Alternative methods to animals testing are the development and implementation of test method that avoid use of live animals or use of less animals in method.
The council directive on protection of animals used for experiments and scientific purpose in article 23
“The commission and member states should encourage
research into development and validation of alternative methods which could provide the same level of information as that obtained in experiment using animals but which involves less animal”.
Alternative methods able to do:
Reduce Refine Replace
collectively called as “The 3Rs Principle”.
Needs for alternative methods
Because in laboratory animals may be:
Poisoned.
Deprived of food water and sleep.
Applied with skin and eye irritants.
Subjected to psychological stress.
Deliberately infected with the infected disease.
3. Introduction
3
Alternative methods to animals testing are the development and
implementation of test method that avoid use of live animals or use of
less animals in method.
The council directive on protection of animals used for experiments and
scientific purpose in article 23
“The commission and member states should encourage
research into development and validation of alternative methods
which could provide the same level of information as that obtained in
experiment using animals but which involves less animal”.
Alternative methods able to do:
•
•
• Reduce
Refine
Replace
collectively called as “The 3Rs Principle”.
4. Needs for alternative methods
4
Because in laboratory animals may be:
• Poisoned.
• Deprived of food water and sleep.
• Applied with skin and eye irritants.
• Subjected to psychological stress.
• Deliberately infected with infected disease.
5. THE 3RS PRINCIPLE
5
The 3Rs principles were defined in 1959 by W.M.S
Russel and R.L Bruch in subject “The Principle of
Humane experimental technique”.
They provide a strategy for rational and stepwise
approach to minimising animals use and suffering
in experiments without compromising the quality
and quantity of scientific work being undertaken.
6. ALTERNATIVETECHNIQUES
The term “alternative” is used to refer to those techniques or
methods that replace the use of laboratory animals altogether
reduce the number of animals required or techniques to minimize
the level of stress endured by the animal.
Techniques can greatly reduce the number of animals needed and
refined protocols can improve design efficiency and quality of
studies and lessen stress and discomfort experienced by lab
animals.
The field of alternative study particularly in vitro toxicology has
evolved into a respected discipline and is attracting competent
and motivated scientist around the world.
It is not possible to replace the whole animal models with in vitro
system to evaluate drugs effects on major organ system.
7. ALTERNATIVETOANIMAL EXPERIMENTS
Continued but modified use of animals 3Rs
In vitro[test tube] method
Tissue culture technique
In silico [computer modellingtechnique]
Computer aided molecular drugdesign[CADD]
Computer assisted learning[CAL]
Microfluidic chips
Quantitative structure activity relationship
Organ on chips
Computer or mathematicalanalysis
8. CONTINUED BUT MODIFIED USE OF
ANIMALS
• Russel and Burch developed 3R’s strategy which include:
1. Refinement: refine experimental methods to decrease
unnecessary pain and trauma toanimals.
2. Reduction: reduce the no. of animals used inthese experiments.
3. Replacement: replace the animal experiments ,
.
.
.
.
.
.
.
.
.
.
.
.
.
.
e.g. computer
stimulation methods, In vitro methods, Cell culture techniques.
9. Methods of refinement
Modified to reduce pain and distress inanimal
Providing relief [pain and distress]bygiving
drugs like analgesics, anesthetics,
tranquillizers and sedatives.
By changing procedure:
Use non invasive technique like MRI
Use less sensitive animal species
Use smaller dose
Improving housing conditions
10. METHODSOFREDUCTION
Good planning of studies
Change in experimental design
Improve methods of data analysis
Sharing research animals
Redesigning studies
To collect as much information aspossible
Share information
METHODS OF REPLACEMENT
Replace higher animals with lower animals
Replace live animals with dummies for teaching and dissection
purpose
ABSOLUTE REPLACEMENT: no need to use animals e.g. cell
lines, tissue of
human or invertebrate cell and tissue
RELATIVE REPLACEMENT: humane killing of animals to
provide cells or tissues for in vitro studies
Substitution of insentient material in place of conscious higher
animals.
