Alternative methods to animal testing: review

Bhupal Noble's University,Udaipur (RAj.)

 Animals are used in science for:
I. Undergraduates teaching to learn physiological
mechanism, anatomy and effect of various drugs
on human body
II. Postgraduate teaching to show effects of various
drugs, to find out the nature of unknown drug
and for bioassay
III. Research to understand the working of body
and processes of disease and health
IV. Research to conduct screening for drugs,
bioassay and for preclinical testing of new drug

 Animal models are used to test possibilities that
would be difficult or impossible to test using the
target species (Humans)
 It is mandatory to do extensive toxicological
studies in animals before the candidate drug gets
approval for clinical trials in humans
 “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.”

 In the laboratory an animal
maybe:
 Poisoned
 Deprived of food, water and sleep
 Applied with skin and eye
irritants
 Subjected to psychological stress
 Deliberately infected with disease
 Brain damaged, Paralysed,
Surgically mutilated
 Irradiated, burned, gassed
 Force fed and electrocuted

 It is not possible to replace whole animal models with in vitro systems
to evaluate drug effects on major organ systems.
 However, techniques can greatly reduce the number of animals
needed, and refined protocols can improve the design efficiency and
quality of studies,and lessen stress and discomfort experienced by
lab animals.
 In order to monitor physiological functions in conscious animals,
survival surgery may be performed to implant catheters, electrodes,
flow probes or other devices.
 While chronically instrumented animal models can reduce the
numbers of animals used per study and reduce numbers associated
with acute procedures, these models are resource-intensive to
prepare and maintain.
 Generally instrumented animal models can be reused in major organ
systems toxicology (MOST) for studies to evaluate more than one
drug.

 Continued but modified use of animals: 3 R’s
 In vitro (test tube) test methods
 Tissue cultures techniques:
 In-silico (Computer Modelling) techniques:
 Computerized patient-drug databases and virtual
drug trials by Computer or mathematical analysis
 Computer assisted learning
 Non-invasive imaging techniques such as MRIs and
CT Scans
 Microdosing
 Microorganism based model

 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’s strategy which includes:
 Refinement- refine experimental methods to decrease
unnecessary pain and trauma to animals
 Reduction- reduce the number of animals used in these
experiments
 Replacement- replace the animal experiments
Eg.: Computer Simulation Models, In-vitro Methods, Cell
Culture Techniques

Modified To Reduce Pain & Distress In Animal:
 - Providing relief (pain & distress) by giving
drugs like analgesics, anesthetics, tranquilizers,
sedatives.
 - By changing procedure
i. Small needle
ii. Use non-invasive techniques like MRI etc.
 -Use less sensitive species
 -Use smaller dose
 - Test can be ended at the earliest feasible time
 - Improve housing conditions

 Good planning of studies
i. - Change in experimental design
ii. - Improve methods of data analysis
 Sharing research animals
 Re-designing Studies
i. - To collect as much information as possible
ii. - Share information
 Avoid duplicative testing
i. - By improving communication and co-operation
in the planning & execution of testing
ii. - Sharing data – avoids unintentional repetitions

 Substitution of insentient material in place of
conscious higher animals
 Replace higher animals with lower animals.
 Replace live animals with dummies for teaching and
dissection purpose.
 Could be relative or absolute
 Absolute replacement: no need to use animals
 Eg,: cell lines, tissue of human or invertebrate cells
and tissues
 Relative replacement: humane killing of animals to
provide cells or tissues for in vitro studies

 Instead of using animals, cell and tissue
cultures can be used to test product
ingredients.
 Cell culture experiments can show, for
example, the lowest concentration at
which an ingredient causes damage to
cells.
 The results enable conclusions to be
drawn about the ingredient’s
compatibility with tissue.
 Cell cultures are now also used
routinely to test substances for
mutagenic properties.
 A familiar example is the Hen’s Egg
Test, which can be used to test for
mutagenic properties as well.
 In vitro biomedical research
entails the maintenance of organs,
tissues (or fragments of organs
and tissues), and cells outside of
the body.
 Tissue cultures are additionally
used to test substances for
compatibility with mucous
membranes.
 Can be grown as independent cell
lines or preserve the architecture
of the entire organ as organ
culture and tissue culture
 Stem cells are also used as invitro
models

 Avian- chick embryos
 Rodents- rats and mice ( wild types and
transgenic): embryonic, post-natal and
adult
 Human cells:
i. Neural progenitor cells from aborted
foetuses and stem cell lines.
ii. Cord blood derived stem cells.

