ALTERNATIVES TO

ANIMAL EXPERIMENTS

Dr Charles V. Adhav II year Resident Dept of PharmacologyT.N.M.C 11th August 2012

History of Animal Experiments

History of Animal Experiments

Present scenario

• Rodents 84%• Fish, amphibians, reptiles 12%• Large mammals 2.1%• Small mammals 1.4%• Dogs and cats 0.3%• Primates 0.1%

Scope of animal experiments• Teaching factual knowledge.• Demonstrating dynamic processes in life.• For all industry sectors (pharmaceuticals, cosmetics)• Compound selection in drug discovery & development.• Research studies – understanding mechanisms, e.g. how certain

chemical classes cause cancer?

Animals in experiments

Advantages

• Active & interactive learning

• Teaching lab skills• Developing responsible

attitude towards animals• Hands-on experience

Disadvantages

• Harmful (animal) use• Student have

conscientious objection.• Not always the most

suitable model• Intensive Labour• Not practical with large

number of students.• Costs of material, animal

& housing high

History of Animal Welfare• 1641: First Legislation forbidding cruelty to animals passed in

the Court of Massachusetts

• 1822- First anti-cruelty law was passed in England. Martin was among the founders of the world's first animal welfare organization, the Society for the Prevention of Cruelty to Animals, or SPCA, in 1824

• 1840 - Queen Victoria gave the society her blessing, and it became the RSPCA

• 1871 - “The British Association for the Advancement of Science” published guidelines for animal experimentation after of the first national laws in UK- Cruelty to Animals Act, 1835

• 1883 - American Antivivisection society came into existence

•  1960 -In India, animals are protected by the Prevention of Cruelty to Animals Act, 1960

The Silver spring monkeys• The Silver Spring Monkeys(1981)

- Macaque Monkeys at Institute of Behavioural Research (IBR), Silver Spring, Maryland were subjected to debilitating surgeries

- Electrocuted, food deprived, chair restrained, forced to try to regain the use of their impaired limbs

- Edward Taub, a psychologist, who had cut afferent ganglia that supplied sensation to the brain from their arms to evaluate Neuroplasticity

- He then used arm slings to restrain either the good or deafferented arm to train them to use the limbs they could not feel

The Case that launched PETA• Ensuing battle over the monkeys' custody saw,

– an amendment in 1985 to the Animal Welfare Act– the transformation of PETA from a group of friends into a national

movement– the creation of the first North American Animal Liberation

Front cell, – and the first animal research case to reach the United States Supreme

Court

• THE BRIGHT SIDE– Based on the concept of neuroplasticity, for people disabled as a

result of brain damage. Known as constraint-induced movement therapy, it has helped stroke survivors regain the use of limbs paralysed for many years, and has been hailed by the American Stroke Association as at the forefront of a revolution

Organizations against experimentation on animals

• Animal Welfare Institute• Humane Society of the United States• Johns Hopkins Center for Alternatives to

Animal Testing• People for the Ethical Treatment of Animals• Psychologists for the Ethical Treatment of

Animals

Arguments against animal experiments

• Animal suffering is excessive due to widespread abuse, regardless of possible benefits

• Supporters of animal rights argue that benefits to human beings cannot outweigh suffering of animals

• Human beings have no moral right to use individual animals in ways that do not benefit that individual

• Animals do not consent to being tested upon

• Animal testing is bad science

Pro Organizations• American Association for Laboratory Animal Science• Americans for Medical Progress• Michigan Society for Medical Research• National Association for Biomedical Research

Motives To Develop Alternatives

Law

Ethics

Scientific

Economy

Public pressure

LAWS• In Europe law clearly states as article 7.2 of EU Directive that:

– "An experiment [animal] shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practically available.“

• In U.S. The Animal Welfare Act (AWA) (1966) is the only federal law that covers animals in research

• In India, The prevention of cruelty to animals Act(1960)states– Experiments on animals are avoided wherever it is possible to do so; as for

example; in medical schools, hospitals, colleges and the like, if other teaching devices such as books, models, films and the likes, may equally suffice

Concept Of 3 “R”s

Russell and BurchThe Principles of Humane Experimental Technique, 1959

A 4th R Exists – Responsibility

Replacement• Substitution of insentient material in place of

conscious higher animals• Comparative substitution :

