This document presents information on alternative methods to animal toxicity testing. It discusses the need for alternatives due to the harms animals face in toxicity testing. The 3Rs principles of reduction, refinement and replacement are explained, which aim to minimize animal use and suffering. The validation process for new alternative methods through organizations like ECVAM is covered. Specific alternative methods mentioned include in vitro tests like the Ames test and HET-CAM test, in silico methods, and mathematical models. The HET-CAM test for eye irritation is described in more detail.

1. Institute of Pharmaceutical Science
Kurukshetra University
Presentation on:Alternative method to animal
Toxicity Testing.
Presented By :
Sachin sharma
M.Pharma 2nd Sem
Pharmacology
Roll No: 5461076 1

2. Alternative Methods To
Animal Toxicity Testing

3. CONTENT
 Introduction
 Need for alternatives to animals
 The 3Rs Principles
 Validation of alternative methods
 Alternative methods
 HET- CAM Method
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4. Introduction
 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”.
 Alternative methods able to do:
• Reduce
• Refine
• Replace
 collectively called as “The 3Rs Principle”.
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5. 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.
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6. The 3Rs Principle
 The 3Rs principles were defined in 1959 by W.M.S
Russel and R.L Bruch in subject “The Principle of
Humane experimental technique”.
 They provide a strategy for rational and stepwise
approach to minimising animals use and suffering
in experiments without compromising the quality
and quantity of scientific work being undertaken.
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7.  Reduction means any decrease in numbers of
animals used to obtain information of a given
amount and precision.
 Refinement means any decrease in the
incidence or severity of procedures applied to
animals.
 Replacement means the substitution of
conscious living animals with non sentient.
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8. Validation of alternative methods
 European centre for validation of alternative method (ECVAM) was
established in 1991.
 ECVAM serves as information centre and to maintain a database on in
vitro test and validated method.
 “Test validation is a process based on principle by which the reliability
and relevance of particular test/method are established for specific
purpose”.
 Validation process carry out by three organisation
• Organisation for economic corporation and development (OECD).
• European centre for validation of alternative methods(ECVAM).
• Interagency co-ordinating committee on validation of alternative
method.
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9.  There are two type of validation studies.
Validation studies
Prospective Retrospective
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10. Prospective validation studies
• Involve generation of new data.
• Composed of six stages.
Research
Method
Development
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11. Pre-validation
Validation
Review
Regulatory
Acceptance
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12. Alternative Method
 In-vitro test
• Toxicity test
• Corrosivity test
Insilico
Mathematical model
• Computer simulation
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13.  Genotoxicity Test
• Ames Test
 Occular toxicity Test
• HET-CAM Test
 Hepatotoxicity Test
• Natural Red Uptake test
 Corrosivity Test
• Transcutaneous Electrical Resistance
Test Method
 HET-CAM: Hen’s egg test- chorio allantoic membrane
Toxicity Test
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14. HET-CAM 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 chorioallantoic membrane is
exposed.
• The test substance is droped 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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15. 0.5(min) 2(min) 5(min)
haemorrhage 5 3 1
Coagulation 9 7 5
Effects Score
Scoring Scheme
Classification scheme
Cumulative score Irritation Assessment
Up to 0.9 Practically none
1 to 4.9 Slight
5 to 8.9 Moderate
9 and above Strong
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