Safety & Efficacy Study of Herbal Drugs

1. SAFETY & EFFICACY STUDY OF DRUGS Afsar fathima

2. Introduction • Herbal drugs have made a great contribution to maintaining human health. • Majority of the worlds population in developing countries still relies on herbal drugs. • WHO supports the use of herbal drugs and encourage the remedies that have been proven to be safe and effective. • A few herbal drugs have withstood scientific testing, but others are used simply for traditional reasons to protect, restore or improve health.

3. INTRODUCTION No evidence to answer question of safety and efficacy about most of the herbal drugs now in use. Still need to be validated scientifically. Toxicity evaluation of herbal drugs to document toxicological profile and boost the export of herbal drugs manufacturing in India.

4. Pre-Clinical Studies OBJECTIVES: To determine whether such studies support clinical use of a herbal drug. To characterize the range of pharmacological action of herbal drug. To evaluate the pharmacologically active natural products and to find their mechanism or actions. Toxicological study: 1. Assess the safety of herbal drugs 2. Define the possible toxicity from short-term and long term use.

5. CPCSEA: Committee for the Purpose of Control and Supervision on Experiments on Animals. • IAEC: Institutional Animal Ethics Committee • Members of IAEC - One biological scientist - Two scientists from different biological discipline - Veterinarian - Scientist in-charge of animal facility - Scientist from out side the institute - Socialogist and CPCSEA Nominee

6. Toxicological investigation • Acute toxicity study • Repeated dose toxicity study • Dermal toxicity test • Mutagenicity test • Carcinogenicity test • Reproductive and development toxicity tests

7. Toxicological investigation • Paracelsus (1493-1541) stated that “All substances are poisons; The right dose differentiates a poison and remedy” this concept is the fundamental principle of toxicology. • WHO- guidelines have given the important criteria to establish the safety profile of the drugs. • Toxicological study results play an important safety assessment for Herbal Drugs, Pharmaceuticals, food additives , pesticides and other chemicals.

8. Toxicity study • Essential for any compounds having biological activity. • Acute toxicity: Adverse effects occurring within a short time following administration of single dose. • Sub- acute toxicity • Chronic toxicity- Period of 90 days or more. • Special toxicity studies- Period of 2 years or more • Carcinogenicity • Mutagenicity • Teratogenicity • Reproductive toxicity

9. Important OECD guideline • Organisation for Economic Co-operation and Development (OECD) • 420 Acute oral Toxicity- fixed dose method • 423 Acute oral Toxicity –acute toxic class method • 425 Acute oral Toxicity-Up and Down method • 402 Acute dermal Toxicity • 404 Acute dermal Irritation/ Corrosion • 403 Acute inhalation Toxicity • 405 Acute Eye Irritation /Corrosion • Skin sensitization • 28 days repeated oral Toxicity studies in rodents • 408 90 days repeated oral Toxicity studies in rodents • 409 90 days repeated oral Toxicity studies in non rodents

10. Important OECD guideline • 410 90 days repeated Dermal Toxicity • 411 90 days inhalation Toxicity study • 412 28/14 days repeated dose inhalation Toxicity study • 413 90 days repeated dose inhalation Toxicity study • 414 Prenatal Developmental Toxicity study • Reproduction /Development toxicity screening test • Combined Repeated dose toxicity study with Reproduction/Developmental Toxicity screening test • Neurotoxicity study in rodents • Carcinogenicity studies • Chronic Toxicity studies • Combined chronic toxicity/carcinogenic studies

11. Experimental Animals • Rats : Sperague dawley (SD)Wistarlsiter HoodedLong evansFischerBrown NorwayLewisHybrid and Congenic RatsMutant Rats (Athymic nude, Zuckar) • GerbilsMongolian gerbil Mice • AlbinoCD1BALB/C • Guinea PigsDunkin Hartley • RabbitNewzealand White • Dogs: Beagles • HamsterGolden Syrian

12. Experimental Animals Rat

13. Experimental Animals Mice

14. Experimental Animals C.S.M.D.R.I.A.S

15. Conversion factor for Animals[Man to Animals] C.S.M.D.R.I.A.S • 20g Mouse = Dose x 0.0026 = Xx50= Dose /Kg/Body wt. • 200g Rat = Dose x 0.018 = Xx5 = Dose /Kg/Body wt. • 400g Guinea pig = Dose x 0.031 = Xx2.5= Dose /Kg/B. wt. • 1.5 Kg Rabbit = Dose x 0.07 = Dose /1.5Kg/Body wt.

16. OECD 423 (Acute toxic class method) www.themegallery.com • PRINCIPLES • Based on a stepwise procedure with the use of a minimum number of animals per step. • Absence or presence of compounds related mortality of the animals dosed at step will determine the next step. No further testing is needed. • Dosing of three additional animals with the same dose. • Dosing of three additional animals with the next higher or the next lower dose level.

