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Presented byShraddha khairnarM pharm Sem 2nd Department of pharmacology

3R

Alternative to Animal Testing

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Moral status of animal in western philosophy

Jeremy Bentham(17481832) The question is not can they reason ? Nor can they talk ? But can they suffer?(1789) Minimise harms Maximise benefits Balance harm against benefits3

1959 The concept of the 3Rs is born

William Russell and Rex BurchPrinciples of Humane Experimental Technique- Published by UFAW,1959 Introduced the concept of the 3Rs

ReplacementReductionRefinement

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What is 3 RRussell and Burch in 1959 proposed that if animals were to be used in experiments, every effort should be made to replace them with nonsentient alternatives

They developed the 3R strategy which includes, Refinement- refine experimental methods to decrease unnecessary pain and trauma to animals Reduction- reduce the number of animals used in these experiments Replacement- replace the animal experiments Eg. computer simulation models, In-vitro methods, cell culture techniques

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Alternative approachThe term alternatives includes all assays, tests, methods, techniques, tools, strategies and approaches etc.To obtain the required information without the use of live animals;

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ReplacementEfficient use of existing informationIn vitro methods Micro-organismInvertebrates animalsPhysical mechanical methodChemical techniqueIn silico methods (Computer ,mathematical model, QSAR)Organ on chip

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Absolute replacement No need to use animals (cell lines, human or invertebrate cells and tissues)No need to test for skin irritation if pH 11.5No need to test for eye irritation if the chemical is a skin irritant. Relative replacement: Humane killing of animals to provide cells or tissues for in vitro studies. Partial replacement: e.g. use of non-animal methods as prescreens in toxicity testing.

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Direct replacement ; e.g. human or guinea pig skin is used in vitro to replace guinea pig tests in vivo

Indirect replacement ; e.g. Limulus amoebocyte lysate(LAL) test or test based on whole human blood is used to replace rabbit pyrogen tests

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In vitro methods In vitro Pyrogen test Embryonic stem cell test Local lymph node assay for skin sensitization Neutral red uptake assay Carcinogenicity test Repeated dose toxicity test Developmental neurotoxicity test

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Advantages of in vitro testsControlled testing conditionsHuman cells and tissues can be usedLack of systemic effectsReduction of variability between experimentsTesting is fast (and cheap)Small amount of test material is requiredTransgenic cells carrying human genes can be usedReduction of testing in animals

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Limitations of in vitro testsGeneral toxicity profile of a chemical cannot be assessed In vivo dose-responses cannot be obtained (for human risk assessment)Systemic effects cannot be evaluatedPharmacokinetics cannot be evaluatedSpecific organ sensitivity cannot be assessedChronic effects cannot be tested

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Safety testing of chemicalsReplacement alternatives in use for testing Genotoxicity:Mutagenicity chromosomal effectsLocal tolerance: Phototoxicity Skin corrosivity Skin irritation Eye irritationToxicological screening: general cytotoxicity

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Eye irritancy- Draize test Measures eye irritancy of concentrated chemicals into animals eye. Consumer Product Safety Commission drew guidelines for draize test included use of anesthetics, reduction of number of animals. Alternatives include test and Isolated Chicken Eye assay (ICE)Bovine Corneal Opacity(BCOP)

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Bovine Corneal Opacity and Permeability (BCOP)Corneal Opacity- Is a disorder of the cornea. This stops light from passing through the cornea to the retina.Topical ocular medications also may cause nerve damage Corneal permeability- Lipophilic compounds and hydrophilic compounds Movement of compounds through corneal tissue

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History Background of BCOPMuir 1984 - First proposed CO Required- Chambers, PS, and PhotocellGautheron et al.(1992) Modified this method.Required- Chambers, Opacitometer, test material, sodium fluorescine solutionCasterton et al (1996) Modified this method by mounted chambers into spectrophotometer.