 In-vitro Pyrogen test
 Embryonic stem cell test
 Local lymph node assay
for skin sensitization
 Clinical skin patch test on
human volunteers
 Neutral red uptake assay
 Carcinogenicity test
 Acute toxicity test
 Repeated dose toxicity test
 Developmental
neurotoxicity test

 Removal of cells, tissues, or organs from an animal or
plant and their subsequent placement into an
environment conducive to growth.
 Types of Tissue Culture:

PrimaryCultureCellLineCulture  Primary cultures are impractical as a source of cells
for high throughput screening b’coz:
i. Difficult to expand the population in culture
ii. Scarcity of human material
iii. High cost
iv. Batch to batch variation
 Once the primary culture is sub cultured, it becomes a cell
line
 A cell line derived by selection or cloning is referred to as
cell strain.
 Cell strain do not have infinite life, as they die after some
divisions.
 Types: a) Finite cell line b) Continuous cell line

 Without animal dissection computer generated
simulations are used to predict the various
possible biological and toxic effects of a chemical
or potential drug candidate.
 For in vivo experimentation only the most
promising molecules obtained from primary
screening are used.
 For example, to know the receptor binding site of
a drug, in vivo experimentation is necessary.

 Computer aided molecular drug design (CADD)
 Quantitative structure activity relationships
 Computer assisted learning (CAL)
 Computer or mathematical analysis
 Micro fluidic chips
 DNA chips
 Organ on chip
 Human on chip

 It is used to predict the receptor binding site for a
potential drug molecule.
 CADD works to identify the probable binding site
and hence avoids testing of unwanted chemicals
having no biological activity.
 We can tailor make a new drug for the specific
binding site and then in final stage animal testing is
done to obtain confirmatory results with the help of
CADD.

 CAL is an interactive computer assisted learning
(CAL) program without involvement of real
experimental tools
 CAL had better problem solving attitude &the cost
was much less than the traditional laboratory
practices.
 Two softwares are curently used in INDIA:
a.) Ex-pharm b.) X-cology

 Translation of biological effect into a mathematical
equation.
 Virtual human organs and virtual metabolism
programmes can now predict drug effects in humans
more accurately then animals can.
 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

 Computer programs which can predict the toxicity
of new chemicals or drugs based on their similarity
to more established compounds.
 Principle that similar chemicals should have similar
biological properties.
 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

 Harvard’s Wyss Institute has created "organs-
onchips” that contain human cells grown in a state-
ofthe-art system to mimic the structure and
function of human organs and organ systems
 The chips can be used instead of animals in disease
research, drug testing, and toxicity testing and have
been shown to replicate human physiology, diseases,
and drug responses more accurately than crude
animal experiments.

 Chips 2 cm wide and 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

 A ‘microdose’ is defined as less than one hundredth
of the proposed pharmacological dose up to a
maximum of 100 μg
 Can be measured in any biological sample including
plasma and urine to determine ADME
 Analysed using an accelerator mass spectrometer
(AMS).
 Early metabolism data can be obtained before going
into human phase 1 trials.
 Allows testing in relevant species.

 Increasing access to the Internet, World Wide Web andkeeping
current with information associated with alternatives to animal
testing is a challenge made easier.
 On in vitro and other alternatives to animal testing Internet and
World Wide Web, resources provided guidance and other
information.
 The toxicological studies of new cosmetics ingredients should be
present at in vitro. Yet, safety evaluation can be based on in vivo
studies performed before the European ban on the use of animals
came into effect. Before the ban,the evaluations of different
cosmetics ingredients performed by the SCCS are mostly based on
in vivo studies.
 At the moment, the total number of in vitro studies is small
compared to that of studies on laboratory animals. The increase in
the use of in vitro methods can be seen in near future.
 Though there are some accepted and validated methods, yet there
are no methods for all the studies required, such as repeat dose
toxicokineticsand toxicity others.Bhupal Noble's University,Udaipur (RAj.)

Alternative methods to animal testing: review

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