– When free living metazoan invertebrates are considered as possible substitutes for vertebrate subjects

Examples 1. The Fruit fly Drosophila has been employed for

titration of antivenene for the venom of an Australian spider

• Larger number of doses can be tried and neutralised than in mice

2. The Octopus has been described as a more suitable subject than the albino rat for studies on the mechanism of visual discrimination

Relative and Absolute replacement

• Relative/ Partial replacement : – Animals are still required though in actual

experiment they are exposed, probably or certainly to no distress at all

– Work on isolated cells, tissues and organs of vertebrates which may be maintained for short periods for acute experiments

• Absolute replacement :– Animals are not required at any stage of the

experiment– Use of metazoan endoparasites, higher

plants, micro-organisms( protozoa, bacteria and moulds), and non living physical and chemical systems

Classification of replacement methods

COMPUTER AIDED LEARNING• Virtually replaces the use of living animals.• Biological phenomenon are adapted to computer models &

basic processes are expressed as mathematical formula.• After the formula is developed, enormous variables can be

introduced to get different responses.• Thus, more accurate the formula, more successful is the

program

Computer models

Pure Computer software

Emulation• Concentrate more on teaching

principles rather than absolute response.

• Eg: Pharma tutor

• Lack quantitative accuracy.

Simulation

• Aim to depict scenarios that are as close to real life.

• Eg: Expharma

• Quantitatively correct data.

ETHICAL: In accordance with 3R principal s of Russel‟a. Replacementb. Reductionc. Refinement

2. Repetition of experiments 3. Unambiguous and complete data4. Built-in self-assessment 5. Saves time & Cost6. Avoid negative student perceptions of “failed” experiments

EMULATION : PHARMA TUTOR• Pharmatutor, devised by Daniel Keller (Keller 1987)• Pharmatutor emulates the effects of several drugs on the

cardiovascular system.• The qualitative effects of epinephrine (adrenaline) on heart rate and

blood pressure can be demonstrated

1. Blood pressure and catecholamine's

2. Blood pressure and acetylcholine

3. Dose response curves

4. Neuromuscular transmission

Pharma tutor• Screen display from the

Pharmatutor program• A menu bar (not shown) allows

different combinations of drugs (including blockers) to be added, the experiment to be halted at any time, and the screen image to be printed

• Screen display from the Rat Blood Pressure program

• The figure shows simulated data of heart rate (upper trace) and carotid artery pressure (lower trace)

SIMULATION :Ex Pharma ProExPharmPro is a computer assisted learning package containing5 programs which simulate animal experiments in Pharmacology.These programs can be used to demonstrate drug on differentanimals systems. Developed by R. Raveendran,Prof of Pharmacology

JIPMER, Pondicherry

The current version of ExPharm consists of five programs:

1. Effects of drugs on rabbit eye2. Bioassay of histamine on the guinea pig ileum 3. Effect of drugs on ciliary motility of frog oesophagus4. Effect of drugs on isolated frog heart5. Effect of drugs on blood pressure (BP)

and heart rate (HR) of dog

Ex pharma pro – main menu

Strathclyde Pharmacology Simulations

• The virtual cat• The virtual rat- drugs on CVS• The virtual rat – phrenic nerve hemi diaphragm preparation.• The virtual NMJ

Strathclyde Pharmacology Simulations

Integrated Course Ware• Interactive tutorial package by

MicroLabs, University of Amsterdam, Academic Medical Centre (The Netherlands).

• Includes integrated topics from animal behaviour, experimental design, anaesthesia of rats, and video footage of animals (rats), Guinea pig ileum in vitro, Phrenic nerve diaphragm in vitro used in pharmacology courses

• Some elements still under construction

Virtual Reality• It creates images that deludes

observers into believing that he/she is another world.

• Operator wears a special helmet in which computer images are projected.

• Sound is produced by stereo phones.• All sensory input from real world is

cut off.• Gloves carrying sensors that analyze

the position of hands and fingers and inturn use this information to adjust visual image.

