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![BIOMEDICAL ETHICS
• BEFORE CONDUCTING ANY TOXICOLOGICAL STUDY OR TAKING ANY
TISSUE/CELLS FROM THE ANIMALS, THE STUDY SHOULD BE APPROVED BY
INSTITUTE ANIMAL ETHICS COMMITTEE (IAES) OR SHOULD SUBSCRIBE TO THE
LOCAL GUIDELINES
[ US FDA, EMA, CPCSEA ]
IN INDIA WE HAVE THE COMMITTEE FOR THE PURPOSE OF CONTROL AND
SUPERVISION OF EXPERIMENTS ON ANIMALS [CPCSEA]](https://image.slidesharecdn.com/toxicitystudy-210422074313/85/Toxicity-study-4-320.jpg)
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Toxicity study
- 1. TOXICITY STUDY DR GAUTAMPANDA 1ST YEAR PG STUDENT PG DEPT OF PHARMACOLOGY
- 2. INTRODUCTION • THE OBJECTIVESOF TOXICITY STUDIES ARE:- • TO DETERMINE LD50 & THERAPEUTIC INDEX OF A NEW DRUG • TO DETECT ANY ACUTE TOXICITY OF THE DRUG • TO DETECT ANY CHRONIC TOXICITY OF THE DRUG • TO DETERMINE GENOTOXICITY • TO DETECT ANY CARCINOGENIC POTENTIAL OF THE DRUG IN LONG TERM TREATMENT • TO DETECT ANY SPECIFIC TOXICITY (REPRODUCTIVE HEALTH)
- 3. • TOXICITY STUDIESARE EXPECTED TO BE PERFORMED IN COMPLIANCE WITH GOOD LABORATORY PRACTICE • TOXICITY STUDIES HELPS TO CALCULATE NO OBSERVED ADVERSE EFFECT LEVEL (NOAEL)
- 4. BIOMEDICAL ETHICS • BEFORECONDUCTING ANY TOXICOLOGICAL STUDY OR TAKING ANY TISSUE/CELLS FROM THE ANIMALS, THE STUDY SHOULD BE APPROVED BY INSTITUTE ANIMAL ETHICS COMMITTEE (IAES) OR SHOULD SUBSCRIBE TO THE LOCAL GUIDELINES [ US FDA, EMA, CPCSEA ] IN INDIA WE HAVE THE COMMITTEE FOR THE PURPOSE OF CONTROL AND SUPERVISION OF EXPERIMENTS ON ANIMALS [CPCSEA]
- 5. TOXICITY STUDIES • ACUTETOXICITY TEST • SUBACUTE TOXICITY TEST • CHRONIC TOXICITY TEST • SPECIAL TESTS OR SPECIFIC TESTS
- 6. ACUTE TOXICITY TEST •SINGLE DOSE • TEST IN WHICH A SINGLE DOSE OF THE DRUG IS USED IN EACH ANIMAL ON ONE OCCASION • TO DETERMINE LD50 OR MEDIAN LETHAL DOSE • LD50 IS THE DOSE WHICH WILL KILL 50% OF THE ANIMALS OF A PARTICULAR SPECIES • LD50 VALUE IS DETERMINED IN A 24-HR TEST USING 2 SPECIES • RODENTS LIKE MICE OR RATS & NON-RODENTS LIKE RABBITS
- 7. • 2 ROUTESOF DRUG ADMINISTRATION IS TO BE USED WITH ONE BEING THE INTENDED ROUTE • DETERMINATION OF GROSS BEHAVIOUR WHICH INCLUDE
- 8. INCREASED MOTOR ACTIVITY DECREASED MOTOR ACTIVITY ATAXIA SEDATION MUSCLERELAXATION HYPNOSIS ANALGESIA ANAESTHESIA ARCHING & ROLLING PTOSIS LACRIMATION EXOPTHALMOS DIARRHOEA SALIVATION: VISCID/WATERY WRITHING RESPIRATION: DEPRESSION STIMULATION FAILURE SKIN COLOUR: BLANCHING CYANOSIS VASODILATATION TREMORS CLONIC CONVULSIONS TONIC EXTENSION STRAUB REACTION PILO-ERECTION MUSCLE SPASM CATATONIA SPASTICITY OPISTHOTONUS HYPERESTHESIA LOSS OF RIGHTING REFLEX
