on gel formation
• Turbidimetric method -based on development of turbidity after
cleavage of endogenous substrate
• Chromogenic method - based on development of color after cleavage
of synthetic peptide - chromogen complex
MONOCYTE ACTIVATION TEST
1. The monocyte activation test
(MAT) uses human
mononuclear cells (e.g.
monocytes) obtained from
human volunteers or from
the blood bank.
2. This test detects pro-
inflammatory and
pyrogenic contaminants not
always detected in the rabbit
pyrogen test or in the LAL
test.
EMBRYONIC STEM CELL TEST (EST)
This is used for the detection of any embryonic toxicity. The embryonic stem
cells develop spontaneously into contracting myocardium.
The different end points are as follows:
1. Inhibition of differentiation of the embryonic stem (ES) cells into
cardiomyocytes
2. Cytotoxic effects on the ES cells
3. Cytotoxic effect on 3T3 fibroblasts
LOCAL LYMPH NODE ASSAY FOR
SKIN SENSITIZATION
PRINCIPLE:
• When a test compound is applied on the skin, it is considered as a sensitiser
when the lymph node draining the site of chemical application reveals a
primary proliferation of lymphocytes as measured by the radioactive labeling.
• This proliferation is proportional to the dose applied that provides a simple
means of obtaining an objective, quantitative measurement of sensitization.
• The ratio of the proliferation in the treated groups to that in the vehicular
controls is the Stimulation Index. The index must be at least three before the
test substance can be further evaluated as a potential skin sensitizer.
SKIN PATCH TESTING
Corrositex
• To determine chemical corrosivity.
• Replaces rabbit test of dermal corrosivity
• Principle- a unique bio membrane and chemical detection system which
becomes colored when exposed to potentially corrosive substance
• Cultured human epidermal keratinocytes mimic human epidermis are
used to measure skin irritation and dermal corrosion.
• Replaced the Draize rabbit skin irritation test
NEUTRAL RED REUPTAKE ASSAY
• Alternative to Draize rabbit eye test for screening of chemicals for eye
irritation potential
• Neutral red penetrates cell membrane and accumulates intracellularly in
lysosomes
• Alteration of cell surface or lysosomal membrane result in decreased uptake
• NRU assay measures the ability of test compound to inhibit uptake of
neutral red dye
• NRU 50 or IC 50 serves as toxicological end point
ADVANTAGES:
• Simplicity, speed, economical, sensitivity.
• Used in acute toxicity testing.
CONTD..
CARCINOGENICITY TESTS
• By using cell transformation assays. Eg-1. Balb/c3T3 assay, Syrian hamster
embryo (SHE)
• These assays are faster, less expensive, and involve fewer animals.
• Alternative to rodent bioassay and transgenic mouse model bioassay for
carcinogenicity assays.
• AMES TEST (Bacterial reverse mutation test) - To test mutagenicity
REPEATED DOSE TOXICITY STUDIES
• Computerized biokinetic modeling is used as a means of predicting the
distribution of chemical among various organs and tissues of the body and
also to predict organ specific toxicity.
• Such predictions are verified quantitatively using cell cultures of specialized
tissues.
IN CHEMICO TESTING METHODS
The toxic potential of substances can sometimes be detected using relatively
simple chemistry based methods and not requiring human cells.
Eg- High performance liquid chromatography.
DIRECT PEPTIDE REACTIVITY ASSAY
• Used to assess whether a chemical or cosmetic will cause allergy.
• The tests works by mimicking a key step in the development of allergies –
the binding of proteins found in the skin to the substance.
• If proteins bind to the substance then it is very unlikely that it will cause an
allergic reaction.
CONTD..