InterNICHE• InterNICHE is the International Network for Humane

Education• InterNICHE began in 1988 as EuroNICHE, and transformed in 2000 to a

global network

• Support progressive science teaching and the replacement of animal experiments by working with teachers to introduce alternatives and with students to support freedom of conscience

• The InterNICHE Alternatives Loan Systems are evolving libraries of multimedia software, models, mannekins and simulators. There is one global Loan System, and other localised libraries in India, Kenya, Mexico, Peru, Russia, South Africa and the Ukraine

InterNICHE in INDIA• Nick Jukes is the current interNICHE co-ordinator• 3000 animals replaced in Gujarat, India

– InterNICHE partners Gujarat Society for the Prevention of Cruelty to Animals (GSPCA)

– Bhavnagar university agreed to end use of over 3000 mice, rats and rabbits for dissections and severe experiments in pharmacology, biochemistry, zoology and health science education.

– Replaced by CAL Pharmacology compilation by Dr. R. Raveendran.

Computer softwares for Research

Virtual Heart1. In 1997, Roche pharmaceuticals

had a new heart drug approved on basis of a virtual heart.– The animal tests were

inconclusive.

2. FDA approval was gained for its calcium channel blocker, Mibefradil.

3. Drug however was withdrawn due to its renal side effects.

Virtual patients• A combination of infinite variables to make complex equations

resembling physiological processes in cells;• Entelos, a firm based in Foster City, California with Novartis

pharma in development of oncology drugs using virtual patients• Pfizer evaluated 125 unique virtual patients, to represent

variability seen in real-world patients, and then ran a simulation to evaluate asthma treatment .

Computer aided learning

Advantages

1. Minimizes use of animals.

2. Helps in learning in better

way; simulations are

prepared considering ideal

situations with no

experimental error.

3. Ensures repeatability of

experiment.

Disadvantages

1. At any time, it cannot

create actual situation of

conduction of experiment.

2. Does not allow to learn the

skills & techniques of

handling.

3. Does not allow to learn

from errors/ changes in

conditions during

experiment.

Conclusions on simulation• Gives a student a ”taste” of experimental pharmacology• Showing in theory how drugs act on pharmacological

preparations• Encouraging students to devise experiments to test

hypotheses• No hands-on experience on techniques

IN-VITRO TECHNIQUES

• Cell culture technique:1. Human neuronal model cell line

2. myeloma cells for antibody production

• Tissue techniques:1. Skin full thickness model

2. Chitosan models

3. Stem cell derived tissues

• Ethically sourced animal cadavers/ tissues

Neuronal cell line and myeloma cells for antobody production

• Antibodies—which are used to research, diagnose, and fight diseases and have traditionally been created by injecting cancer cells into mice—can now be produced using DNA that's made in a laboratory or taken from human cells

•ReNcell VM is an immortalized human neural progenitor cell line with ability to readily differentiate into neurons and glial cells

•ReNcell VM was derived from the ventral mesencephalon region of human fetal brain.

•By retroviral transduction with the v-myc oncogene, this cell line grows rapidly as a monolayer on laminin with a doubling time of 20-30 hours

Phenion® Full Thickness Skin model

• Phenion® FT Skin Models are produced from human primary keratinocytes and fibroblasts isolated from neonatal skin. Cells are neither pooled nor genetically modified

• Suitable for– long-term observation of up to 9 days after maturation– Topical or systemic application– Repeated dosing– Measurement of epidermal & dermal markers on protein

&molecular biological level– Vitality and efficacy testing

The following endpoints can be analyzed

Epidermal differentiation Cell viability Anti-inflammatory effects Anti-aging effects Extracellular matrix

Chitosan Models• Derived by deacetylation of chitin from crustaceans.• Substitute for animal/ human epidermal sheets used for in

vitro permeation of polar/ non-polar drugs.• Chitosan films which simulate the flux of model drugs like

5-Fluorouracil & Indomethacin across rat, rabbit & human epidermal sheet have been developed.

• Used for local drug delivery of various plant extracts (Thymus vulgaris, Croton lechleri) to test their anti-microbial activity against peri-odontal pathogens.

• They are biocompatible, biodegradable and have adhesive property.

• Can be substitute for animal models.

