OECD Guidline on acute and chronic toxicity

brief idea about acute and chronic toxicity

1. GANDHI SANKET J. 1st YEAR M.PHARM Dept. of pharmacology R. C. Patel Institute of Pharmaceutical Education and Research ACUTE ORAL CARCINOGENICITY CHRONIC TOXICITY STUDY OECD GUIDELINES

2. CONTENTS 2 INTRODUCTION TO TOXICOLOGY OECD GUIDELINE FOR ACUTE ORAL TOXICITY LD50 LD50/LC50 Methods to calculate LD50 Limitation of LD50 How OECD GUIDELINES More Humane? Alternatives to use of Animal in AOT Description of Whole AOT Guidelines along with Its Sighting Study (Guidelines no. 401,420,423,425) OECD GUIDELINES FOR CARCINOGENICITY (451) (Principle ,Housing – Feeding,Objectives , Procedure,Result,DiscussIon ) OECD GUIDELINES FOR CHRONIC TOXICITY (452) (Principle ,Housing – Feeding,Objectives , Procedure,Result,DiscussIon )

3. WHAT IS TOXICOLOGY ? Toxicology is the scientific study of adverse effects that occur in living organisms due to chemicals. It involves observing and reporting symptoms, mechanisms, detection and treatments of toxic substances, in particular relation to the poisoning of humans. 3

4. ADVERSE DRUG EFFECTS Any undesirable &/ or unintended effects of drug, 1. Predictable (type A reactions) 2. Non-predictable (type B reactions) SIDE EFFECTS Unwanted but often unavoidable effects at therapeutic doses. SECONDARY EFFECTS - Indirect consequences of primary action of drug. TOXIC EFFECTS – Are results of excessive pharmacological effect of drug due to over dosage or prolonged use. 4 (Barar,2005; Ghosh , 2005)

5. ANIMAL TOXICITY TESTS Acute toxicity 14 days Sub-acute (repeated doses) toxicity 28 days Sub-chronic toxicity 3 months Chronic toxicity 6 months to 2 Special toxicity e.g. Carcinogenicity 5 (Curtis,2003;Ghosh,2005)

6. ACUTE ORAL TOXICITY 6

7. ACUTE TOXICITY SYMPTOMS Tremor Salivation Muscle spasm Convulsion Lacrimation Weight loss Diarrhoea Altered Respiration Loss of righting reflex 7 ( Ghosh ., 2005)

8. WHAT IS LD 50 ? LD50 represents the individual dose required to kill 50 percent of a population of test animals. It is an index determination of medicine and poison’s virulence. lower the LD50 dose, the more toxic the pesticide. WHAT IS LC 50 ? The concentrations of the chemical in air that kills 50% of the test animals during the observation period is the LC 50  value. Other durations of exposure (versus the traditional 4 hours) may apply depending on specific laws. 8 (P.A. Botham et al. 2004)

9. LD 50 /LC 50 9 9

10. METHODS TO DETERMINE LD 50 Karber’s method Miller and Tainter method Lorke’s method Fixed dose method UP and down method 10 OECD,1987; Ghosh , 2005)

11. KARBER`S METHOD This method is also known as ARITHMATIC method . Simplest & rapid. It does not involve any plotting of dose-response curve. It is useful method particularly when number of animals is small. The interval mean of the no. of dead in each group of animals was used as well as the differences between doses for the same interval. The number of animal in every group must be equal. 11

12. LD 50 CALCULATION BY KARBER`S METHOD Group Dose Mg/kg No. of animal Dose difference (a) dead Mean mortality (b) Product ( a.b ) 1 64 10 - - - 2 71 10 7 2 1 7 3 81 10 10 4 3 30 4 90 10 9 9 6.5 58.5 5 100 10 10 10 9.5 95 L D 50 = 100 – {€ a.b /n} n is the number of animals 12

13. MILLER AND TAINTER METHOD Also called as Graphical method Simple & accurate enough. The observed % mortality is converted into probit ( deviation from the mean) by referring to the table. Percentage dead for 0% & 100% are corrected. The probit value thus obtained are plotted against log dose. And then dose corresponding to probit 5 (50%) is found out. The LD 50 may be determined from the graph if the line is straight enough. 13

