The document discusses alternative methods to animal toxicity testing, highlighting the development and implementation of non-animal testing methods to reduce animal suffering and improve efficiency. It provides a historical context, reviews relevant legislation and regulatory bodies, and outlines various alternative testing approaches, such as in vitro cell culture techniques and in silico computer simulations. The advantages and limitations of these methods are also noted, emphasizing a shift towards more humane and efficient testing practices.

1. SUBJECT : TOXICOLOGICALSCREENINGMETHODS(II)
TOPIC : ALTARNATIVE METHODS TO ANIMAL TOXICITY TESTING
SAMIR PANDA
(I I ND SEM, M.PHARM)
DEPARTMENT OF PHARMACOLOGY

2. INTRODUCTION
• Alternatives animal testing are development and implementation of test
methods that avoid the use of live animals.
• Human biochemistry, physiology, pharmacology, and endocrinology and
toxicology has been derived from animal models.10-100 millions of animals are
using for experimentation in a year.
• 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
• Minor animal suffering:- observing animals in behavioral studies, single blood
sampling, Immunization without adjutants, etc.
• Moderate animal suffering:- repeated blood sampling, recovery from general
anesthesia, etc.
• Severe animal suffering:- LD50% test, starvation vaccine potency tests, etc.
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3. HISTORY
• In 1824 the organization for animal rights: royal society for the prevention of
cruelty to animals
• In 1876, an act for prevention of cruelty to animal was formed in the UK
• In India prevention of cruelty to animals (PCA) Act came in 1960 and amended
in 1982
• In the late 1990s, the committee for the purpose of control
• and supervision of experiments on animals (CPCSEA) came into existence.
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4. Regulatory agencies
• 1989:- ZEBET in Germany.
• 1993:- ECVAM (European).
• 1997:- ICCVAM (US) consists of 15 research and regulatory agencies
among which the Environmental Protection Agency 1999(EPA), the
Food and Drug Administration 2001 (FDA), and the Agency for Toxic
Substances and Disease Registry 2003 (ATSDRO).
• 2005:- JaCVAM (Japanese).
• 2011:- BraCVAM (Brazil)
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5. LEGISLATION
• EU directive 2010/63/EU:- on January 1,2013, EU directive 2010/63/EU
member states EU cosmetic regulation:- It establishes prohibitions against.
(a) testing finished cosmetic products and cosmetic ingredients on animals
(testing ban),
(b) marketing in EU finished cosmetic products.
• EU chemical policy: REACH the Registration, Evaluation, Authorization and
Restriction of Chemicals came In force in 2007.
• EU test methods regulation: development and validation of alternative
techniques.
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6. REASONS FOR DEVELOPMENT OF
ALTERNATIVE ANIMAL TESTING
• Economic and efficiency play a key role. The "In vitro" testing provide toxicity
information in a cost effective and time saving manner. These can also increase
the efficiency of whole study and decrease the number of animals required for
toxicity.
• 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.
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7. INTERNATIONAL ORGANIZATION
ICATM
• International cooperation on alternative test methods(ICATM): on April 27,2009
• United states, Canada, japan and EU.
• Reduction of the number of animals required for consumer product safety testing
worldwide.
OECD
• Organization for Economic Co-operation and Development.
• More than 35 member countries worldwide.
• Test with OECD test guidelines and principles of Good Laboratory Practice
(GLP), shall be accepted.
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8. ACCORDING TO OECD
• Complete replacement of the animal experiment TG 428 skin
absorption: in vitro method
• TG 430 In vitro skin corrosion:-transcutaneous electrical
resistance(TER)
• TG 431 In vitro skin corrosion:- human skin model test
• TG 432 In vitro 3T3 NRU phototoxicity test
• TG 437 Bovine corneal opacity and permeability test method for
identifying ocular corrosives and severe irritants. It is evaluated by
ICCVAM, ECVAM, JACVAM.
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9. • TG 439 Invitro skin irritation: reconstructed human epidermis (RhE)
test method
• acute toxic class method
• Reduction in the number of animals and stress of the laboratory
animals
• G 420 acute oral toxicity --fixed dose procedure
• 1423 Acute oral toxicity acute toxic class method
• TG425 Acute oral toxicity up and down procedure
• TG 429 skin sensitization local lymph node assay
• TG 486 Acute inhalation toxicity
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10. ALTERNATIVE TESTS
• In silico computer simulation methods