Embryonic stem cell test

• Cytotoxic effects on embryonic stem cell tests• Cytotoxic effects on the 3T3 fibroblasts• In the embryonic stem cell test ,

differentiation of embryonic stem cells into specialised cardiomyocytes in vitro within 10 days are used to assess the embryotoxic potential of test compound

using Light microscope or

objective molecular endpoints

Fever in a test tube• In Vitro pyrogen test :

– Lumulus amoebocyte Lysate test :• Principle : the lipopolysaccharides cause extracellular

coagulation of the blood (haemolymph) of the Horseshoe Crab (Lumulus polyphemus)

• More sensitive than Rabbit pyrogen test but gives false negative results and also does not detect pyrogens other than bacterial endotoxins, virus and fungi.

– Monocyte activation test :• Principle : uses mononuclear cells from human

volunteers or from blood bank for detection of pyrogens

• Detects pro-imflammatory and pyrogenic contaminants not always detected in rabbit pyrogen test and LAL test

Tests for occular toxicity• Draize Eye Irritation test:

– Developed by Draize in 1944 where 0.1 ml of liquid or 0.1 gm solid test substance is instilled into lower conjunctival sac of one of eyes in rabbit.

– Other eye serves as control and the test eye is not washed but examined at 1, 24,48, 72 hours

– Eye examined for corneal opacity, redness of iris, chemosis and discharge of cojunctiva

Alternatives to Draize test• 4 Alternative tests devloped and accepted by regulatory

processes for validation:– HET- CAM Assay on the embryonated chicken egg– BCOP test on isolated bovine cornea obtained from slaughter houses– In-Vitro tests

• Isolated rabbit eye (IRE) • Isolated chicken eye (ICE) from animals sacrificed for other purposes

Phototoxicity testsIn vitro 3T3 NRU Phototoxicity test:

1. Objective :- used to identify the phototoxic potential of a test substance

induced by the excited chemical after exposure to light

2. Method :- Balb/c 3T3 cells are maintained in culture for 24 h for formation of monolayers.

- 2 of 96-well plates per test chemical are pre-incubated with 8 concentrations of test substance and with positive control and negative control for 1 h.

- Then one of the two plates is irradiated (+UV) for 50 min. with 1.7 mW/cm & other is kept in the dark.

- In both plates the treatment medium is then replaced by culture medium & after 24 h of incubation cell viability is determined by Neutral Red uptake.

Tests for skin corrosivity• Animal tests:

– A test substance is applied to the shaved bare skin (about 6 cm2) of healthy young adult albino rabbits and the area is covered with gauze (OECD Test ; Acute Dermal Irritation/Corrosion)

– The substance is removed after four hours and the rabbit's skin is observed at specific times for irritant responses for as many as 14 days

Validated methods

In Vitro tests for Skin corrosivity • In vitro tests:

– Reconstructed human skin (EPISKIN) : first validated by the European Coalition on the Validation of Alternative Methods (ECVAM) as a complete replacement for animal tests

– Rat skin Transcutaneous electrical resistance (TER) : Effect of corrosive material on isolated rat skin TER(<5kw) and effect on penetration of sulforhodamine dye through skin is measured by optical density measurements and compared with HCl and H2O as +ve and –ve controls

– Corrositex® uses a biomembrane and chemical detection system that changes color when in contact with corrosive substances

Ehically Sourced Animal Cadavers & Tissues

• Animal cadavers and tissue obtained from:1. Animals that have died naturally or in accidents2. Animals that have been euthanized secondary to natural, terminal disease or serious

non-recoverable injury.• Not ethical:

1. Animals harmed or killed to provide cadavers or tissues.2. Places where harming or killing is common.

• Used for training surgical techniques such as intestinal anastomosis.• Raises student’s skills to a sufficient level in preparing for apprentice work in

live animals.• Sources:

1. Body Donation Program2. Animal Tissue Banks

Source of animal tissues• Body Donation Programs

– Clients of the vet clinics consent to donate cadaver of their companion animal to teaching, after the animal’s natural death or euthanasia.

– ‘Willed body’ , ‘Client Donation’ or ‘Educational Memorial’• Animal tissue banks

– Collection and immediate use of fresh tissue.– Animal blood from healthy companion animal volunteers, stored for life-saving transfusion.

Preservation & Storage• Freeze drying: Emptying of blood, skinning or removal of fur and

embalming fluids & formalin• Silyophilisation: silicone impregnation• Plastination: water and lipid within cadaver are replaced by plastic polymers

which are hardened; resulting in real specimens.

