Alternative methods to animal toxicity testing are needed because animal testing can cause suffering. Some alternative methods include computer modeling, cell and tissue cultures, and organ chips. Computer modeling uses computer programs to simulate biological effects and predict toxicity without animal testing. Cell and tissue cultures grow isolated cells and tissues in vitro to study toxicity. Organ chips are microfluidic devices that contain living cells arranged to simulate organ-level functions, allowing tests on human-relevant systems without whole animals. These alternative methods can help reduce and replace animal use in toxicity testing.

1. ALTERNATIVE METHODS
TO ANIMAL TOXICITY
TESTING
Saif Imtiyaz
M Pharm Pharmacology
Jamia Hamdard

2. PRESENTATION OUTLINES
1. Introduction…………………………………………….. 01- 02
2. The Rs Principles……………………………………… 03
3. Need For Alternatives To Animals…………………. 04
4. Alternative Methods…………………………...……… 05
5. Different alternative methods……………………….. 06- 22
6. References……………………………………………… 23

3. Introduction
● Animal models have been used to develop human biochemistry, physiology, pharmacology,
endocrinology, and toxicity. Every year, 10-100 million animals are used for testing.
● Animals used experimentation distributed among zebra- fish to primates. Vast majority of
animals are sacrificed at end of research programme.
● The use of animals can be further subdivided according to the degree of suffering
a. Minor animal suffering:- observing animals in behavioral studies, single blood sampling ,
immunization without adjuvants, etc.
b. Moderate animal suffering:- repeated blood sampling , recovery from general anesthesia ,
etc.
c. Severe animal suffering:- LD50% test , starvation vaccine potency tests, etc.
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4. ● 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”.
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5. The 5 R’s Principle
● Alternative methods able to do: Reduce Refine Replace ;
collectively called as “The 3Rs Principle”.
● The 3Rs principles were defined in 1959 by W.M.S Russel
and R.L Bruch. They provide a strategy for rational and
stepwise approach to minimizing animals use and
suffering in experiments without compromising the quality
and quantity of scientific work being undertaken.
● Reuse & Rehabilitate- The 4th & 5th R of Research implies
addition of ‘responsibility’ to the original three R’s, reflects
integrity, honesty, and scientific correctness in appropriate
and reasonable use of laboratory animals.
5 R’s
REDUCE
REHABILIT
ATE
REUSE
REPLACE
REFINE
3

6. 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 infected disease
Economic and efficiency
• Invitro testing human cell lines have been useful in securing relevant information for human risk
assessment thereby opening up opportunities to explore responses to existing and emerging
therapies human cell lines can be used for the tumor and some other chronic disease. 4

7. Alternative Techniques
● 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.
● 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
● 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.
5

8. Alternative to Animal Experiments
Continued but
modified use of
animals 5Rs
In vitro [test tube]
method
Tissue culture
technique
In silico
[computer
modelling
technique]
Computer aided molecular drug design [CADD]
Computer assisted learning [CAL]
Microfluidic chips
Quantitative structure activity relationship
Organ on chips
Computer or mathematic analysis
In silico
[computer
modelling
technique] 6

9. 1. Continued But Modified Use Of Animals
Russel and Burch developed 3R’s strategy which include:
Refinement
• Refine
experimental
methods to
decrease
unnecessary
pain and
trauma to
animals.
Reduction
• Reduce the
no. of
animals used
in these
experiments
Replacement
• replace the
animal
experiments
e.g.
computer
stimulation
methods, In
vitro
methods,
Cell culture
techniques
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10. Methods Of Refinement
 Providing relief [pain and distress] by giving drugs like analgesics,
anesthetics, tranquillizers and sedatives.
 By changing procedure
Modified to
reduce pain and
distress in
animal
Use non
invasive
technique like
MRI
Use less
sensitive animal
species
Use smaller
dose
Improving
housing
conditions
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11. Methods Of Reduction
•Good planning of
studies Change in
experimental design
Improve methods of
data analysis
•Sharing research
animals
•Redesigning
studies to collect as
much information as
possible
•Share information.
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12. 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
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13. 2. Invitro Models
11
• In vitro testing is the scientific analysis of the
effects of chemical substances on cultured
bacteria or mammalian cells, organ, tissues,
enzymes receptors enzymes.
• In vitro (literally 'in glass') testing methods are employed primarily : ̵ ̵ toidentify potentially
hazardous chemicals to confirm the lack of certain toxic properties in the early stages of the
development of potentially useful new substances such as therapeutic drugs, agricultural
chemicals and food additives.
• In vitro testing methods can be more useful and cost-effective than toxicology studies in
living animals (which are termed in vivo or "in life" methods).

