OECD Guideline 420: Acute oral Toxicity - Fixed Dose Procedure

Acute Oral Toxicity - Fixed Dose Procedure As per OECD Guideline 420

1. ACUTE TOXICITY STUDIES AS PER OECD GUIDELINES Prepared by :- Khushboo Thakur m.Pharm sem II Department of pharamacology Ssr college of pharmacy, sayli, silvassa.

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 and/or unintended effects of drug Predictable (type A reactions) 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 result of excessive pharmacological effect of drug due to over dosage or prolonged use

4. 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 yrs. Special toxicity e.g. Carcinogenicity

5. ACUTE ORAL TOXICITY TEST GUIDELINE 401: ACUTE ORAL TOXICITY – CONVENTIONAL ACUTE TOXICITY 420: ACUTE ORAL TOXICITY- FIXED DOSE PROCEDURE 423: ACUTE ORAL TOXICITY – ACUTE TOXIC CLASS METHOD 425: ACUTE ORAL TOXICITY – UP-AND-DOWN PROCEDURE

6. OECD GUIDELINES FOR ACUTE ORAL TOXICITY NUMBER TITLE ORIGINAL ADOPTION MOST RECENTLY UPDATED 401 Acute oral toxicity- Conventional acute toxicity test 12 May, 1981 Date of deletion: 17 December 2002 420 Acute oral toxicity- fixed dose procedure 17 July, 1992 8 February, 2002 423 Acute oral toxicity- acute toxic class method 22 March, 1996 8 February, 2002 425 Acute oral toxicity- up and down procedure 21 September, 1998 16 October, 2008

7. 420: Acute Oral Toxicity- Fixed Dose Procedure INTRODUCTION The original Guideline 420 was adopted in July 1992. It is the first alternative to the conventional acute toxicity test, described in T est G uideline 401. Revision was considered timely – differ in harmonised LD50 Cut-off values Testing in one sex (usually females) is now considered sufficient

8. Traditional methods for assessing acute toxicity use death of animals as an endpoint. In 1984, a new approach to acute toxicity testing was suggested by the British Toxicology Society - Approach avoided using death of animals as an endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose levels. The statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models in a series of studies.

9. Found way which- in vivo and modelling studies have demonstrated Uses fewer animals Causes less suffering Is reproducible Able to rank substance in a similar manner

10. INITIAL CONSIDERATIONS Use of only moderately toxic doses. Doses that are known to cause marked pain and distress, due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed (considered in the interpretation).

11. The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the following: identity and Chemical structure of test substance, Its physical and chemical properties, Results of any other in vivo or in vitro toxicity test on the substance, Toxicological data on structurally related substances or similar mixtures, Anticipated use of the substance. Helps to rank and classify acc. t o “Globally Harmonised System” (GHS) .

12. PRINCIPLE OF TEST Groups of animals of a single sex are dosed in stepwise procedure using the fixed dose 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. Clinical signs and conditions associated with pain, suffering, and impending death.

13. Groups of animals may be dosed at higher or lower fixed doses, It depend on the presence or absence of signs of the toxicity or mortality. 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 death occur at lowest dose.

14. DESCRIPTION OF THE METHOD Selection of animal species Mostly rat is preferred as rodent species. Normally females are used. For males – adequate justification should be provided. Healthy young adult animals of commonly used laboratory strains should be employed. Female should be nulliparous and non–pregnant. At the commencement of i ts dosing, should be between 8 to 12 weeks old, and weight should fall in an interval within ±20% of the mean weight.

15. Housing and feeding conditions Temperature = 22°C ± 3°C Relative Humidity = 50-60 % Light = Artificial (cycle of 12 hrs. light, 12 hrs. dark) Feeding = conventional lab. diets with unlimited drinking water supply Animal grouped = Group caging but no. of animals per cage must not interfere with clear observations of each animal.

16. Preparation of animals Randomly selected Marked to permit individual identification, and Kept in their cages for at least 5 days prior to start of dosing allow for acclimatization.

17. Preparation of doses Test substances should be administered in a constant volume over the range of doses. In case of a liquid end product or mixture is to be tested – use undiluted test substance i.e., at a constant concentration In either case, the maximum dose volume for administration must not exceeded In rodents maximum volume 1 ml/100 gm of body wt. For Aqueous solution – 2 ml/100gm of body wt.

18. Order of administration – Liquid Suspension Emulsion Vehicle other than water – toxicological characteristics should be known Preparation prior to administration

19. PROCEDURE Administration of doses Test substance administered by gavage using a stomach tube or a suitable intubation canula. Fasted overnight prior to dosing Weigh animal and administer the test substance Withheld food for further 3-4 hrs. in rats or 1-2 hrs. in mice

20. Sighting study Purpose of the sighting study is to allow selection of the appropriate starting dose for the main study. Test substance is administered to single animals in a sequential manner following the flow charts. The starting dose for the sighting study is selected from the fixed dose levels of 5, 50, 300, and 2000 mg/kg as a dose expected to produce evident toxicity.

