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Tocixity studies

toxicity studies

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Tocixity studies

  1. 1. W E L C O M E
  2. 2. TOXICITY STUDIES Dr. K.J.KARTHIKA FINAL YEAR P.G SCHOLAR5 DEPT. OF RASASHASTRA AND BHAISHAJYAKALPANA GAVC, TRIPUNITHURA
  3. 3. INTRODUCTION •Toxicology is classically defined as the study of poisons. •It is concerned mainly with the study of adverse effects of xenobiotics. •Casarett 1996 defined it as a science that defines the limits of safety of chemical agents for human and animal population.
  4. 4. HISTORY OF TOXICITY STUDY •Paracelsus( father of toxicology) determined specific chemicals responsible for the toxicity of plants and animals. •“ All substances are poison ,there is none which is not a poison. The right dose differentiates a poison and a remedy.”- Paracelsus ( dose response relation ship). •Mathieu Orfila ( father of modern toxicology) Determined relationship between poison and their biological effects.
  5. 5. NECESSITIES •Toxicological screening is very important for the development of new drugs and extension of the therapeutic potential of existing molecules. •Benefit risk ratio can be calculated. •Prediction of therapeutic index/ safety window (median lethal dose/ median effective dose)
  6. 6. TYPES OF TOXICOLOGY STUDIES •Systemic toxicity studies - Single dose study (Acute) - Repeated dose study( Sub acute and chronic) •Reproductive toxicity studies - Male fertility - Female reproduction and Developmental studies •Local toxicity studies •Hypersensitivity studies •Genotoxicity studies •Carcinogenicity studies
  7. 7. Animals used for toxicity studies •Rats and mice are generally used but for certain classes of chemicals, such as agrochemicals and pharmaceuticals, the tests may also be conducted in non-rodent animals such as the beagle dogs, pigs, marmosets or macaques . •Some regulatory agencies require that at least two species be used, one of them to be selected from rodents and the other from non- rodents. •In at least one of the species, males and females should be used. •In the case of rodents, each group should consist of at least five animals per sex. In the case of non-rodents, each group should consist of at least two animals per sex.
  8. 8. HOUSING AND FEEDING CONDITIONS. • The temperature in the experimental animal room should be 22ºC (+ 3ºC). •The relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning the aim should be 50- 60%. •Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. •For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. •Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.
  9. 9. Route of administration: •Ordinarily, the oral route is sufficient as this is the normal route of clinical administration. However, some regulatory agencies suggest in addition a parenteral route of administration. •In cases where it is proposed to administer the herbal preparation to a human subject by the parenteral route, it may be sufficient to use this route alone for animal testing.
  10. 10. Biomedical ethics Before conducting toxicity studies approval form Institutional Animal Ethics Committee(IAEC) is essential.
  11. 11. Guidelines for toxicity / safety profile evaluation Of Ayurveda & Siddha plant drugs(CCRAS)
  12. 12. Acute Toxicity Test evaluation: Acute toxicity refers to those adverse effects occurring following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours. AIM 1. Determination of Therapeutic Index and LD50. 2. Define Intrinsic toxicity of a Chemical. 3. Provide information for the design and dose selection for prolonged studies. 4. Provide valuable information to the clinician for the prediction, diagnosis and treatment for acute over dose of chemicals. 5. Classification and labeling of chemicals for regulatory purposes.
  13. 13. Sl.No. Test Groups No. of Animals 1. VC (Control) 10 (5M + 5F) 2. Therapeutic Dose (TD) 10 (5M + 5F) 3. Average Dose (TD x 5) 10 (5M + 5F) 4. Highest Dose (TD x 10) 10 (5M + 5F) Test dose : Single dose Route of administration : Oral Duration : 10 days
  14. 14. OBSERVATIONS: a) Mortality : To be observed on 24, 48 & 72 hours b) Clinical signs: Monitoring of convulsions, lethargy, sleep, coma, salivation, diarrhoea •Cage side examination (daily) •Skin colour, fur, eyes & mucous membrane (daily) •Spontaneous and voluntary motor activity on every ½ hour, 1 hour, 2 hours, 4 hours and 24 hours after drug administration. •0,1,7th day spontaneous motor activity •Necropsy - In case of animal dies
  15. 15. SUB ACUTE TOXICITY •This study is conducted to determine organs affected by different dose levels. •Three dose levels are normally used. -High dose (elicit definite signs of toxicity but not to kill many of the animals) - Low dose (induce no toxic effect.) -Intermediate dose. • Doses are generally selected on the basis of information obtained in acute toxicity studies using both LD50 and the slope of the dose response curve. •The duration of sub acute toxicity studies depend on intended duration of the test substance.
