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Alternative methods to animal toxicity testing
1. Alternative methods to animal toxicity testing
Presented By- PRIYA CHHIKARA
M. Pharmacy 1st year (Pharmacology)
190000803001
DEPARTMENT OF PHARMACEUTICAL SCIENCES
CHAUDHARY BANSI LAL UNIVERSITY
BHIWANI-127021
3. 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.
4. 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
5. Regulatory agencies
1989:- ZEBET in Germany
1993:- ECVAM ( European)
1997:- ICCVAM (US) consists of 15 research and regulatory agencies
among which environmental protection agency 1999( EPA) , the food
and drug administration 2001 (FDA) , and the agency for toxic
substances and disease registry 2003(ATSDR0)
2005:- JaCVAM (Japanese)
2011:- BraCVAM (Brazil)
6. philosophy
Alternative tests are part of philosophy known as the “3’R philosophy “
Replacement, reduction, refinement
Replacement:- refers to the preferred use of non- animal methods over animal methods whenever it is
possible to achieve the same scientific aim.
Reduction:- refers to methods that enable researchers to obtain more information from the same number of
animals.
Refinement:- refers to methods that minimize potential pain, suffering, or distress, and enhance animal welfare
for the animals used in experiment.
Research initiatives and legislation
Research initiatives implemented to achieve 3R principle in research institutes and colleges
SEURAT-1[EU] USDA[US]
EUROECOTOX[EU] AWIC[CAD]
AXLR8[EU]
7. 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
8. 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.
9. 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
10. 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 photo toxicity test
TG 437 Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe
irritants. It is evaluated by ICCVAM, ECVAM, JaCVAM.
TG 439 In vitro skin irritation:---- reconstructed human epidermis (RhE) test method
Reduction in the number of animals and stress of the laboratory animals
TG 420 acute oral toxicity –-------------fixed dose procedure
TG 423 Acute oral toxicity –------------ acute toxic class method
TG 425 Acute oral toxicity –------------- up and down procedure
TG 429 skin sensitization –------------- local lymph node assay
TG 436 Acute inhalation toxicity ------- acute toxic class method
11. Registration, Evaluation, Authorization, And
Chemicals (REACH)
REACH was established after 7th EU directive
REACH is the European community regulation on chemicals and their safe use
The law was enacted on June, 1st 2007
The aim of REACH is to improve the protection of human health and the environment through more
effective and earlier identification of the intrinsic properties of chemical substances. It also enhance
innovation and competitiveness of the EU chemicals industry
Since 2004, the European union has rejected the practice of using guinea pigs in producing cosmetics for
the beauty market. During 2008 and 2009, ECVAM expanded its activities related to the development
validation, validation, and optimization of alternative methods
ECVAM has also become highly active in the regulation of alternative methods(ECVAM,2010). Therefore,
several international organizations support the use of alternative methods for toxicological tests, as well
as extensive chemical and pharmaceutical support, because in addition to the issue of ethics, the financial
nature of this shift is quite favourable(brown,2003)
12. Alternative tests
In silico computer simulation methods
In vitro cell culture techniques
Using fewer animals
Microdosing
Epidemiologic and clinical studies and genetic
monitoring
13. 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
Vitic:- it is chemical based intelligent toxicity database
These software tools includes illustrations on:-
I. The use of data entry and editing tools for the sharing of data and knowledge with in 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
14. The use of proprietary data to validate predictions
Recent studies shows that the use of “in silico” method in the way forward for assessing the genotoxicity and
potential carcinogenicity /repeat dose general toxicology of large no. 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
These software developed by FDA with proprietary databases
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).
15. In silico design are:- CADD
Protein based methods:- make use of the structure of the target protein or receptor.
Ligand based methods:- based on the known inhibitors or ligands.
Receptor based methods
Uses the 3D structure of the biological target. Obtained through X- ray crystallography.
If experimental structure Is not available, create a homology model of the target, based on the experimental
structure of a related protein.
Homology model include steps:-
Template identification and initial alignment
Alignment correction, backbone generation , loop modeling, side chain modeling , model optimization , model
validation.