14. Various Test In Vitro Methods
● In vitro pyrogen test
● Embryonic stem cell test
● Carcinogenicity test
● Neurotoxicity test
Avian chick embryo
Rodents[rat and mice, wild
type, transgenic , embryonic,
post natal , adult
Human cells [neural
progenitor cells from
aborted fetus and stem
cell lines]
Source Of Tissue For In Vitro Methods
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15. 3. 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
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16. 4. In silico [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 structure activity relationship
Computer assisted learning[CAL]
Computer or mathematical analysis
Organ on chips 14

17. A. Computer Aided Drug Design [CADD]
● It is the inventive process of finding new medications based
on the knowledge of a biological target.
● It involves the design of molecules that are complementary in
shape and charge to the biomolecular target with which they
interact and therefore will bind to it.
● It is used to predict the receptor binding site for a 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
analyze drugs.
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18. ● Drug design with the help of
computers may be used at any of the
following stages of drug discovery:
I. hit identification using virtual screening
(structure- or ligand-based design)
II. hit-to-lead optimization of affinity and
selectivity (structure-based design,
QSAR, etc.)
III. lead optimization: optimization of other
pharmaceutical properties while
maintaining affinity.
● Advantages of CADD
I. Time
II. Cost
III. Accuracy
IV. information about the disease
V. screening is reduced
VI. Database screening
VII. less manpower is required
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19. Categories of software
Databases & Draw Tools
Molecular Modeling & Homology Modeling
Binding site prediction & Docking
Ligand design Screening -QSAR
Binding free energy estimation
ADME Toxicity 17

20. B. Quantitative Structure Activity Relationship
● A quantitative structure-activity relationship (QSAR) is a mathematical relationship which
correlates measurable or calculable molecular properties to some specific biological activity
in terms of an equation
● 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.
● QSAR has been widely used in medicinal chemistry as support in drug’s discovery and
development process as well as in study of harmful and poisonous substances in
toxicological chemistry. QSAR attempts to find consistent relationship between biological
activity and molecular properties, so that these “rules” can be used to evaluate the activity of
new compounds
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21. ADVANTAGES OF QSAR
● It gives quantifying the relationship between structure and activity with their physiochemical
property basis.
● Possible to make predictions of designed compounds before the chemical synthesis of novel
analogues.
● It may help to understand the interactions between functional group of designed molecules
and their activity of target enzyme or protein.
DISADVANTAGES OF QSAR
● Due to biological data experimental error it may give false correlations.
● If training set of molecule is less, the data may not reflect the complete property and it cannot
be used to predict the most active compounds.
● In some 3D QSAR study ligands binding receptor or protein may not be available
19

22. C. Organ On Chips
● It is a multichannel 3-D micro fluidic cell culture chip which simulates the activities,
mechanisms, physiological response of entire organs.
● These micro devices are translucent , they provide a window to watch inner workings of
human organs.
● 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

23. How will these organs on chips help the pharmaceutical
industries??
● Replace animal models.
● Help the laboratories reach the stage of clinical trials.
● Comparison studies of drugs on human and animals.
● To study the effect of drug on its main action of site and also other organs.
● Study of toxicity of drugs and cosmetics.
● To study about cancer cells and produce new drugs in cancer treatment.
● Ensure better regulatory decision-making.
● Develop vaccines and drugs to counter bioterrorism threats.
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24. D. HET-CAM (Hen's egg-chorioallantoic membrane) TEST
● 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 Chorio
Allantoic-Membrane is exposed.
● The test substance is dropped 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.
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25. References
● Fundamentals of Experimental Pharmacology. M.N.Ghosh. 7th edition, 2020.
● Practical Manual of Pharmacology. Dinesh Badyal. 1st edition, 2021.
● Alternative Methods to Animal Experiments in Toxicity Testing. Nuhoğlu Öztürk, Zeyno
& Aksoy, Abdurrahman. 7th International Congress on Veterinary and Animal
Sciences (2022).
● Animal Use In Pharmacology Education And Research: The Changing Scenario.
Dinesh K. Badyal and Chetna Desai. Indian Journal of Pharmacology, May-June,
2014.
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