IN SILICO METHODS
1. Computer aided molecular drug design
2. Quantitative structure activity relationships
3. Computer assisted learning
4. Computer or mathematical analysis
5. Organ on chips
6. DNA chips / DNA Microarrays
7. Human on chip
DRUG DISCOVERY PROCESS - CADD
COMPUTER ASSISTED LEARNING
CAL deals with a range of software packages which simulate the animal
experiments
Two softwares are curently used in india
1. Expharm - developed by JIPMER, India
2. X-cology
EX-PHARM
Contains programs on
1. Effect of drugs on the rabbit eye
2. Bio assay of histamine using guinea pig ileum
3. Effect of drugs on the frog heart
4. Effect of drugs on dog blood pressure and heart rate
5. Effect of drugs on the ciliary movement of frog
esophagus
The user can conduct experiment and collect data
Each program can be run in two modes
a) tutorial mode,
b) examination mode
X-COLOGY
a) Video demonstrations of different procedures like isolation and
mounting of animal tissues
b) Screen interactive interface to study the effects of various drugs on
the isolated tissues
c) Content is classified into three sections
1. Experimental animals
2. Equipment
3. Experimental technique – procedure to carry out bioassay and experiments
on whole animals
 COMPUTER OR MATHEMATICAL
ANALYSIS
1. Translation of biological effect into a
mathematical equation.
2. Virtual human organs and virtual
metabolism programmes can now predict
drug effects in humans more accurately
then animals can.
3. Computers design the molecular structure
of drugs to target specific receptors
Eg- Protease inhibitors were designed by
computers and tested in tissue culture and
computer models bypassing animal tests
 QUANTITATIVE STRUCTURE ACTIVITY
RELATIONSHIPS
1. Computer programs which can predict the toxicity of new chemicals
or drugs based on their similarity to more established compounds.
2. Principle that similar chemicals should have similar biological
properties.
3. Greater computer power and the ability to generate large databases
have facilitated the development of these methods and a wide range of
models now exist that cover a variety of toxicities
QSAR AND 3D-QSAR SOFTWARES
1. Tripos - CoMFA, VolSurf
2. MSI - Catalyst, Serius
DOCKING SOFTWARES
1. DOCK - Kuntz
2. LigandFit - MSI Catalyst
DNA MICROARRAY / DNA CHIPS
The use of miniaturized microarrays for gene expression profiling was first reported
in 1995, and a complete eukaryotic genome Saccharomyces cerevisiae on a
microarray was published in 1997.
PRINCIPLE:
• The principle of DNA microarrays lies on the hybridization between the
nucleotide. Using this technology the presence of one genomic or cDNA
sequence in 1,00,000 or more sequences can be screened in a single hybridization.
• The property of complementary nucleic acid sequences is to specifically pair with
each other by forming hydrogen bonds between complementary nucleotide base
pairs.
CONTD..
ORGAN ON CHIPS
• Chips 2 cm wide contain a series of tiny chambers - each
containing a sample of tissue from different parts of the body.
• The compartments are linked by microchannels through which a
blood substitute flows
• The test drug is added to the blood substitute and circulates
around the device
• Sensors in the chip feed back information for computer analysis
• This can be used to study the disease process and drug
metabolism
HUMAN ON CHIPS
ADVANTAGES OF ORGAN CHIPS
1. Replace animal models.
2. Help the laboratories reach the stage of clinical trials.
3. Comparison studies of drugs on human and animals.
4. Study on interactions of pathogens and organ cells, and also the mechanisms
of some virus attacks.
5. To study the effect of drug on its main action of site and also other organs.
6. Study of toxicity of drugs and cosmetics.
7. To study about cancer cells and produce new drugs in cancer treatment.
8. Ensure better regulatory decision-making.
9. Develop vaccines and drugs to counter bioterrorism threats.
DISADVANTAGES
1. Every property of an organ cannot be measured.
2. It is some times not possible to study complete organ.
3. Drugs action during pregnancy cannot be studied.
THE MAN WHO CREATED ORGAN
CHIPS
Don Donald Ingber
Wyss insttitute, at Harvard
university druing 2009 - 2011
SUMMARY
REFERENCES
Fundamentals of Experimental Pharmacology - M.N.Ghosh, 6th edition

Practical Manual of Experimental and Clinical Pharmacology Bikash

Medhi - 2nd edition