These involve molecular docking of each compound in the chemical database into the binding site of the target
and predicting the electrostatic fit or flexibility between them and energy of binding.
Energy of binding= energy of complex- energy of ligand- energy of receptor
Flexibility= score of ligand fit- score of flexible docking
16. Ligand based strategy
In the absence of the structural information of the target, ligand based method make use of the information
provided by known inhibitors for the target receptor.
Structures similar to the known inhibitors are identified from chemical databases by variety of methods
Some of the methods widely used are similarity and substructure searching, pharmacophore matching or 3D
shape matching.
QSAR
QSAR is statistical approach that attempts to relate physical and chemical properties of molecules to their
biological activities. Used to predict the activity of new analogues
employs statistics and analytical tools to investigate the relationship between the structures of ligands and
their corresponding effects.
Various descriptors like molecular weight, number of rotatable bonds log P etc. are commonly used.
Many QSAR approaches are in practice based on the data dimensions
It ranges from 1D QSAR to 6D QSAR
3D QSAR:- CoMFA and CoMSIA
17. CoMFA:- Comparative molecular field analysis
Biological activity of a molecule is dependent of 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
18. Pharmacophore mapping
Pharmacophore is a part of a molecular structure that is responsible for
a particular biological or pharmacological interaction that it undergoes.
It is a 3D description of a pharmacophore, developed by specifying the
nature of the key pharmacophoric features and the 3D distance map
among all the key features. A pharmacophore map can be generated
by superposition of active compounds to identify their common
features.
Based on the pharmacophore map either de novo design or 3D
database searching can be carried out.
19.
20. In vitro cell culture techniques
In vitro cell culture is currently the most successful , and promising , alternative to animal use .
Isolated cell, 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 tumor
cells. Genetic microarrays are being used to predict liver toxicity by measuring gene expression in
human liver cells.
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
21. Common cell lines
Human cell lines
MCF-7 breast cancer
HL 60 leukemia
HEK-293 human embryonic kidney
HeLa Henrietta lacks
Primate cell lines
Vero African green monkey kidney epithelial cells
Cos-7 African green monkey kidney cells
And others such as CHO from hamster, sF9 and sF21 from insect cells
22. 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 effect could not be studied
Transportation of material not easy
Application of in vitro test
In vitro genetic toxicology testing
Bacterial mutation assay:- measure genetic damage
Chromosome aberration assay in cultured cells:-
evaluation of damage to chromosome on the basis of
direct observation
Comet or Single cell gel(SCG) electrophoresis:- detects
DNA damage and repair in individual cells. Damage is
represented by increase in DNA fragments in the form of
streak similar to tail of the comet. Comet assay can be
conducted into both “in vivo and in vitro”. The length and
fragments contents is directly proportional to amount of
DNA damage.
In vitro pharmacologic activity testing
In vitro drug disposition studies
Metabolic stability
23. Using fewer animals
International conference on harmonization (ICH) guidance approached “in vivo” genotoxicity testing method
by using few animals to detect toxicity.
1. Utilizes the general principles of toxicology
2. Factors drug metabolism, toxicokinetic and saturation of defense mechanism are considered in evaluating
genotoxicity
3. Steady state plasma concentration studies
Microdosing
Micro dose is defined as less than 1/100th of the dose of a test substance to yield a pharmacological effect of the
test substance.
Accelerator mass spectrometry(AMS) is most sensitive detection for micro dosing studies.
Advantages:-
a. Select the best lead compound supported by clinical data
b. Cost-effective approach and Save time and money
24. Epidemiologic, clinical studies and genetic
monitoring
Epidemiology:- it is the study of factors affecting health and illness of human
populations. It serves as the foundation and reasonable way of evaluating
interventions made in the interest of public and preventive medicine.
Clinical trials and genetic monitoring:-
1. To identify individual species or population.
2. To quantify changes in population genetic metrics' over time
Genetic monitoring thus used to detect changes in species abundance/ diversity
has become important part
clinical trials – medical research are used to determine whether new drug treatment
are both effective and well tolerated in humans phase 1,2,3, and 4 trials to evaluate
the effect of drug