Microfluidic Chips• 2 cm wide, containing tiny chambers

each having tissues from different parts of the body.

• Compartment linked by micro-channels through which blood substitute flows.

• Test drug added to the blood substitute circulates in the device.

• Sensors in the chip give feedback for computer analysis.

• Mimics the micro-physiology of body

In vitro methods

1. Provides controlled conditions, minimizes effect of non-experimental variables on study outcome.

2. Potential uses are limitless; expand with tissue harvesting knowledge & improved control facilities.

1. Expensive 2. At suboptimal

environment, there is loss of entire study result

3. Requires training and financial inputs

Advantages Disadvantages:

Micro-Organisms• Ames test

1. Salmonella typhimurium cultures are used to screen compounds.

2. Can detect 80-90% of carcinogenic chemicals studied.

3. Formerly required animals

• Fungi : 1. Cunninghamela elegans,

capacity to metabolize wide variety of drugs.

2. Anti-coagulants, diuretics, anticonvulsants & hemorheologic agents have been tested.

Plant analysis• A recent study of effect of environmental

contaminants & pharmaceuticals on Brassica juncea.

• Demonstrated drug induced defense responses & activation of detoxification mechanism due to oxidative stress.

• Limited use due to presence of rigid cell wall.

• Arabidopsis thaliana , currently the most popular model plant related to mustard plant. Its short generation time facilitates rapid genetic studies, many phenotypic and biochemical mutants have been mapped. Arabidopsis was the first plant to have its genome sequenced

• Tobacco BY-2 cells is suspension cell line from tobacco (Nicotiana tabaccum). Useful for general plant physiology studies on cell level

NON-LIVING SYSTEM

Chemical methods• MNT: dilutions of Serum-virus

mixture inoculated intra-cerebrally into the mice.

• CIET: dog sera + virus in the cathode well & indicator sera in the anode well. Negative & positive controls tested simultaneously.

• Both MNT & CIET results have been found to be comparative

Endopeptidase Assay: • Developed and validated at the National Institute for Biological Standards and Control (NIBSC) for the detection of botulinum type B neurotoxin • Has been validated to verify potency as a measure of consistency of clinical samples containing BoNT serotypes A, B, C, E, and F•Replacement for LD50 : dose which will kill half of the test animals at a defined time-point

Different chemical methods

•TRF – time resolved fluorescence• MALDI- matrix-assisted laser desorption/ionization

PHYSICAL SYSTEMS• Synthetic training objects designed to simulate organs, limbs or whole animals.• Models: objects designed to appreciate the anatomy• Mannequins: life-like representations for clinical skill training• Simulators: computerised mannequins, surgical training devices, and suture

trainers.

Rat tail vein simulator• Silicone rat, with weight of a real

adult, and consists of a simulated blood solution and 3 different tails, which can be used to practise venepuncture skills.

• Opaque latex tail designed to replicate haematoma, which demonstrates to trainee damage that can be inflicted from poor technique.

• Translucent silicone tail material, which enables trainee to see simulated blood in vessel & observe the needle penetration and blood withdrawal.

Reduction• Refers to methods that enable researchers to obtain comparable levels of

information from fewer animals, or to obtain more information from the same number of animals

• Reduction in the number of animals is desirable in any procedure, however directly humane ,which employs large number of animals in one laboratory

– ANIMAL SHARING– IMPROVED STATISTICAL DESIGN– PHYLOGENETIC REDUCTION– BETTER QUALITY ANIMALS

• Performing pilot studies to determine some of potential problems in an experiment before numerous animals are used

Animal sharing• Within the institution.• Allow surgical procedures on animals to be euthanatized.• If two studies involve sham operated group, by means of

identical routes, identical environment & standard control diets, these animals can be shared.

• Centralized process from Institution Animal care program to heed various needs of investigators.

Improved statistical design• Improper study design : inappropriate number of animals used.• Design: maximize the analysis of data from an animal used.• Protocols : serial sacrifice, group sequential testing, cross over designs can reduce

the number.• Epidemiological surveys: use of previously exposed species data to study lifestyle

factors• Performing pilot studies to determine some of potential problems in an

experiment before numerous animals are used• Consulting with a statistician to use only the numbers of animals required to

achieve significance • Performing appropriate literature searches and consulting with colleagues to

ensure that experiments are not duplicated

Phylogenetic reduction• One of the replacement techniques also.• Projects designed for use of one of the myriad

invertebrates instead of non-human primates.• Animals should be least advanced on the

phylogenetic ranking.• Disadvantages:

– Less knowledge about maintenance & use of species down the phylogenetic ladder.

