Oral toxicity

acute , sub-acute and chronic oral toxicity study according to oecd guidelines

1. KARNATAKA COLLEGE OF PHRMACY ( DEPARTMENT OF PHARMACOLOGY) PHARMACOLOGICAL AND TOXICOLOGICAL SCREENING METHODS PRESENTATION TOPIC : Oral toxicity SUBMITTED TO : Dr NAGARATHNA P.K.M PRESENTED BY: DIANA MORIA

2. 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. ADVERSE DRUG EFFECTS Any undesirable &/ or unintended effects of drug, 1. Predictable (type A reactions) 2 . Non-predictable (type B reactions)

4. WHAT IS LD50? LD50 represents the individual dose required to kill 50% 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.

5. LC50 The concentrations of the chemical in air that kills 50% of the test animals during the observation period is the LC50 value. Other durations of exposure (versus the traditional 4 hours) may apply depending on specific laws

6. LD50/LC50

7. METHODS TO DETERMINE LD50 Karber’s method Miller and Tainter method Lorke’s method Fixed dose method UP and down method

8. ANIMAL TOXICITY TESTS Acute toxicity 14 days Sub-acute (repeated doses) toxicity 28 days Sub-chronic toxicity 3 months Chronic toxicity 6 months to 2yrs Special toxicity e.g. Carcinogenicity

9. ACUTE ORAL TOXICITY

10. Initial consideration:- Test substances :- at doses that are known to cause marked pain and distress due to corrosive or severely irritant actions, need not be administered. Moribund animals shall be humanely killed .

11. Principle of the test It is the principle of the test that, based on a stepwise procedure with the use of a minimum number of animals per step(3) sufficient information is obtained on the acute toxicity of the test substance . .

12. Description of the method Selection of animal species:- The preferred rodent species is the rat, although other rodent species may be used. Normally females are used . should be nulliparous (8-12weeks) wt _/+20 Male can also be use if they shows better sensitivity to the TS.

13. Housing and feeding conditions

14. Preparation of animals The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least 5 days prior to dosing to allow for acclimatisation to the LC.

15. Preparation of dose TS:- should be administered in a constant volume . Liquid sample should be use in undiluted form at constant conc. Other vehicles can also be use s/no dose Dosage form 1 1ml/100gBW liquid 2 2ml/100gBW Aqueous solution

16. Administration of dose TS is given in a single dose using dose by gavage using stomach tube . Its given in a smaller fraction over a period not exceeding 24hours Animal should fast prior to dosing

17. Number of animals and dose level s 3 animals are use for each step Dose level to be used as the starting dose is selected from one of the above 4 fixed levels 5,50.300,2000mg/kg If there is no information about the compound it is recommended to start using 300mg/kg

18. Limit test The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be nontoxic. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds, taking into consideration the identity and % of components known to be of toxicological significance

19. OBSERVATIONS Observe the animal individually at for 30mins for period of 24hrs Special attention at the 1 st 4hrs daily for 14days Time in which toxicity appears or disappears is important and recovery period.

20. OBSERVATIONS

21. DATA AND REPORTING Data : all data should be summarize in a tabular form containg the following information Time course of death No of animal used No of animals found death No of animals killed for human reason Time of death

22. report Should contain the follow Test substance Vehicle Test animal Test condition

23. Result Tabulation of response data and dose level for each animal are noted . BW , time of sacrifice and time and date of death. Histopathology and necropsy finding .

24. 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.

25. 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.

26. ACUTE ORAL TOXICITY GUIDLINE NO. 420 (FIXED DOSE PROCEDURE ) As 1st alternative to conventional acute toxicity test. Testing in 1 sex usually females is considered sufficient. Uses fewer animals. Causes less suffering to the animals. Uses only moderately toxic doses, doses expected to be lethal should be avoided

27. Initial consideration Principle of test substance Description of method Housing and feeding Preparation of animal Preparation of dose Dose administration

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

29. Sighting study Purpose : to allow selection of appropriate starting dose For the main study. Single dose is administered in a sequential manner. Sighting study is completed when decision on starting dose is done

30. Main study No of animal and dose level Usually 5 animals are use Limit test Observation same report

31. ACUTE ORAL TOXICITY GUIDLINE NO.425 (UP AND DOWN METHOD) Up and down testing approach was 1st described by Dixon and Mood. Bruce in 1985 proposed to use it for acute toxicity determination of chemicals . Estimates confidence intervals for LD50

32. initial consideration In procedure (main test ) 1-animal dosed at a time, at minimum of 48hrs 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 1st dose a step below the level of the best estimate of LD50 .

33. PRINCIPLE of the main test consist of a single ordered dose progression in which animal are dose and kept for minimum of 48hrs First animals receive dose step below level of best estimate of LD50 if animal survives next dose is increase by factor of 3.2times original dose And decreases if animal dies.

34. Description of method Limit test for 2000 Limit test for 4000 Limit test for main test

35. Calculation of LD50 for main test Its calculate using likelihood method , following dixion .assume as death or delay death L=L1L2………Ln Li=1-f( Zi ) if animal survives Li =f( zi ) if animal dies Where Zi = ⟮log (di)- µ ] ̸ 𝜕 di]  

36. Computation of confidence interval After main test and estimation of LD50 its possible to compute the interval estimates for LD50. The wide confidences interval should be narrow If main test is repeated should be close to the original value Test report

37. COMPARISON OF GUIDELINES OF LD50

38. Hyposalivation / Xerostomia Xerostomia , also known as dry mouth and dry mouth syndrome, is dryness in the mouth, which may be associated with a change in the composition of saliva, or reduced salivary flow, or have no identifiable cause

39. Lichenoid Reaction/Lichen Planus It is an immune mediated process where Tcells mediate the destruction of the basal cells of the epithelium, Its while associated with erythema and ulcer Often occurs in buccal mucosa, tongue and gingiva.

40. Aphthous -like and Non– Aphthous -like Ulcers :-idiopathic aphthous ulcersusually begins in the 1 st two decades of life,appears as ovoid ulcers usually 1cm/ lesswith yellowish fibrinous member.

41. Bullous Disorders:- Its rare in oral cavity ,usually occurs on skin. Occurs due to autoimmunity Antipsychotics .spironolactone and sulphonamide others

42. Thank You