cell cultures,in vitro studies,stem cells,various substitutes for in vivo experiments

1. ALTERNATIVES TO
ANIMAL
EXPERIMENTS
. DR. SIBI P I
PROFESSOR
Division of Pharmacology
Dept. of Pharmaceutical Sciences, CPAS
Cheruvandoor,Ettumanoor, Kerala, India,

2. Definition
“Alternatives” or “Substitutes” is
defined as anything from absolute
to partial replacement of live
animals in biomedical research and
testing.

3. Introduction
❖ Animals are used in science for:
➢ Undergraduates teaching
➢ to learn physiological mechanism,
➢ anatomy
➢ effect of various drugs on human body
➢ Postgraduate -effects of various drugs, -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 -extensive toxicological studies in
animals- 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 may
be
➢ 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. 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

7. 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

8. 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

9. 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

10. 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

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. 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

13. Responsibility
•The 4th
R of Research implies addition of ‘responsibility’ to the
original three R's of Russell and Burch.
•It has grown into a new era of performance-based outcomes,
which reflects integrity, honesty, and scientific correctness in
appropriate and reasonable use of laboratory animals.
•This ensures that animal life is required and necessary for
biomedical advancement

14. Alternatives
❖ Physico-chemical techniques
❖ Microbiological systems
❖ Epidemiological surveys
❖ Plant analysis(Toxicity assays in Plants)
❖ Stem cells
❖ Microdosing
❖ DNA chips
❖ Microfluidic chips
❖ Human tissues
❖ New imaging technologies
❖ Post marketing survelliance

15. Physico-chemical techniques
These help to identify human responses to chemicals and
biological substances
eg:-Chitosan films as a substitute for animal and human
epidermal sheets -in vitro permeation of polar and
non-polar drugs
capable of stimulating the flux of model
drugs-5-FU,Indomethacin across rat, rabbit and human
cadaver epidermal sheets
local delivery of systems of various plant
extracts-Calendula officinalis,Porphyromonas gingivalis-
due to their properties-Biocompatibility,biodegradabilityand
adhesion ability

16. Chitosan films

17. Microbiological systems
Based on the capability of chemicals to induce
mutating changes in a cell’s DNA eg:-Ames test
-detect 80-90% of all carcinogenic chemicals that
have been studied.
Cunninghamella
elegans-Anti-coagulants,Diuretics,Anticonvulsants
,hemorheologic agents have been tested using
this fungi
.
Bacteria Vibrio vulnificus to study the modulation
of the toxic RtxA1which induces acute

18. Ames’s test

19. Cunnighamella elegans vibrio vulnificus

20. 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

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

22. Avian-Chick embryos
Avian (chick) embryos are an established and
accessible model organism making them ideal
for studying developmental processes. Chick
embryos can be harvested from the egg and
cultured allowing real-time observations and
imaging

23. Neural progenitor cells from
aborted foetuses

25. 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
❑ Organs-on-Chips

26. 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

27. 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

29. 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

31. Organs-on-Chips
A variety of cell-based tests and tissue models can be used to asses
the
safety of drugs, chemicals,
cosmetics
consumer products.
Example:
CeeTox developed a method - to cause a skin allergy in humans tha
incorporates
MatTek’sEpiDerm™ Tissue Model—a 3-dimensional, human
cell–derived skin
model that replicates key traits of normal human skin.
It replaces tests in which experimenters injected guinea pigs or mice
with a substance or applied it to their shaved skin to determine an

32. Lung-on-a-chip
It can mimic the function of alveoli – the gas exchange units
in your lungs. This thumb drive-size device has two
channels: an air-filled upper channel lined with human
alveolar epithelial cells, and a lower channel lined with blood
vessel cells and a flowing medium with human blood cells in
it.

34. organ on a chip

35. 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

36. 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

37. Embryonic stem cell test

38. 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

39. Local lymph node assay

40. 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.

41. Skin patch tests

42. 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

43. Neutral red uptake assay

44. 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

46. Syrian hamster embryo assay

47. 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

48. 3D CELL MODELS-STEM CELLS

49. 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

51. 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

52. ◻ Tetrahymena pyriformis

53. Salmonella typhimurium

54. 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

55. ◻ Direct peptide reactivity assay

56. 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

57. Computer aided molecular drug
design

58. 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

59. 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

60. 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

61. 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

62. 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

63. Technologies to obtain antibodies without the use
of animals
‘Phage display’ technology
Example:
The antibody adalimumab, for example, is an approved drug
that recognises and binds to a particular biomolecule in the
body to reduce inflammation, helping alleviate symptoms
associated with many different conditions such as arthritis,
psoriasis and Crohn's disease.

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

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

67. 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

68. Microfluidic chips

69. 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

71. 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

72. 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.

73. 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

74. 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

75. 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