– Ability of the institute to provide the facilities for such species

Better Quality Animals• Cost & quality of animals be correlated during purchase.• Possibility of animal loss or data compromise after intrusion of

disease will be minimized.• Animals of unknown health status should never share the same

environment or lab equipment as healthy ones

Local Lymph Node Assay • LLNA - an in vivo method will not eliminate the use of animals in the

assessment of allergic contact sensitizing activity• It however has, potential to reduce number of animals required and offers a

substantial refinement (less pain & distress) of the way in which animals are used for allergic contact sensitization testing

• Principle – In LLNA, sensitizers induce proliferation of lymphocytes in lymph nodes draining site of test substance application

• Proliferation is proportional to dose and to potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitization

Acute oral toxicity test• Principle : based on a stepwise procedure with use of a minimum number of

animals per step, sufficient information is obtained on the acute toxicity of test substance to enable its classification

• Method : substance is administered orally to a group of experimental animals at one of defined doses.

• The substance is tested using a stepwise procedure, each step using three animals • Absence or presence of compound-related mortality of animals dosed at one step

will determine the next step, i.e.;– no further testing is needed,– dosing of 3 additional animals, with the same dose– dosing of 3 additional animals at next higher or next lower dose level

Refinement• Refinement refers to methods that alleviate or minimize potential pain,

suffering or distress, and enhance animal welfare for the animals still used

• Ways to achieve refinement are classified broadly as :1. Decreased Invasiveness

2. Improved Control of Pain

3. Improved Instrumentation

4. Improved control of techniques

Decreased Invasiveness• Use of Magnetic Resonance Imaging(MRI),

Ultrasound( USG)• Vascular canula , permits repeated

sampling & injecting of drugs• Minimally invasive laproscopy  and

procedures aseptically to prevent infection

Improved Pain Control• While submitting protocol to IAEC,

investigator should consider alternatives to procedures causing pain.

• Provision for use of analgesics, tranquilizers & anesthetics unless the design does not allow

Improved Instrumentation• In vivo Imaging System (IVIS)  Core

– The Xenogen IVIS Spectrum Instrument with Fluorescence Kit, Anesthesia System, and Living Image Acquisition/Analysis Package provides the  capacity to non-invasively monitor up to (5 mice) at once with the capacity to monitor in vivo multiple fluorescent reporter and bioluminescent reporters simultaneously

• VisualSonics micro-ultrasound

– imaging system with integrated anesthesia-delivery

and physiological monitoring

– for small animal model research

• Availability of micro/nanoelectronics, fibre optics.

• Use of catheters or wireless transmission systems from implants within the animal body.

Improved Control of Techniques1. Under expertise of veterinarian.2. Properly executed techniques are less distressful using

proper handling techniques for animals3. Receiving adequate training prior to performing a procedure4. Ensuring that drug doses are correct and that the drugs

used are not expired5. Ensuring that procedures to be performed on the animal are

reasonable for that species

Refinement

• Setting earliest possible endpoint for experiment i.e. if needed information can be gathered before animal experiences any ill effects from experiment, this should be defined as the endpoint and animal subsequently euthanized

– Ex: if measuring toxicity of a compound or survival following implantation of a neoplasm

• a pilot study may determine that– once certain clinical signs are seen, or – a tumor achieves a certain size, the time course until debilitation or

death are predictable.• Subsequent experiments may then utilize the earlier endpoint

of tumor size or clinical signs of toxicity, rather than death as the endpoint.