- 9. SUBACUTE TOXICITY TEST •DAILY DOSES • TEST IN WHICH ANIMALS (RATS & DOGS) ARE DOSED DAILY • STARTING AT AROUND EXPECTED THERAPEUTIC LEVEL & INCREASING STEPWISE EVERY 2-3 DAYS UNTIL TOXIC SIGNS ARE OBSERVED • HAEMATOLOGICAL & BIOCHEMICAL MONITORING ARE CARRIED OUT • BLOOD LEVEL OF THE COMPOUND IS CHECKED TO ENSURE ITS ABSORPTION • THE ANIMALS ARE MAINTAINED AT THE MAXIMUM TOLERATED DOSE FOR 2-3 WEEKS • TO ALLOW DEVELOPMENT OF ANY PATHOLOGICAL CHANGES
- 10. • THE ANIMALSARE THEN KILLED & A FULL PATHOLOGICAL & HISTOLOGICAL EXAMINATION IS DONE • DAILY OR WEEKLY BODY WEIGHT CHANGES ARE RECORDED • INHIBITION OF GROWTH RATE IN YOUNG MALE RATS (21 DAYS OLD WEIGHING 40-50GM)
- 11. • TO DETERMINETHE MAXIMUM TOLERATED DOSE • TO INDICATE THE NATURE OF TOXIC REACTIONS, SO THAT WE CAN DESIGN SUITABLE CHRONIC TOXICITY STUDIES
- 12. CHRONIC TOXICITY TEST •DAILY DOSES • TEST IN WHICH 2 SPECIES, RODENT (RAT) & NON-RODENT (DOG OR A PRIMATE) ARE DOSED DAILY FOR 6 MONTHS • 3 DOSE LEVELS ARE CHOSEN
- 13. CHRONIC TOXICITY TEST •HIGH DOSE LEVEL • IDEALLY AT LEAST 10 TIMES THE EXPECTED MAXIMUM CLINICAL DOSE • THE HIGH DOSE WILL PRODUCE SIGNIFICANT RETARDATION OF GROWTH OR SOME PATHOLOGICAL CHANGES • LOW DOSE LEVEL • ABOUT TWICE THE EXPECTED MAXIMUM CLINICAL DOSE • 3RD DOSE IS FIXED MIDWAY BETWEEN THE HIGH & LOW DOSE
- 14. • PARAMETERS TOBE MEASURED AT REGULAR INTERVALS (AT LEAST EVERY 14 DAYS) • BODY WEIGHT • FOOD INTAKE • RENAL FUNCTION • HEPATIC FUNCTION • HEMATOLOGY • PULSE RATE • BLOOD PRESSURE • BLOOD LEVEL OF THE COMPOUND
- 15. • AT THEEND OF THE TEST, AUTOPSY IS PERFORMED • VITAL ORGANS ARE WEIGHED & EXAMINED FOR GROSS & HISTOLOGICAL CHANGES • LONG TERM TREATMENT IN MAN MUST BE PRECEDED BY 3-12 MONTHS OF CHRONIC TOXICITY STUDIES IN ANIMALS
- 16. SPECIAL TESTS • EFFECTON FERTILITY/REPRODUCTIVE HEALTH • TERATOGENIC POTENTIAL • CARCINOGENICITY • GENOTOXICITY
- 17. ACUTE TOXICITY TEST •TO DETERMINE LD 50 & THERAPEUTIC INDEX • THERAPEUTIC INDEX IS THE RATIO BETWEEN THE PHARMACOLOGICALLY EFFECTIVE DOSE & THE LETHAL DOSE ON THE SAME STRAIN & SPECIES • LD50/ED50 • THE GREATER THE INDEX, THE SAFER THE COMPOUND
- 18. • BECAUSE OFSPECIES VARIATION SEVERAL SPECIES OF ANIMALS ARE USED TO DETERMINE LD50 • LD50 WITH CONFIDENCE LIMITS IS ESTABLISHED ON ONE COMMON LABORATORY SPECIES, MICE OR RATS • THE LD50 DOSE IS THEN ADMINISTERED TO GUINEA PIGS, RABBITS, CATS, DOGS, ON WEIGHT BASIS OR ON THE BASIS OF RELATIVE SURFACE AREAS • THE DOSE TO BE ADMINISTERED TO A PARTICULAR SPECIES ON THE BASIS OF SURFACE AREA CAN BE EXTRAPOLATED BY REFERRING TO THE FOLLOWING TABLE