17. DESCRIPTION OF THE METHOD • Preparation of the animals • Randomly select the animals and keep in their cages for at least 5 days prior to start of the test to allow of acclimatization of the laboratory conditions. • Preparation of dose • If vehicles other than water than toxic characteristics of vehicle should be known (gum acacia, SCMC, tween 20&80, honey, vegetable oils, palm jaggary etc.) • Procedure • Three animals in each step • Doses: Starting dose from- 5, 50, 300, 2000 mg/kg b/w. If no information available regarding lethality of drug than dosing start from 300 mg/kg, bw.

18. Administration of Doses • Rodents • Volume not to exceed 1 ml/100gm b.w. however for aqueous solution 2 ml/100g b.w. can be consider. • Rats • Overnight fasting prior to dosing but free assess to drinking water. • Mouse • 3-4 hrs fasting prior to dosing but free assess to drinking water. • After administration of drugs, food may be withdraw for 3-4 hrs in rats and 2 hours in mice.

19. Limit test • When information suggests that mortality is unlikely at the higher starting dose level 2000 mg/kg, orally, then conduct the limit test. • Limit test carryout at dose of 2000 mg/kg body weight with six animals (three animals per step) • Exceptionally and additional dose of 5000 mg/kg can be consider when justify by specific regulatory need.

20. Observation • Observe for 14 days. Closely observe for first four hours after the dosing and than at least once daily thereafter up to 14 days. • Parameters: • Mortality, Changes in skin, eyes, mucus. • Behavioural pattern changes • Respiratory, Circulatory, ANS, CNS (stimulants or depressant) • Observation of tremors, convulsions, salivation, diarrhea, sleep and coma • Body weight – At least once in a week • Pathology – Gross necropsy and microscopic examination if organs showing any changes.

21. OECD 423

22. Acute Oral Toxicity – Fixed dose procedure (OECD 420) Fixed doses 5, 50, 300, 2000 mg/kg, orally (exceptionally and additional dose of 5000 mg/kg can be consider when justify by specific regulatory need). The initial dose select from the sighting study is the dose expects to produce some sign of toxicity without causing severe toxic effects. Further groups of animals may be dose at higher or lower fixed dose depending on the presence or absence of toxicity. Limit test perform at 2000 mg/kg bw, when information indicating test material is likely to be nontoxic or toxicity above regulatory limit dose.

23. OECD 420 Animal: Rat of female sex preferable and 10-12 weeks old. Number : Total of five animals use for each dose level. Dose: single dose Rat: overnight fasting Mice: 3-4 hours fasting Time interval between dosing: Period of 3 or 4 days between dosing at each dose level is recommended. Onset, duration and severity of toxic sign. Observation parameters same as OECD 423.

24. OECD 420 Sighting Study

25. OECD 420 Main Study

26. Acute Oral Toxicity – up and down method (OECD 425) • This is a sequential test uses a maximum of 5 animals. • Dose one animal at the test dose. • Animal dies, conduct the main test to determine the LD50 . • Animal survives, dose four additional animals sequentially observe 14 days. • MAIN TEST • The main test consist of single ordered dose progression in which single animals are dose in sequence usually at 48 hour intervals (dosing is determine by the onset, duration and severity of toxic signs).

27. OECD 425- MAIN TEST The first animal dose below best preliminary estimate of LD50. If the animal survives. The dose for second animal is increase by (a factor of) 3.2 times the original dose. If the first animal dies or appears morbid than dose for second animal is decrease by (a factor of) 3.2 times the original dose. 3.2 is default factor corresponding to a dose progression of one half log unit. Dosing would be select from the sequence dose 1.75, 5.5, 17.5, 55, 175, 550, 2000, (or 1.75, 5.5, 17.5, 55, 175, 550, 1750 and 5000 for specific regulatory needs). If no information available regarding lethality of drug than dosing start from 175 mg/kg, bw.

28. OECD 425- Limit test • Limit test carryout at 2000 mg/kg, orally (Exceptionally 5000 mg/kg) when information indicating test material is likely to be nontoxic or toxicity above regulatory limit dose. • The LD50 less than the test dose (2000 mg/kg) when 3 or more animals die. • LD50 is greater than the test dose (2000 mg/kg) when 3 or more animal survive. • Observation:14 days same as OECD 423

29. 28 day repeated dose oral Toxicity study (OECD-407) Animal species: Rat or Mice Age : Nine weeks old. Housing condition Temperature: 220 c ± 30c Humidity: 50-60 % Lighting: 12 hr light 12 hr dark light Feed : laboratory feed with water ad libitum House: individually, small groups in same sex, not more than five/cage