4th R- Responsibility• CPCSEA in 1998 notified in the gazette of India the "Breeding of and

Experiments on Animals (Control and Supervision) Rules 1998“

• CPCSEA; has enabled creation of a common platform of discussion for scientists and animal activists for humane and progressive solutions for use of animals in experimentation

• CPCSEA has made it a national policy that personnel using experimental animals have a moral responsibility towards these animals after their use

• Costs of after-care/rehabilitation of animals post experimentation are to be a part of research costs and should be scaled in positive correlation with level of sentience of animals

• CPCSEA ordered the confiscation of 37 monkeys who had been horribly abused for years from the National Institute of Virology, Pune

4th R - Responsibility

ECVAM report classified alternatives

The following groups of alternatives have been identified 1. models, mannequins and mechanical simulators2. films and interactive videos3. computer simulations and virtual reality systems4. self-experimentation and human studies5. plant experiments6. observational and field studies7. waste materials from slaughterhouses and fisheries8. in vitro studies on cell lines9. dead animals from a humane and ethical source (for example, animals which have

died naturally or which have been killed humanely after scientific procedures)10. clinical practice

Alternative methods approval

Steps of ECVAM validation process

Alternative methods approved• Acute oral toxicity ( reduction in total animals )• Eye irritation

– Isolated chicken eye test– BCOP

• Genotoxicity : In vitro mammalian cell micronucleus test• Phototoxicity : 3T3 NRU phototoxicity test• Pyrogenicity : Invitro tests like mononuclear cell IL6 assay• Skin corrosion : CORROSITEX, Epiderm , Episkin• Skin irritation : Modified EpiDerm SIT and SkinEthicTM RHE assay• Skin Absorption : In vitro test• Skin sensitization : Local lymph node assay(LLNA) and reduced LLNA• Vaccine potency and safety testing

Methods in validation1. Carcinogenecity 

• three in vitro Cell Transformation Assays (CTA) using Syrian Hamster Embryo cells (SHE) and the BALB/c 3T3 Mouse Fibroblast cells for the prediction of the carcinogenic potential

2. Eye irritation• Cytosensor Microphysiometer • Fluorescein Leakage 

3. Monoclonal antibody production: In vitro production 4. Hematotoxicity

• Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay for predicting acute neutropenia in humans

5. Reproductive toxicity• Whole Rat Embryo embryotoxicity assay

Institutes researching (and organizations funding) alternatives to

animal testing• Center for Alternatives to Animal Testing

• UCDavis Center for Animal Alternatives

• Physicians Committee for Responsible Medicine

• Dr Hadwen Trust

• National Centre for the Replacement, Refinement and Reduction of Animals in Research

• Canadian Council on Animal Care Three Rs Microsite

• European Centre for the Validation of Alternative Methods (ECVAM), an online database of toxicology non-animal alternative test methods

Indian scenario• Alternatives to Animal Experimentation Laboratory, Department of

Pharmacology, Jawaharlal Nehru Medical College, Muslim University, Aligarh– response of drugs are demonstrated and taught by computer simulation exercises. This is the

first such lab in India. In addition, a guide to alternatives to animal experiments in pharmacology is prescribed

• Mahatma Gandhi-Doerenkamp Center for Alternatives to Use of Animals in Life Science Education, India

• ECVAMs first congress in India – was co-sponsored by Indian co-sponsor is the International Centre for Alternative Research

and Education (I-Care), an arm of the People for Animals, aided by The University of Meenakshi Academy of Higher Education and Research, the Indian Council of Medical Research (ICMR) and the Medical Council of India

Latest initiative• In a letter dated January 13, 2012, the CPCSEA,

Min. of E&F, Govt. of India, has called on the Ministry of HRD, Dept of Higher Education, the Ministry of H&FW, UGC , Pharmacy Council of India, and the MCI – to discontinue dissection and animal

experimentation associated with the teaching of medical, pharmacy and other UG & PG courses in life sciences in universities & colleges and to introduce use of

– alternatives to animal experimentation

Problems Related to the Introduction of Alternatives

1. Some teachers are resistant to change and need to be convinced of the benefits of using alternatives.

2. Initial investment of time and money is needed for the integration of an alternative into a course

3. Information about potential alternatives is not widely disseminated.

4. Quality of the material available varies considerably.

5. Technical, financial, and other factors which restrict the use of alternatives

Conclusion• Animal testing cannot be replaced completely

until complex human simulations are available• It can be minimised by use of simulators at

academic level.• Despite guidelines by ECVAM, few industries

follow alternative techniques instead of animal testing in India.

• Alternatives can substantially reduce drug development time & cost in near future ,but only when a lot of investment and time is put in the process now