1. ALTERNATIVES
TO ANIMAL
EXPERIMENTS
Dr.Roopali Somani
P.G Resident
M.R.M.C

2. Overview
 Introduction
 Need for alternative to animals
 Laws and regulations
 Refinement
 Reduction
 Replacement
 In vitro methods
 In Silico methods
 Advantages and disadvantages
 Summary

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

4. Introduction
 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.”

5. Need for alternatives
 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

6. Need for alternatives
Disadvantages of animal
experiments
 Pain, distress and
unethical behaviour to
animals
 Requirement of skilled
manpower
 Time consuming protocols
 High cost
 Translation rates of
animal experiments are
absymal

7. Alternatives to animal experiments
 Continued but modified use of animals
 In vitro (test tube) test methods and models based on
human cell and tissue cultures
 Computerized patient-drug databases and virtual
drug trials
 Computer models and simulations
 Computer assisted learning
 Non-invasive imaging techniques such as MRIs and
CT Scans
 Microdosing

8. Laws and regulations
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

9. Laws and regulations
Year Law
2001
Indian Council of Medical Research (ICMR)
“Guidelines for use of Laboratory animals in Medical
Colleges”
2009
MCI amendment-Recommends to use alternatives to
replace 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

10. Continued but modified use of
animals
 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
 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

11. Methods of Refinement
Setting the earliest possible end point
Using appropriate analgesics and anaesthetics for painful procedure
Use proper handling technique for animals
Adequate training prior to performing experiment
Ensure drug doses are correct and drugs are not expired
Perform surgeries and procedure aseptically to prevent infection,

12. Methods of Reduction
 Perform pilot studies
 Design studies to use animals as their own
controls eg- Cross over study
 Gather data for more than one experiment
concurrently
 Consult with statistician and use minimum
number of animals
 Minimise variables such as disease, diet, stress,
genetics
 Use appropriate species of animals

13. Replacement
 Substitution of insentient material in place of
conscious higher animals
 Could be relative or absolute
 Replace higher animals with lower animals
 Replace live animals with dummies for teaching
and dissection purpose
 Use computer simulation and in vitro methods
 Use cell culture and tissue culture

14. In vitro models
 In vitro biomedical research entails the
maintenance of organs, tissues (or fragments of
organs and tissues), and cells outside of the body.
 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

15. Source of tissue for
in-vitro methods
 Avian- chick embryos
 Rodents- rats and mice( wild types and
transgenic): embryonic, post-natal and adult
 Human – 1. Neural progenitor cells from aborted
foetuses and stem cell lines.
2. Cord blood derived stem cells
Types of in vitro systems- cell culture
1. Cell lines
2. Primary culture
3. Organ architecture preserved

16. In vitro methods
 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

17. In vitro pyrogen test
 Rabbit pyrogen test is
replaced with
A. Limulus amoebocyte
lysate(LAL)
B. Monocyte activation test
 Based on the response of
human leukocytes which
release inflammatory
mediators in response to
pyrogen contamination

18. Limulus amoebocyte
lysate(LAL)
 Principle- Lipopolysaccharides cause extracellular
coagulation of blood( Haemolymph) of horseshoe crab
Lumulus polyphemus
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

19. Monocyte activation test
Uses human
mononuclear cells
obtained from
human volunteers
or from blood bank
Very specific and
sensitive
Detects pro-
inflammatory
contaminants
Better than LAL and
rabbit pyrogen test

21. Embryonic stem cell test
 Used for detection of any embryonic toxicity
 Principle- the capacity of stem cells(rodent cell
line D3) to develop into specialized contracting
heart cells in vitro within 10 days is assessed using
light microscopic evaluation
 End points –
1. Inhibition of differentiation
2. Cytotoxic effect on the ES cells
3. Cytotoxic effect on 3T3 fibroblasts

22. Embryonic stem cell test
 Metabolism studies using human microsomal
enzymes or cell lines can predict if a non toxic
chemical is likely to be metabolized to a toxic form
or vice-versa
 Positive result classifies the chemical as likely to be
hazardous for development and reproduction
 Better alternative to study cancer, liver and
cardiac toxicity

23. LOCAL LYMPH NODE ASSAY
 Used to test the potential of test compound for skin
sensitization
 Principle- a test compound is considered as a
sensitiser when the lymph node draining the site
of chemical application reveals a primary
proliferation of lymphocytes as measured by
radioactive labelling in test and vehicle groups
 Proliferation is proportional to dose applied
 Stimulation index- ratio of proliferation in test
groups to that of control
 Index must be atleast 3