  16. 16. Species Animals No. Age (wks) Weight (gms) Duration of experiment Male Female TC Exp. Imm. Exp. Post Exp. Mice (Swiss) 24 6x4* 24 6x4* 4-6 18-20 15 days oral 50% 15th day 50% 30th day Rat Wistar 24 6x4* 24 6x4* 4-6 60-90 15 days oral 50% 15th day 50% 30th day TC-Test compound exposure Imm Exp – Immediate (48 hours) after last exposure – instant effect Post exp – Post-exposure (15 days) after last exposure – reversibility of toxicity if any
  17. 17. Sl.No. Test Groups No. of Animals 1. VC (Control) 12 (6M + 6F) 2. Therapeutic Dose (TD) 12 (6M + 6F) 3. Average Dose (TD x 5) 12 (6M + 6F) 4. Highest Dose (TD x 10) 12 (6M + 6F) TEST GROUPS Route of Administration: Oral Duration: 30 days
  18. 18. Pre-experimentation phase I. Acclimatization of animals •Period – 7 days (Recording of body weight and food intake twice in a week) •Urine qualitative test (Ames multiple sticks) •Fecal consistency (Filter paper technique)
  19. 19. Experimentation phase I)Test compound exposure multiple dose (once daily for 15 days) and dosage schedule (as recommended by sponsor) II)Mortality 6/12/24 hours III)Body weight (Twice in a week) IV)Food consumption (Daily) V)Fecal consistency VI)Cage side activity VII)Neurological examination VIII)Urine qualitative test
  20. 20. IX) Haematology - Twice (15th day/30th day) (Hb, RBC, WBC, Platelet count, differential count) X) Clinical chemistry – Twice (15th day/30 day) (Blood glucose, total protein, serum Creatinine, SGOT, SGPT). XI) Histopathology -Liver, Kidney, Lungs, Spleen, Ovaries, Testis, Stomach, Intestine
  21. 21. General comments •Metal free pellet diet and water. •One rat and two mice in each cage •Test compound, vehicle control, its dosage regimen will be supplied by sponsor (ISM) •Scoring of cage side activity, neurological activity according to OECD guidelines.
  22. 22. Observations: a. Mortality: To be observed on 24, 48 and 72 hrs. b. Clinical Signs: • A careful cage side examination will be made daily. changes in: - Skin, fur, eyes and mucous membrane. - Convulsions, lethargy, sleep, coma, salivation, diarrhoea and date of death.
  23. 23. CHRONIC TOXICITY STUDIES This study is basically to determine the organs affected and to check whether the drug is potentially carcinogenic or not. This test extends over a long period of time and it involves large groups of laboratory animals. Species No. of Animals Age (wks) Weight (gms) Duration of experiment Male Female TC Exp. Term. Exp. Mice (Swiss) 40 10 x 4* 40 10 x 4* 4 15-18 90 days 100% Rat Wistar 40 10 x 4* 40 10 x 4* 4 60-80 90 days 100% TC-Test compound exposure Term. Exp – Termination of experiment immediately after last exposure (48 hours) euthanization of animals for collection of vital organs.
  24. 24. Sl.No. Test Groups No. of Animals 1. VC (Control) 20 (10M + 10F) 2. Low Dose/Now effect Dose 20 (10M + 10F) 3. Intermediate Dose 20 (10M + 10F) 4. Highest Dose 20 (10M + 10F) Test groups Route of drug administration : Oral Duration of drug administration : 90 days( or more)
  25. 25. Expected period of Clinical use Administration period for the toxicity study Single administration or repeated administration for less than one week 2 weeks to 1 month Repeated administration, between one week to four weeks 4 weeks to 3 months Repeated administration, between one to six months 3 to 6 months Long term repeated administration for more than six months 9 to 12 months As a rule, the test substance should be administered seven days a week. Administration periods for the toxicity study must be recorded in each result. The following table reflects commonly used ranges of administration periods:
  26. 26. Pre-experimentation phase I. Acclimatization of animals •Period – 7 days (Recording of body weight and food intake twice in a week) •Urine qualitative test (Ames multiple sticks) •Fecal consistency (Filter paper technique)
  27. 27. Experimentation phase I)Test compound exposure - multiple dose (once daily for 90 days) and dosage schedule (as recommended by sponsor) II)Mortality 6/12/24 hours III)Body weight (Twice in a week) IV)Food consumption (Twice/weekly) V)Fecal consistency VI)Cage side activity VII)Neurological examination
  28. 28. •Urine qualitative test (30/60/90 days) •Haematology - Twice (30/60/90 days) (Hb, RBC, WBC, Platelet count, differential count) •Clinical chemistry – Twice (30/60/90 days) (Blood glucose, total protein, serum creatinine, SGOT, SGPT). •Histopathology – Liver, heart, brain, lung, kidney, spleen, sciatic nerve, testes/ ovaries
  29. 29. 1. General observations and examinations: •General signs should be observed daily. •Body weight: before the start of drug administration, at least once a week for the first three months of administration and at least once every four weeks thereafter. •Food intake: before the start of drug administration, at least once a week for the first three months of administration and at least once every four weeks thereafter. If the test substance is administered mixed in the food, the intake should be measured once a week. .