21. Also, evidence from in vivo and in vitro data from the same chemical and from structurally related chemicals. In absence of such information, the starting dose will be 300 mg/kg. At least 24 hrs. will be allow b/w the dosing of each animal. All animals should be observed for at least 14 days.

22. In the case, where an animal tested at the lowest fixed dose level (5 mg/kg) in the sighting study dies, The normal procedure is to terminate the study and assign the substance to GHS category I. For further confirmation use supplementary procedure.

23. MAIN STUDY Numbers of animals and dose levels Select dose from sighting study. Perform study on a total of five animals of one sex at each level including animals tested in sighting study. Time interval b/w dosing at each level determined by– Onset, duration and severity of toxic signs Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals.

24. To observe delayed toxicity – A period of 3 or 4 days b/w dosing at each dose level is recommended. In case of inconclusive response – time interval may be adjusted as appropriate. Fixed dose of 5000 mg/kg – procedure outlined in Annex 4.

25. Limit Test Performed when information indicating that the test material is likely to be nontoxic. i.e., having toxicity only above regulatory limit doses Information about the toxicity of the test material gained from knowledge about similar tested compound. Using the normal procedure test will be done.

26. OBSERVATIONS At least once during the first 30 min. Periodically during the first 24 hrs. with special attention given during first 4 hrs. And daily thereafter for a total of 14 days. However, duration of the observation should not be fixed rigidly. Times at which signs of toxicity appear or disappear are important, especially if there is a tendency for toxic signs to be delayed.

27. Observation should include Change in skin and fur, eyes, and mucous membranes, and Respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to observation: Tremors, convulsions, salivation, diarrhoea, lethargy, sleep, and coma.

28. Body weight Shortly before administration Thereafter weekly At the end

29. Pathology All animals subjected to gross necropsy. Including those that die during the test or are removed from study

30. DATA AND REPORTING Data Should include individual animal. All data summarised in tabular form, showing For each test group the number of animals used. The no. of animals displaying signs of toxicity. The no. of animals found dead during the test or killed for humane reasons. Time of death of individual animals. A description and the time course of toxic effects and reversibility, and Necropsy finding.

31. Test report Test substance : Physical nature, purity, and where relevant, physic-chemical properties (including isomerisation), identification data, including CAS No. Vehicle (if appropriate): Justification for choice of vehicle, if other than water

32. Test animals: Species/strain used, Microbiological status of the animals, when known, No., age, and sex of animals (including, where appropriate, a rationale for males instead of females), Source, housing conditions, diet, etc. Test report

33. Test conditions: Details of test substance formulation, including details of the physical form of the material administered, Detailed of the administration of the test substance including dosing volumes and time of dosing, Details of food and water quality (including diet type/source, water source), The rationale for the selection of the starting dose. Test report

34. Test report Results: Tabulation of response data and dose level for each animal (i.e., animals showing signs of toxicity including mortality, nature, severity and duration of effects), Tabulation of body weight and body weight change, Individual weights of animals a t the day of dosing, in weekly intervals thereafter, and at time of death or sacrifice, Date and time of death if prior to scheduled sacrifice, Time course of onset of signs of toxicity and whether these were reversible for each animal, Necropsy finding and histopathological findings for each animal, if available

35. Test report Discussion and interpretation of results conclusions

36. EXCEPTIONAL TESTING (Dose >2000 mg/kg) If reliable evidence is already available that indicates the LD50 to be in the range of category 5 values. Other animal studies or toxic effects in humans indicate a concern for human health of an acute nature. Based on mortality incidences while testing schemes of Annex 3. Assignment to a more hazardous category is not warranted.

37. Reliable information is available indicating significant toxic effects in humans, Any mortality is observed when tested up to category 4 values by oral route Where expert judgement confirms significant clinical signs of toxicity, when tested up to category 4 values, except for diarrhoea, piloerection or an ungroomed appearance , Where expert judgement confirms reliable information indicating the potential for significant acute effects from the other animal studies.

38. Testing at dose above 2000 mg/kg Sighting study Annex 2 are extended to include a 5000 mg/kg dose level 5000 mg/kg A B C Second animal to be tested at 2000 mg/kg Selection of 5000 mg/kg as the main study starting dose Selection of 5000 mg/kg as the main study starting dose

39. Main study Sequential procedure presented in annex 3 are extended to include a 5000mg/kg dose level Main study with 5000 mg/kg A (≥ 2 deaths) B (evident toxicity and/or ≤ 1 death ) C (no toxicity) Testing of a 2 nd group at 2000 mg/kg Substance being unclassified acc. to GHS Substance being unclassified acc. to GHS