14. LD 50 CALCULATION BY GRAPHICAL METHOD Group Dose Mg/kg Log dose Dead/total % Dead corrected % dead probit 1 64 1.81 0/10 2.5 - 2 71 1.85 2/10 20 20 4.16 3 81 1.91 4/10 40 40 4.75 4 90 1.95 9/10 90 90 6.28 5 100 2.00 10/10 100 97.5 - corrected formula for 0 % = 100(0.25/n) corrected formula for 100% = 100{(n –0.25) /n} 14

15. % 1 2 3 4 5 6 7 8 9 - 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66 10 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12 20 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45 30 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.72 40 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97 50 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23 60 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50 70 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81 80 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23 90 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33 15 TRANSFORMATION OF PERCENTAGES TO PROBITS (GHOSH,. 2005) 15

16. GRAPHICAL METHOD ---------------- --------------------- . . . . . 1.8 1.9 2.0 3 4 5 6 7 - - - - - - - - Log dose PROBIT 16

17. LORKE‘S METHOD Phase 1: 3 groups of mice are prepared .one dose was given to each group i.p The treated mice were monitored for 24hr for mortality and general behavior . Phase 2: After 24 hr 3-4 groups of one mouse were given doses based on the findings of phase 1. I.p . The mice were again monitored for 24 hrs .The geographic mean of least dose that killed mice and the highest dose that did not kill mice was taken as the median lethal dose 17

18. ALTERNATIVE METHOD (1) FIXED DOSE PROCEDURE This method does not use death as an end points instead it uses the observation of clear signs of toxicity developed at one of a series of fixed dose levels to estimate the LD50 (2) UP AND DOWN METHOD ( Staircase method ) Two mice were injected with a particular dose and observed for a period of 24 hrs for any mortality . 18

19. The subsequent doses were then increased by a factor 3.2 if the dose was tolerated ,or if not then decreased by a factor of 0.5. The max. Non lethal and the min lethal doses were thus determined using only 15 mice. A final and more reliable LD50 assay was planned using at least 3 or 4 dose levels within the range of max non lethal and min doses 19

20. LD 50 LIMITATIONS LD 50  is somewhat unreliable and results may vary greatly between testing facilities due to factors such as the genetic characteristics of the sample population, animal species tested, environmental factors and mode of administration There can be wide variability between species as well; what is relatively safe for rats may very well be extremely toxic for humans ( cf.   paracetamol toxicity), and vice versa. For example, chocolate, comparatively harmless to humans, is known to be toxic to many animals. When used to test venom from venomous creatures, such as snakes, LD 50  results may be misleading due to the physiological differences between mice, rats, and humans. 20

21. HOW OECD GUIDELINES FOR ACUTE TOXICITY MORE HUMANE ? A humane endpoint can be defined as the earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death. OECD Test Guidelines do not require death as an endpoint. Animals humanely killed during the test will be regarded as dosage-dependent deaths. Three alternative test methods (Guidelines 420, 423, and 425) to the traditional acute oral toxicity test have been adopted by the OECD. One of these, the Fixed Dose Procedure (Guideline 420). 21

22. CONTINUE… Refinement of the traditional acute oral test in that it requires fewer, but fixed, dosage groups to be tested, and thus fewer animals. It also employs non-lethal endpoints to determine the toxicity of the test substance. Two other methods, the Acute Toxic Class Method (Guideline 423) and the Up-and-Down Procedure (Guideline 425), use impending death as the only endpoint. Series on Testing and Assessment: Testing for Human Health in that guidline no. 19 contains Guidance document on the recognition, assessment and use of clinical signs as humane endpoints 22

23. CONTINUE… GUIDLINES NUMBER OF ANIMAL PER TEST NUMBER OF DEATH PER TEST AIM 401 Up to 25 Up to 12 - 420 5-7 1 Endpoint-evident toxicity 423 Average 7 2-3 - 425 6-9 2-3 Stopping criteria to limit number of animals used Animal welfare considerations, All three alternatives contain requirement to follow OECD Document on Humane Endpoints. 23

24. SOME ALTERNATIVES TO THE USE OF ANIMALS IN TESTING in vitro (test tube) test methods and models based on human cell and tissue cultures. computerized patient-drug databases and virtual drug trials. computer models and simulations. stem cell and genetic testing methods. non-invasive imaging techniques such as MRIs and CT Scans. microdosing (in which humans are given very low quantities of a drug to test the effects on the body on the cellular level, without affecting the whole body system). 24