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BCOP (Gautheron et al. (1992) -OECD TG 437)Principle Corneal opacity is measured amount of light transmission, Permeability is measured amount of sodium fluoresceine dye passes across cornea

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Method-1) Isolation and mounting cornea Eagles minimum essential medium Incubation 1 hour at 320 C

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2) Initial opacity Initial reading using opacitometer3) Dosing and rinsing- cornea exposed to materials to be tested, opacity is recorded for each cornea.4) Permeability Determination- Sodium fluorescein dye

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Score calculationIn vitro irritation score = corrected opacity value+(15*corrected OD490 value)

In vitro irritation scoreIn vitro irritation scale0-3Non eye irritant3.1-25Mild eye irritant25.1-55Moderate eye irritant>55.1Corrosive eye irritant(Strong base)

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Demerits-1) Does not differentiate compounds on the basis ofthe mechanism.2) It matters more that the measured damage actually happened rather than why it happened

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EpiDerm and EpiSkin (OECD TG 404)Principle- The irritant chemicals are cytotoxic to the cells in the underlying layers. The amount of inflammatory mediators are released by the epidermis Utilizes three-dimensional (3D) skin model

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Method EpiDermTM and EpiSkinTM validated for in vitro skin irritation test Course of a 4 day time period1) Tissue pre-incubation(Day 0)2) Tissue exposed to chemicals. Positive and negative control (Day 1)3) Medium exchanged(Day 2)4) Tissue post-incubation (Day 3)-5) MTT viability assay

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Toxicity test on 3-D skin model. Control (A) and after addition of 20% SDS for 2 sec (B), 30 sec (C) and 90 seconds (D)

Apply the sample to be tested directly onto the surface of the skin model.The quantitative determination is carried out by evaluating the resultant metabolites by photometrically.

AdvantagesCan be used for distinguishing between corrosive and non-corrosive chemicals.Considered to be safety applicable to the safety evaluation of chemicals.replacement for the in vivo Draize rabbit skin corrosivity test

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Skin corrosion- Corrositex (OECD 435)PrincipleA test material is evaluated based on its penetration through the biobarrier membrane into CDS. The time required to break through is determinedThe Corrositex kit, manufactured byIn Vitro International(IVI) Evaluating corrosivity of acid or base29

Method -Qualification of Test Material-Each test material added into vial containing CDSTest materials must cause a color change will qualify for testingCategorization- Resultant color compared to colorChart provided. Category 1 and 2

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Biobarrier Preparation-Solubilized Biobarrier matrix powderprepared membrane refrigerated.

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Biobarrier Qualification with Positive Control Top of vial Positive control monitored until color change Break through time is compared to historical data rangeDosing of the test material Test material is added monitored For 10 min., repeated if no color changed

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WhyUse Corrositex ?

Time Savings: Corrositextesting can provide a Packing Group determination in a matter of minutes and no more than 4 hours, unlike animal testing which can take 2 - 4 weeks.Accuracy:More accurate than pH testing and is packing group specific.Cost Savings: Reduced shipping charges, additional cost savings workplace safety.

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Transcutaneous Electrical Resistance Test (OCDE TG 430)Principle-Corrosive material cause to loss of normal stratum corneum integrity and barrier function which measured as electrical resistance Dye binding steps determines the strength of ionic permeability34

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Skin Discpolytetrafluoroethylene tube0 RingMgSO4Spring clipElectrodes

Receptorchamber

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Method-Preparation of the skin discs- 28-30 days old animal killedskin removed, skin disc diameter 20 mmDiscs placed at end of PTFE end, press O ring to hold skinDiscs submerged into chamber containing MgSO4 solutionElectrical resistance of each discs is measured before test

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38Application of the test and control substances-10M HCL and dist. H2O suggestedControl and test applied for 24 hrs at 20-23oC and Washing

TER measurements-

The electrodes are placed on either side of the skin discThe distance between the inner electrode and the skin disc should be constant and minimal (1-2 mm).The outer electrode is positioned inside the chamber If resistance >20kohm, wash and fresh MgSO4 added and resistance measured repeated.

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Dye Binding Method-To determine the TER values obtained were the result of increased skin permeability, or skin corrosion sulforhodamine B dye

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Interpretation of ResultThe mean TER results

The mean dye binding results

41Control Substance Resistance range (k)

Positive 10M HCL 0.5-1.0

Negative Distilled water 10-25

Control Substance Dye content range (g/disc) Positive 10M HCL 40-100 Negative Distilled water 15-35

AdvantagesThe Rat Skin TER method offers animal welfare advantages, including animal use refinement and reductionThis method reduces the number of animals used as skin from one humanely killed rat may be used to test up to five chemicals

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43methodsTest purposeValidation AuthorityEpiskin SITSkin irritationECVAMEpiDerma SITSkin irritationECVAM

Modified EpiDerm SITSkin irritationECVAM

SkinEthic RHESkin irritationECVAM

LabCyst EPI-MODEL 24Skin irritationECVAM

S7B Guideline:-

The non-clinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals.