24. Skin patch tests
 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

25. Skin patch tests

26. Neutral red uptake 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

27. Neutral red uptake assay

28. Carcinogenicity test
 By using cell transformation assays
 Eg-1. Balb/c3T3 assay
2. 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

29. Stem cell models
 Can be used for toxicological screening and also
as invitro models of disease
 Disease genes are inserted into embryonic stem
cells, induced to differentiate into human disease
tissue which is used for screening of drugs
 Eg- Genes from a Parkinsons patient were
introduced in embryonic stem cells which grew
into a model of Parkinsons disease and is used for
screening potential drugs
 Alzheimers and Diabetes models

30. 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

31. Microorganism based model
 Tetrahymena pyriformis—a ciliate protozoan
being used to study the effects of anesthetics on
metabolism
 Salmonella typhimurium—bacteria used in
mechanistic studies in genetics as well as the Ames
mutagenicity/carcinogenicity test

32. IN CHEMICO TESTING
 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

33. In silico models
 Computer aided molecular drug design
 Quantitative structure activity relationships
 Computer assisted learning
 Computer or mathematical analysis
 Microfluidic chips
 DNA chips
 Organ on chip
 Human on chip

34. Computer aided molecular drug
design

35. Computer assisted learning (CAL)
 CAL deals with a range of
software packages which
simulate the animal
experiments
 Two softwares are curently
used in india
 Expharm- developed by
JIPMER, India
 X-cology

36. expharm
 Contains programs on
 Effect of drugs on the rabbit eye
 Bio assay of histamine using guinea pig ileum
 Effect of drugs on the frog heart
 Effect of drugs on dog blood pressure and heart rate
 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

37. X-cology
 video demonstrations of different procedures like
isolation and mounting of animal tissues
 Screen interactive interface to study the effects of
various drugs on the isolated tissues
 Content is classified into three sections
 Experimental animals
 Equipment
 Experimental technique – procedure to carry out
bioassay and experiments on whole animals

38. Trauma man
 Computer programme
 Simulates hemorrhaging, fractures, amputations
and burns
 Is used for military training and training medical
students
 Combat Trauma Patient Simulator similar to
trauma man

39. Computer or mathematical analysis
 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

40. Recent trend
 Researchers are working on a “virtual human”
which is designed to predict drug metabolism and
metabolite interaction with any given organ

41. Identify disease
Isolate protein
Find drug
Preclinical testing
GENOMICS, PROTEOMICS & BIOPHARM.
HIGH THROUGHPUT SCREENING
MOLECULAR MODELING
VIRTUAL SCREENING
COMBINATORIAL CHEMISTRY
IN VITRO & IN SILICO ADME MODELS
Potentially producing many more targets
and “personalized” targets
Screening up to 100,000 compounds a
day for activity against a target protein
Using a computer to
predict activity
Rapidly producing vast numbers
of compounds
Computer graphics & models help improve activity
Tissue and computer models begin to replace animal testing

42. Microfluidic chips
 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

43. Micro dosing studies
 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

44. Quantitative structure activity
relationships
 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

45. Advantages
 Alternative scientific tests are often more reliable
than animal tests.
 The use of human tissue in toxicity testing is more
accurate than the animal models.
 Cruelty-free products are more environmentally
friendly.

46. Summary
 Ethical concerns and dismal rate of translation with
animal experiments have led to development of
alternate methods
 3R concept-
 Reduce- reduce the number of animals used
 Refine- refine the experimental procedure
 Replace- wherever feasible replace the animal
experiment

47. Summary
In vitro methods In chemico In silico
Pyrogen tests
• LAL
• Monocyte activation test
Teratogenicity
• Embryonic stem cell test
Skin sensitizer
• Local lymph node assay
Skin irritation
• Corrositex
• Epiderm
• Episkin
• Skin ethic RHE
Eye irritation
• Neutral red uptake assay
Carcinogenicity
• Cell transformation assays
Stem cell models
• LUHMES for parkinsons
HPLC
Direct peptide reactivity assay
 CAL- EXPHARM
X-Cology
Trauma man
 Computer aided molecular
drug design
 Microfluidic chips
 Quantitative structure
activity relation ships
 Human studies
Microdosing studies

48. References
 Fundamentals of Experimental Pharmacology. M.N.Ghosh. 6th edition.
 Practical Manual of Pharmacology. Dinesh Badyal. 1st edition.
 A Review on Alternatives to Animal Testing Methods in Drug Development.
Ranganatha N, I. J. Kuppast. International Journal of Pharmacy and
Pharmaceutical Sciences.
 Animal use in pharmacology education and research: The changing scenario.
Dinesh K. Badyal, Chetna Desai. Indian Journal Of Pharmacology