- 20. TO DETERMINE THEABSOLUTE DOSE FOR A SPECIES IN THE COLUMN, THE ABSOLUTE DOSE ADMINISTERED TO THE SPECIES IN A ROW IS MULTIPLIED BY THE FACTOR PRESENT AT THE INTERSECTION OF RELEVANT ROW & COLUMN FOR EXAMPLE, THE LD50 OF CROTALABURNINE (CL) IS 83MG/KG IV IN MICE ABSOLUTE DOSE IN A 20 GM MOUSE IS 1.66MG EXTRAPOLATED TO DOG (12KG) BY SURFACE AREA, THE EFFECT MIGHT BE EXPECTED AT A DOSE OF 206.17MG (1.66*124.2) LD50 IS THEREFORE 17.18MG/KG
- 22. INFANTS LESS THAN6 MONTHS OF AGE • THE LD50 IN NEW BORN RATS <24 HOURS OF AGE IS COMPARED WITH THE LD50 OF MATURE RATS IN ORDER TO ASSESS ANY DIFFERENCE IN SENSITIVITY DUE TO AGE
- 23. DESIGN OF ACUTETOXICITY TEST • USUALLY PERFORMED FIRST ON MICE • THE COMPOUND IS ADMINISTERED ONCE ORALLY OR PARENTERALLY AT VARIOUS DOSE LEVELS TO GROUPS OF 5-10 MICE • INCLUDE BOTH THE SEXES, EQUAL IN NUMBER • OVERNIGHT FASTING (18 HOURS) • AT LEAST 3-4 DOSE LEVELS CAUSING <50%, BUT NOT 0% & >50%, BUT NOT 100% MORTALITY SHOULD BE USED • IV ROUTE IS PREFERRED OVER INTRAPERITONEAL ROUTE
- 24. • SOLVENT SHOULDBE ISOTONIC SALINE • USUAL VOLUME OF IV INJECTION SHOULD BE 1-10 ML/KG, MAX 50ML/KG • SLOW & UNIFORM SPEED OF INJECTION WILL AVOID UNDUE KILLING BY A DRUG • ESPECIALLY FOR THOSE ACTING ON THE CNS OR CVS • CONVULSIONS DURING INJECTION IS FELT AS TREMOR IN THE TAIL OR OBSERVED AS PADDLING OF THE FEET • SUDDEN RESPIRATORY ARREST IS ALMOST ACCOMPANIED BY RISING OF THE HEAD
- 25. • INJECTION ISREPEATED IN FRESH MICE TAKING CARE TO INJECT VERY SLOWLY
- 26. STAIRCASE METHOD • 2MICE ARE INJECTED WITH A PARTICULAR DOSE • THEN OBSERVED FOR 24 HOURS FOR ANY MORTALITY • IF THE DOSE WAS TOLERATED, SUBSEQUENT DOSES ARE INCREASED BY A FACTOR OF 1.5 • AND IF THE DOSE WAS LETHAL, SUBSEQUENT DOSES ARE DECREASED BY A FACTOR OF 0.7 • MAXIMUM NON-LETHAL & MINIMUM LETHAL DOSES ARE THUS DETERMINED APPROXIMATELY USING ONLY ABOUT 10 MICE
- 27. • THEN AFINAL & MORE RELIABLE LD50 ASSAY IS PLANNED USING 3-4 DOSE LEVELS WITHIN THIS RANGE WITH LARGER NUMBER OF ANIMALS IN EACH GROUP • FOLLOWING DRUG ADMINISTRATION THE ANIMALS SHOULD BE PLACED SEPARATELY IN GLASS CYLINDERS FOR CLOSE OBSERVATION • ANIMALS SHOULD NOT BE GROUPED TOGETHER IN THE SAME CAGE • AGGREGATION OF MICE HAS BEEN SHOWN TO AFFECT LD50 • EX AMPHETAMINE
- 28. A NUMBER OFINFORMATION SHOULD ALSO BE RECORDED FOR FUTURE REFERENCE SOURCE OF THE ANIMAL SEX AGE BODY WEIGHT INJECTION TIME ROUTE USED SOLVENT USED