25. ICCVAM The need for alternatives to the traditional use of animals in toxicity testing was officially recognized by the U.S. government in 1993 with passage of the NIH Reauthorization Act. Requirements under the Act lead to the establishment of committee called the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). To Providing guidance to test method developers To Induce use of alternative tests that encourage the reduction, refinement, or replacement of animal test methods Evaluating recommendations from expert peer reviews of alternative toxicological test methods  25

26. OECD GUIDELINES FOR ACUTE ORAL TOXICITY Number Title Original adoption Number of updates Most recently updated 401 Acute oral toxicity- conventional acute toxicity test 12 may 1981 1 Date of deletion: 20 December 2002 420 Acute oral toxicity- fixed dose procedure 17 July 1992 1 17 December 2001 423 Acute oral toxicity- acute toxic class method 22 march 1996 1 17 December 2001 425 Acute oral toxicity- up and down procedure 21 September 1998 2 23 March 2006 26

27. DESIGN OF ACUTE TOXICITY 14 days study. Study on at least two species. One rodent –mice/rat. One non rodent –usually rabbit. Dose administered orally & parenterally . Various dose levels to groups of both sexes. Dose selection such that causing less than 50% but not 0% and more than 50% but not 100% mortality. 27 (Ghosh,2005)

28. ACUTE ORAL TOXICITY OECD GUIDELINES NO.401 (CONVENTIONAL ACUTE TOXICITY METHOD ) In a study of toxic characteristics of substance, acute oral toxicity testing is initial step. Gives information on health hazards. Test substance administered orally, in graduated doses to several groups of experimental animals. One dose used per group. 28 (OECD,1987)

29. CONTINUE… At least 5 rodents at each dose level of same sex are used. Observations for effects & death are made. After completion of study in one sex, study in another sex is carried out. Studies suggested in rodents but can be adopted for studies in non-rodents. 29

30. ACUTE ORAL TOXICITY GUIDLINE NO. 420 (FIXED DOSE PROCEDURE) New approach in 1984 by British toxicology society based on administration of series of fixed dose levels. Instead of death, clear signs of toxicity to animals as end point. Adopted as 1 st alternative to conventional acute toxicity test. Testing in 1 sex usually females is considered sufficient. Uses fewer animals. 30 (OECD, 2001)

31. CONTINUE… Reproducible procedure. Causes less suffering to the animals. Uses only moderately toxic doses, doses expected to be lethal should be avoided. 31

32. PROCEDURE Procedure involves 2 step study 1- sighting study 2-main study 32

33. FLOW CHART FOR SIGHTING STUDY 33

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35. FLOW CHART FOR MAIN STUDY 35

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37. ACUTE ORAL TOXICITY GUIDLINE NO.423 ( ACUTE TOXIC CLASS METHOD) No sighting study. 3 animals of single sex per step. On avg. 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. Not intended to allow the calculation of precise LD 50 . Death of a proportion of animals as the major end point (response). Ld 50 cut off values are indicated . 37 (OECD, 2001)

38. PRINCIPLE Stepwise procedure with the use of minimum no. of animals per step. Substance administered orally to 2 groups of animals at defined doses . 3 animals per step of single sex (normally females). Compound related mortality determines the next step. Report 38

39. FLOW CHART 39

40. CONTINUE… 40

41. CONTINUE… 41

42. ACUTE ORAL TOXICITY GUIDLINE NO.425 (UP AND DOWN METHOD) Up and down testing approach was 1 st described by Dixon and Mood. Bruce in 1985 proposed to use it for acute toxicity determination of chemicals . Estimates confidence intervals for LD 50 . 42 (OECD2006)

43. CONTINUE … In procedure (main test ) 1-animal dosed at a time, at minimum of 48 hrs interval. Suggested starting dose is 175 mg/kg or can be selected from 1.75, 5.5, 17.5, 55,175,550,2000mg/kg . Animal receives 1 st dose a step below the level of the best estimate of LD 50 . 43

44. CONTINUE … Depending upon the outcome for the previous animal, the dose for the next animal is adjusted up or down. 5 reversal in 6 consecutive animals when obtained test is terminated. No. of animals limited to 15. Report AOT 425 START PROG. 44