30. OECD-407 Preparation of doses: Administration by gavages or via the diet or drinking water in volume of 1ml/100gm for suspension and 2ml/100gm b.w for aqueous. If Vehicles use other than water the toxic properties of the vehicle is must be known. Number of animal: 10 animals per group (5 female and 5 male) Dosage: At least three test groups and the control group should be use. Limit test: 1000 mg/kg/day for 28 days daily

31. OECD-407 Observation :28 days - General clinical observation at least once a day - Morbidity and mortality – at least twice daily - Changes in skin , eyes, mucous membrane, secretion and excretion. - ANS activity- lacrimation, piloerection , pupil size, respiratory pattern, grip strength, motor activity assessment. - Body weight: weekly - Food consumption : weekly - Water consumption :weekly

32. OECD-407 Hematology parameters: Haematocrit, Hb, RBC, WBC, DC, platelet , clotting time. Clinical Biochemistry Liver and kidney function test Plasma or serum Na ,K, glucose, cholesterol, urea, creatinine, SGOT, SGPT, total protein, albumin, ALP, bilirubin, Gamma glutamyl transpeptidase, Lipid profile. Urine analysis Last week of the study. Volume, appearance , specific gravity, pH , protein glucose and blood cells.

33. OECD-407 Metabolic profiles Calcium, phosphate and triglycerides. Pathology: Liver, kidney, adrenals, testes, thymus, spleen, brain, stomach, intestine, breast, lungs, urinary bladder, peripheral nerve, bone marrow etc.

34. 90 day repeated dose oral Toxicity study (OECD-408) Animal species: Rat or Mice Age : Nine weeks old. Housing condition Temperature: 220 c ± 30c Humidity: 50-60 % Lighting: 12 hr light 12 hr dark light Feed : laboratory diet with water ad libitum House: Individually, small groups of same sex, not more than five/cage

35. OECD-408 Preparation of doses: Administration by gavages or via the diet or drinking water in volume of 1ml/100gm for suspension and 2ml/100gm b.w for aqueous. If Vehicles use other than water than toxic properties of the vehicle is must be known. Number of animal: 10 animals per group (5 female and 5 male) Dosage:At least three test groups and the control group should be use. Limit test: 1000 mg/kg/day for 90 days daily Observation: Same as OECD 407

36. Acute Dermal Toxicity Study OECD-402 Prerequisites – Solid or liquid test substance – Chemical identification of test substance – Purity (impurities) of test substance – Solubility characteristics – Melting point/boiling point – PH

37. OECD-402 C.S.M.D.R.I.A.S Acute dermal toxicity is the adverse effects occurring within a short time of dermal application of a single dose of a test substance. Principle : The test substance is applied to the skin in graduated doses to several groups of experimental animals, one dose being used per group.

38. OECD-402 C.S.M.D.R.I.A.S Preparations 24 hours before the test, fur should be removed from the dorsal area of the trunk of the test animals by clipping or shaving. Care must be taken to avoid abrading the skin, which could alter its permeability. Not less than 10 per cent of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering

39. OECD-402 C.S.M.D.R.I.A.S Animals: Adult rat - 200-300 gm Rabbits- 2-3 kg Guinea pigs- 350-450 gm Number and sex: 5 animals ( female or male) Housing --- individual housing ---Other standard condition as per previous OECD guidelines Dose level --- At least three dose level with control and vehicle control if necessary Limit test A limit test at one dose level of at least 2000 mg/kg bodyweight Observation : 14 days.

40. 21/28 –day Repeated dose dermal Toxicity study (OECD- 410) Prerequisites - Solid or liquid - Purity - Solubility - pH - Stability, including stability in vehicle - Melting point/ boiling point

41. OECD- 410 Principle The test substances apply daily to the skin in graduate doses to several groups of experimental animals, one dose per group for 21/28 days. Animals: Adult rat - 200-300 gm Rabbits - 2-3 kg Guinea pigs - 350-450 gm Number and sex: 10 animals (5 female and 5 male)

42. OECD- 410 Housing --- individual housing --- temperature 220 c (±30c) --- relative humidity 30- 70% preferable 50 to 60 % --- lighting 12 hr light: 12 hr dark --- laboratory animal feed + ad libitum supply of drinking water Dose level --- At least three dose level with control and vehicle control if necessary

43. OECD- 410 Limit test: one dose level at least 1 gm/kg ,b.w, dermal. Application: Test drug apply not less than 10% of the body surface area uniformly for 6 hours for a day. Observation : Sign and toxicity ANS CNS Behavior pattern Food consumption weekly Weight variation weekly

44. OECD- 410 Clinical examination Haematology parameters: Haemocrit, Hb, RBC, WBC, DC, clotting time, prothrombin time, platelet count, MCH, MCHC etc. Clinical biochemistry: Liver and Kidney function test, Lipid profile, protein, albumin, Na, K, Cl, Ca, glucose etc. Pathology Gross necropsy Histopathology of organs showing gross changes

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