- 29. • A COMPARISIONOF IV LD50 & WITH THE ORAL VALUE GIVES AN INDICATION OF THE DEGREE OF ABSORPTION OF THE COMPOUND FROM THE GI TRACT • SIMILAR VALUES WILL INDICATE COMPLETE ABSORBABLITY • HIGHER ORAL LD50 WOULD INDICATE POOR ABSORPTION
- 30. FROM LAB TOMAN • PREDICTABILITY OF THE DRUG RESPONSE IN MAN CAN BE IMPROVED BY • INCLUDING ADDITIONAL SPECIES • USING RANDOM BRED ANIMALS
- 31. DOSE RELEVANCE INCONTEXT TO TOXICITY COMMOMLY USED TERMS LD50 (SINGLE ORAL DOSE PER KG IN RATS) PROBABLE LETHAL DOSE FOR MAN EXTREMELY TOXIC 1MG OR LESS 65MG HIGHLY TOXIC 1-50MG 4GM MODERATELY TOXIC 50-500MG 30GM SLIGHTLY TOXIC 0.5-5 G 250 GM PRACTICALLY NON-TOXIC 5-15G 1KG RELATIVELY HARMLESS 15GRAM OR MORE >1KG
- 32. CALCULATION OF LD50 •GRAPHICAL METHOD • ARITHMETICAL METHOD
- 33. GRAPHICAL METHOD • MILLER& TAINTER • OBSERVED PERCENTAGE MORTALITY IS CONVERTED INTO PROBIT • THE VALUES THUS OBTAINED ARE PLOTTED AGAINST LOG DOSE • LD50 & ITS STANDARD ERROR MAY BE DETERMINED FROM THE GRAPH IF THE LINE IS STRAIGHT ENOUGH
- 35. • DETERMINATION OFIV LD50 OF CROTALABURNINE IN MICE • RESULTS ARE PRESENTED IN THE TABLE
- 37. • BEFORE PLOTTING,PERCENTAGE DEAD FOR 0 & 100 ARE CORRECTED • PROBIT VALUES ARE PLOTTED AGAINST LOG DOSES • THE DOSE CORRESPONDING TO PROBIT 5(50%) IS CALCULATED • THE PROBIT FUNCTION IS THE QUANTILE FUNCTION ASSOCIATED WITH THE STANDARD NORMAL DISTRIBUTION, WHICH IS COMMONLY DENOTED AS N(0,1)
- 39. • APPROXIMATE S.E.OF LD50 = (LOG LD84-LOG LD16)/√2N • N IS THE TOTAL NUMBER OF ANIMALS EMPLOYED IN THE 2 GROUPS
- 40. ARITHMETICAL METHOD • KARBER’SMETHOD • THE INTERVAL MEAN OF THE NUMBER DEAD IN EACH GROUP OF ANIMALS • THE DIFFERENCE BETWEEN THE DOSES FOR THE SAME INTERVAL • THE PRODUCT OF THE INTERVAL MEAN & THE DOSE DIFFERENCE • RESULTS FROM DOSES LARGER THAN THE LEAST DOSE LETHAL TO ALL IN A GROUP & FROM DOSES SMALLER THAN THE MAXIMAL TOLERATED DOSE ARE EXCLUDED • THE SUM OF THE PRODUCT IS DIVIDED BY THE NUMBER OF ANIMALS IN A GROUP
- 41. • THE RESULTINGQUOTIENT IS SUBTRACTED FROM THE LEAST LETHAL DOSE TO GET LD50
- 43. CONCLUSION • DRUG DEVELOPMENTIS A COMPLEX PROCESS AND WE MUST UNDERSTAND THE ENORMOUS COST BEHIND IT • NOT JUST THE MONEY, BUT ALSO THE LIVES OF ALL THOSE RATS N MICE N RABBITS N DOGS JUST TO KEEP US HEALTTHY • OUR STUDIES SHOULD BE HUMANE AS WELL AS COST EFFECTIVE
- 44. REFERENCE • FUNDAMENTALS OFEXPERIMENTAL PHARMACOLOGY, 7TH EDITION, M.N.GHOSH
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- 47. KEEP SMILING ALWAYS KEEPTHE CHILD INSIDE YOU ALIVE!!!