45. COMPARISON OF GUIDELINES OF LD 50 No 401 420 423 425 Sighting study Absent Present Absent Absent Preferred dose 2 to 5 000mg/kg per animal 5 to 5000 mg/kg per animal 5 to 5000 mg/kg per animal 1 st animal dose less than estimated Ld 50 outcome by factor 3.2 Drug effect Dose causing high mortality Dose causing evident toxicity Dose causing mortality Stopped if no reversal occur by reaching selected upper limit Grouping of animal 5 animals per group Group of 5 animal of 1 sex 3 animals per group 1 animal per group Estimation of end point Up to 12 animal expected to die 1 animal expected to die 2,3 animal expected to die 2,3 animal expected to die Limitation It used high animal mortality as endpoint , at high dose, so this method require reduction or refinement. It used evident toxicity as endpoint instead of death dose level close to lethal dose not always obtained Finding of a delayed death may require additional lower dose level to be used No limitation 45 (R. L. LIPNICK et al.1995)

46. LIMITATION OF METHODS USED FOR TOXICITY STUDY Indicated that all are likely to perform poorly for chemicals with shallow dose-response slopes Because Guideline 420 uses evident toxicity as an endpoint instead of death. Unusually test substances may cause delayed deaths (5 days or more after test substance administration) mostly in case of guidline no.425 However, both in Guideline 420 and 423, the finding of a delayed death may require additional lower dose levels to be used or a study to be repeated. 46

47. CARCINOGENICITY STUDY The test substance is administered daily in graduated doses Observed closely for signs of toxicity and for the development of neoplastic lesions. Died or are killed animals are necropsied and at the conclusion surviving animals are also killed and necropsied . PRINCIPLE ROUTES OF ADMINISTRATION 451 451 47 www.OECD.org

48. OBJECTIVES OBJECTIVES 48

49. HOUSING AND FEEDING 49

50. ANIMAL TO BE USED, Rodents/non-rodents used 12 months study for rodents 90 days study for non-rodents Mostly mice are used due to short life-span and susceptibility Healthy animals, acclimated to laboratory conditions for 7 days and not been subjected to previous experimental procedures the weight variation of animals used should not exceed ± 20 % of the mean weight Animals should be randomly assigned 50

51. DOSE GROUP AND DOSAGE 51 3 Dose levels short-term repeated dose Toxicological existing data Toxicokinetics existing data Range finding studies Highest dose level to identify the principal target organs and toxic effects (avoid severe toxicity, morbidity, or death) STUDY DURATION 51

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53. Observations 53

54. RESULT 54

55. DISCUSSION 55

56. CHRONIC TOXICITY STUDY The test substance is administered daily in graduated doses to several groups of experimental animals for a period of 12 months During the period of administration the animals are observed closely for signs of toxicity. Animals which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are also killed and necropsied . 452 452 PRINCIPLE ROUTES OF ADMINISTRATION 56 www.OECD.org

57. PREPARATION OF DOSE AND DURATION OF STUDY The test substance is normally administered orally, by gavage or via the diet or drinking water. When the test substance is administered by gavage to the animals this should be done using a stomach tube or a suitable intubation cannula , at similar times each day. Normally the volume should be kept as low as practical, and should not exceed 1 ml/100g body weight, except in the case of aqueous solutions where 2 ml/100g body weight may be used. While this Test Guideline primarily is designed as a 12 month chronic toxicity study, the study design, also allows for and can be applied to either shorter (e.g. 6 or 9 months) or longer (e.g., 18 or 24 months) 57

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59. parameters Body weight, food/water consumption and food efficiency. (At least once a week for the first 13 weeks) Haematology and clinical biochemistry (10 females and 10 males) Total erythrocyte ,leucocytes and platelets count, erythrocytes morphology ,glucose ,urea , creatinine albumine ,total protiens count ,total cholesterol count, total bile level , presence of enzymes Appearance,Volume , Specific gravity,pH , Bilirubine,Glucose , Occult blood Urine analysis 59

60. Survival data; urinalysis tests Outcome of any investigations of neurotoxicity or immunotoxicity Terminal body weight; organ weights Necropsy findings; A detailed description of all treatment-related histopathological findings Absorption data if available Body weight/body weight changes Toxic response data by sex and dose level, including signs of toxicity Duration of clinical observations Ophthalmological examination Haematological tests Clinical biochemistry tests RESULT 60

61. General considerations such as housing, feeding, dose preparation, route of drug administration, histopathology, pathology, data collection, analyzing of data and results submission same as for OECD-451. 61