• In vitro cell culture techniques
• Using fewer animals
• Microdosing
• Epidemiologic and clinical studies and genetic monitoring
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11. IN SILICO COMPUTER
SIMULATION METHODS
• There are many software available in the public domain. Computers can predict
the toxicity of chemicals, including:
• Their potential to cause cancer or birth defects
• Based on their molecular structure
• Lhasa limited:- promotes the sharing of data and knowledge. It has developed
software tools such as:-
• DEREK (Deductive estimation of risk based on existing knowledge)
• Meteor:- knowledge based expert system that predict toxicity and metabolism of
chemical
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12. • Vitic:- its chemical-based intelligent toxicity database
• These software tools include illustrations on:-
• The use of data entry and editing tools for the sharing of data and
knowledge within the organization
• II. Use of proprietary data to develop non-confidential knowledge that can be
shared between organization
• III. The use of shared expert knowledge to refine predictions
• IV. The sharing of proprietary data between organizations through the formation
of data sharing.
• The use of proprietary data to validate predictions
• Recent studies show that the use of the "in silico" method is the way forward for
assessing the genotoxicity and potential carcinogenicity/repeat dose general
toxicology of large numbers of chemicals.
• There are a number of other in silico methods which have been developed and
validated, for example:-
• Leadscope, mcase, topkat, Toxfree, Osiris, some QSAR models
• this software was developed by the FDA with proprietary databases
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13. INSILCO METHODS IN DRUG
DISCOVERY
Molecular docking, QSAR, pharmacophore mapping –
Molecular docking Docking is the computational determination of binding
affinity between molecules(protein structure and ligand).
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14. • COMFA:- Comparative molecular field analysis
 The biological activity of a molecule is dependent on the
surrounding molecular fields(steric and electrostatic fields)
• COMSIA:- Comparative molecular similarity index analysis
Include more additional field properties:- steric, electrostatic,
hydrophobic, hydrogen bond donor, hydrogen bond acceptor Can
offer a more accurate SAR than COMFA
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15. IN VITRO CELL CULTURE TECHNIQUES
• In vitro cell culture is currently the most successful, and promising, alternative
to animal use. Isolated cells, tissues and organs can be prepared and maintained
in culture by methods that preserve properties and characteristics of the same
cells, tissues and organs in vivo. Cell culture was 1st successfully undertaken by
Ross Harrison in 1907.
• Cells and tissue cultures are used to screen new therapies and to test for product
safety
• Different types of cell grown in culture includes connective tissue elements such
as fibroblasts, skeletal tissue, cardiac, epithelial tissue(liver, breast, skin, kidney)
and many different types of tumour ells. Genetic microarrays are being used to
predict liver toxicity by measuring gene expression in human liver cells.
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16. • Cultured cells have also been developed to create monoclonal
antibodies thereby replacing use of animals required to undergo a
procedure likely to cause pain and distress
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 Advantages of in vitro studies:-
• Small quality of test substance is needed.
• Experiment is under controlled conditions
• Result is obtained quickly.
• No major infrastructure is required.
 Disadvantages of in vitro studies:-
• In vitro dose-response not available
• No systemic effect is studied
• Organ specificity lacking
• Chronic and long-term effects could not be studied
• Transportation of material not easy
QUESTIONS
Discuss the alternative methods
to animal toxicity testing ?

17. REFERENCE
1. Araújo GL de, Campos MAA, Valente MAS, Silva SCT, França FD,
Chaves MM, et al. Alternative methods in toxicity testing: the current
approach. Braz J Pharm Sci [Internet]. 2014;50(1):55–62.
2. Alternative methods to animal toxicity testing [Internet]. Slideshare.net.
[cited 2023 Sep 14]. Available from:
https://www.slideshare.net/SanchitDhankhar/alternative-methods-to-
animal-toxicity-testing-249329802
3. Goldberg AM, Frazier JM. Alternatives to animals in toxicity testing. Sci
Am [Internet]. 1989;261(2):24–31.
4. Vinardell M, Mitjans M. Alternative methods to animal testing for the
safety evaluation of cosmetic ingredients: An overview. Cosmetics
[Internet]. 2017 [cited 2023 Sep 14];4(3):30.
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