  30. 30. 2.Haematological examination: •For rodents, blood samples should be taken before autopsy. •For non-rodents, blood samples should be taken before the start of the drug administration, at least once during the administration period (for studies of longer than one month), and before autopsy. •For both haematological and blood chemistry examinations, it is desirable to include as many parameters as possible.
  31. 31. 3.Renal and hepatic function tests: Since the liver and kidneys are the usual organs of metabolism and excretion, potentially toxic agents easily affect them; their functions should be monitored in long term toxicity studies. For rodents, a fixed number of animals from each group should be selected and urinalysis should be performed before the start of drug administration, and at least once during the administration period.
  32. 32. 4.OTHER FUNCTION TESTS: •If appropriate, ECG and visual, auditory tests should be performed. •For rodents, ophthalmological examination should be performed on a fixed number of animals from each group at least once during the administration period; •for non-rodents, examination should be performed on all animals before the start of drug administration and at least once during the period of administration.
  33. 33. 5. Animals found dead during the examination should be autopsied as soon as possible. A macroscopic examination should be made of organs and tissues. In addition, where possible, organ weight measurements and histopathological examinations should be performed in an attempt to identify the cause of death and the nature (severity or degree) of the toxic changes present.
  34. 34. •All survivors should be autopsied at the end of the administration period or of the recovery period after taking blood samples for haematological (including blood chemistry) examinations. • organs and tissues should be examined macroscopically and organ weights measures. •Histopathological examinations of the organs and tissues of animals receiving lower dosage should also be performed •Histopathological examination of all rodents will further improve the chances of detecting toxicity.
  35. 35. OECD Guidelines
  36. 36. Five Organisation for Economic Cooperation and Development (OECD) Test Guidelines (TGs 402, 403, 420, 423, and 425) describe acute systemic testing. 1. Fixed Dose Procedure (OECDTG 420) 2. AcuteToxic Class method (OECDTG 423) 3. Up‐and‐Down Procedure (OECDTG 425) 4. Acute DermalToxicity OECDTG 402, 5. Acute inhalation toxicity OECDTG 403.
  37. 37. Fixed dose procedure •5 animals of a single sex are dosed in a stepwise procedure one animal each,using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered). •The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. •Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. •This procedure continues until the dose causing evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.
  38. 38. AcuteToxic Class method (OECDTG 423) •Three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. • The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. •When available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight), then a limit test should be conducted. •When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight. Exceptionally, and only when justified by specific regulatory needs, the use of additional upper dose level of 5000 mg/kg body weight may be considered.
  39. 39. Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead
  40. 40. Up‐and‐Down Procedure (OECDTG 425) •The main test consists of a single ordered dose progression in which animals are dosed , one at a time , at a minimum of 48-hour intervals. • The first animal receives a dose a step below the level of the best estimate of the LD50 . If the animal survives, the dose for the next animal is increased by [a factor of] 3.2 times the original dose ; if it dies, the dose for the next animal is decreased by a similar dose progression. •Dosing is stopped when • 3 consecutive animals survive at the upper bound; • 5 reversals occur in any 6 consecutive animals tested • LD 50 is calculated.
  41. 41. Six OECDTestGuidelines describe short‐term repeat‐dose toxicity testing: Repeated Dose 28‐day OralToxicity Study in Rodents (TG407) Repeated Dose 90‐Day OralToxicity Study in Rodents (TG 408) Repeated Dose DermalToxicity: 21/28‐day Study (TG 410) Subchronic DermalToxicity: 90‐day Study (TG 411) Repeated Dose InhalationToxicity: 28‐day or 14‐day Study (TG 412) Subchronic InhalationToxicity: 90‐day Study (TG 413)
  42. 42. Repeated Dose 28‐day Oral Toxicity Study in Rodents (TG407) • The test substance is orally administered daily in graduated doses to several groups of experimental animals (rat 5 of each sex ), one dose level per group for a period of 28 days. •A 28 day study provides information on the effects of repeated oral exposure and can indicate the need for further longer term studies. •The data derived from using the TG should allow for the characterization of the test substance toxicity, for an indication of the dose response relationship and the determination of the No- Observed Adverse Effect Level (NOAEL). •Satellite or control groups maintained.
  43. 43. Repeated Dose 90‐Day OralToxicity Study in Rodents (TG 408) •The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 90 days. •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.
  44. 44. THANK YOU

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