11. INVITROMODELS
Instead of using animals, cell and tissue culture can be used to test
product ingredients .
Cell culture are now also used routinely to test substance for mutagenic
properties.
Can be grown as independent cell lines or preserve the
architecture of entire organ as organ culture and tissue culture.
Stem cells are also used as in vitro models.
SOURCE OF TISSUE FOR IN VITRO METHODS
Avian chick embryo
Rodents[rat and mice], wild type, transgenic , embryonic,
post natal , adult
Human cells [neural progenitor cells from aborted foetus and
stem cell lines]
12. VARIOUS TEST IN VITRO METHODS
In vitro pyrogen test
Embryonic stem cell test
Carcinogenicity test
Neurotoxicity test
TISSUE CULTURE TECHNIQUES
Tissue culture is in vitro maintenance and propagation of isolated cells,
tissues or organs in an appropriate artificial environment.
APPLICATION OF ANIMAL CELL CULTURE
Toxicity testing
Cancer research
Virology
Genetic engineering
Gene therapy
Drug screening and development
13. INSILICO[COMPUTER MODELLING]
TECHNIQUES
• Without animal dissection computer generated stimulation are
used to predict the various possible biological and toxic effects
of a chemical or potential drug candidate.
VARIOUS TYPES IN SILICO MODELS
Computer aided molecular drug design [CADD]
Quantitative structureactivity relationship
Computer assistedlearning[CAL]
Computer or mathematical analysis
Organ on chips
14. COMPUTERAIDEDDRUG DESIGN[CADD]
It is used to predict the receptor binding site fora potential
drug molecule.
CADD works to identify the probable binding site and
hence avoid testing of unwanted chemicals having no
biological activity.
Computational approach to discover, develop and analyse
drugs.
15. COMPUTERAIDEDDRUG DESIGN[CADD]
It is used to predict the receptor binding site
fora potential drug molecule.
CADD works to identify the probable binding
site and hence avoid testing of unwanted
chemicals having no biological activity.
Computational approach to discover, develop
and analyse drugs.
16. COMPUTERASSISTED LEARNING [CAL]
CAL is an interactive computer assisted
learning program without involvement of real
experimental tools.
The cost is much less than the
traditional laboratory practices.
Two software are currently used inIndia:
Ex- pharm
X- cology
17. QUANTITATIVESTRUCTURE ACTIVITY
RELATIONSHIP
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 havesimilar
biological properties.
QSAR has been widely used in medicinal chemistry as
support in drug’s discovery and development process as
well as in study of harmful andpoisonous substances in
toxicological chemistry.
18. COMPUTEROR MATHEMATICALANALYSIS
Translation of biological effect into a mathematical equation.
Computer design the molecular structure ofdrugs to target
specific receptors.
E.g. Protease inhibitors were designed by computers
and tested in tissue culture and computer models by
passing the animal test.
19. ORGAN–ON-CHIPS
Organ-on-chips that contain human cells grown in a state-
of the-art system to mimic the structure and function of
human organ and organ system.
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.
20. MICROFLUIDICCHIPS
Chips 2cm wide and contain a series of tiny chambers each
containing a sample of tissuefrom different parts of body.
Sensors in the chip feed back information for computer
analysis.
This can be used to study the disease processand drug
metabolism.
21. HET-CAMTEST
21
• The fresh fertile white leghorn eggs are used.
• The eggs are held in optimized incubation condition.
• On day 10 inner egg membrane is removed , after careful
removal the living vascular ChorioAllantoic-Membrane is
exposed.
• The test substance is droped over CAM in a volume of 0.2-
0.3 ml and irrigated after 20 sec. with 5ml warm water.
• The CAM, the blood vessels, including capillary system, and
albumin are examined and scored for irritant effects 0.5, 2,
5min after test compound application.
22. Effects Score
0.5(min) 2(min) 5(min)
haemorrhage 5 3 1
Coagulation 9 7 5
ScoringScheme
22
Classification scheme
Cumulative score Irritation Assessment
Up to 0.9 Practically none
1 to 4.9 Slight
5 to 8.9 Moderate
9 and above Strong