Alternative methods to animal toxicity testing

we can avoid use of animals by using invitro methods

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

2. content introduction History Regulatory agencies Philosophy International organizations REACH Alternative tests

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:- LD 50 % 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 Restriction of Chemicals (REACH) REACH was established after 7 th EU directive REACH is the European community regulation on chemicals and their safe use The law was enacted on June, 1 st 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:- The use of data entry and editing tools for the sharing of data and knowledge with in organization Use of proprietary data to develop non-confidential knowledge that can be shared between organization The use of shared expert knowledge to refine predictions 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. 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 1 st 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

20. 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

21. 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

22. Using fewer animals International conference on harmonization (ICH) guidance approached “in vivo” genotoxicity testing method by using few animals to detect toxicity. Utilizes the general principles of toxicology Factors drug metabolism, toxicokinetic and saturation of defense mechanism are considered in evaluating genotoxicity Steady state plasma concentration studies Microdosing Micro dose is defined as less than 1/100 th 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:- Select the best lead compound supported by clinical data Cost-effective approach and Save time and money

23. 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:- To identify individual species or population. 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