S7B reached step 4 in June 2005 and will be implemented in 2006.44

Objective of guidelineThis guideline describes a non-clinical testing strategy for assessing the potential of a test substance to delay ventricular repolarization. This guideline includes information concerning non-clinical assays and integrated risk assessments

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QT interval :-The QT intrval is the time from the start of Q wave and end of T wave. It represent the time taken for the ventricular depolarisation and repolarisation.46

QT interval is inversely proportional to heart rate. Abnormally prolong QT interval associated with an increased risk of ventricular arrythmia especially Torsed de point.47

An in vitro IKr assay48 a) In vitro Ikr assay :- Evaluates the effects on the ionic current through a native or expressed IKr channel protein, such as that encoded b hERG :- ( Human ether -a-go-go related gene) these gene encode the pore forming subunit of delayed rectifire channel in humany hERG .

European Centre for the Validation of Alternative Methods

Established in 1991 (Directive 86/609/EEC )Located at the Joint Research Centre, Ispra, ItalyPart of the Institute for Health and Consumer Protection (IHCP)49

Methods of Reduction Perform pilot studies Design studies to use animals as their own controls eg- Cross over study Gather data for more than one experiment concurrently Consult with statistician and use minimum number of animals Minimise variables such as disease, diet, stress, genetics Use appropriate species of animals50

Methods of Refinement Setting the earliest possible end point Using appropriate analgesics and anaesthetics for painful procedureAdequate training prior to performing experiment Use proper handling technique for animals Perform surgeries and procedure aseptically to prevent infection, Ensure drug doses are correct and drugs are not expired

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Types of refinement methods in animal testing:Using pharmaceutical -Use of anaesthetics, analgesics and pain- relievers can be helpful to relieving pain or distress. Using technology - Ultrasound and magnetic resonance imaging (MRI) can both be considered refinement techniques.

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Reducing psychological distress - providing animals with larger cages and toys can make a significant difference in the animal's emotional well-being.Reaching an end point - euthanasia will be employed53

Procedure with care

Subcutaneous injection Intravenous in injection Intramuscular injection 54

4-Rs India adopted 4-Rs (Reduction, Refinement, Replacement and Rehabilitation)Rehabilitation is the moral obligation to feel responsible for the welfare of the large animals after their use for scientific pursuit.

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7-RsHans Martin Sass German philosopher proposed 7-RsReduction, Refinement, Replacement, Rehabilitation, Respect, Review and Relate

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Globalisation of RDr. David B. Morton, expanded this concept to the Fifteen r's 8. Reconsider 9. Read 10. Reflect 11. Reason 12. Record 13. Reward 14. Reappraise 15. Resolve Reduce Refine Replace Respect Recognize Relieve Refuse57

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References1 Guideline, P.B.T., 2001. OECD guideline for the testing of chemicals.The Hershberger,6012 Kolle, S.N., Kandrov, H., Wareing, B., van Ravenzwaay, B. and Landsiedel, R., 2011. In-house validation of the EpiOcular eye irritation test and its combination with the bovine corneal opacity and permeability test for the assessment of ocular irritation.Alternatives to Laboratory Animals-ATLA,39(4), p.365..3 Kandrov, H., Liebsch, M., Spielmann, H., Genschow, E., Schmidt, E., Traue, D., Guest, R., Whittingham, A., Warren, N., Gamer, A.O. and Remmele, M., 2006. Assessment of the human epidermis model SkinEthic RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431.Toxicology in vitro,20(5), pp.547-559.4 Eskes, C., Detappe, V., Koter, H., Kreysa, J., Liebsch, M., Zuang, V., Amcoff, P., Barroso, J., Cotovio, J., Guest, R. and Hermann, M., 2012. Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations.Regulatory Toxicology and Pharmacology,62(2), pp.393-403.5 Eskes, C., Detappe, V., Koter, H., Kreysa, J., Liebsch, M., Zuang, V., Amcoff, P., Barroso, J., Cotovio, J., Guest, R. and Hermann, M., 2012. Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations.Regulatory Toxicology and Pharmacology,62(2), pp.393-403.58