Acute and chronic toxicity studies in animals

Acute and Chronic Toxicity Studies in Animals

1. ACUTE AND CHRONIC TOXICITY STUDIES IN ANIMALS Dr.Swaroopa MD Pharmacology, 1 st year PG, Rangaraya Medical College. 1

2. 2 Introduction Objectives of toxicity studies General principles Types Systemic toxicity studies Lethality testing and calculation Conclusion References CONTENTS

3. 3 Father of Toxicology – Paracelsus Father of Modern Toxicology – Mathieu Orfila All substances are poisons; there is none which is not a poison. The right dose differentiate a poison and a remedy.

4. 4 TOXICITY STUDIES --- INTRODUCTION Casarett 1996 defined toxicology as a science that defines the limits of safety of chemical agents for human and animal populations. Toxicity studies/screening ( hyperpharmacology ) is very important for the development of new drugs & for the extension of the therapeutic potential of existing molecules apart from pharmacodynamics and pharmacokinetic studies. During toxicity studies, always two or more species are preferred as there is species to species variation in the pharmacological responses.

5. 5 Before conducting any toxicological studies in animals or collecting tissue/cell lines from animals, the study should be approved by the Institutional Animal Ethics Committee or CPCSEA ( Committee for the Purpose of Control and Supervision of Experiments on Animals. BIOMEDICAL ETHICS

6. 6 OBJECTIVES OF THE TOXICITY STUDY To identify any toxic substance prior to clinical use. Qualitative and Quantitative assessment of drug use. Mostly used to examine specific adverse events or specific end points such as cancer, cardiotoxicity, skin/eye irritation. Different types of dose identification can be done. (MLD, LD 50, MTD, ED 50, NOAEL) Prediction of therapeutic index (LD 50 / ED 50 ) Benefit-risk ratio can be calculated. To relate the toxicological findings to clinical safety. To support in selecting species, treatment regimen and designing subsequent non clinical toxicity studies.

7. 7 GENERAL PRINCIPLES In a large set-up, conducting toxicity studies should comply the following important requirements: Should follow the Good Laboratory Practices(GLP). Studies should be performed by suitably trained and qualified staff. Instruments should be calibrated and standardized properly and of adequate capacity. Standard operating procedures(SOPs)should be followed. Documentation must be proper and anticipate the results. All documents, histology slides and paraffin tissue blocks should be preserved for a minimum of 5years after marketing of the drug.

8. 8 TYPES OF TOXICITY STUDIES: Systemic Toxicity studies Male fertility studies. Female Reproduction and Developmental Toxicity Studies. Teratogenicity studies. Perinatal studies. Local toxicity studies Geno toxicity studies. Carcinogenicity studies. Single dose toxicity study(Acute toxicity studies) Repeated-dose toxicity study(subacute/chronic toxicity study).

9. 9 SYSTEMIC TOXICITY STUDIES in animals

10. 10 ACUTE TOXICITY STUDY Acute toxicity/single dose study – study of the effect of a single dose on a particular animal species. Testing carried out in 2 different animal species (one rodent and one non rodent) Route of administration : as intended for humans. Doses :administered at different dose levels, at least three-graded doses. Given in a single bolus or by infusion within 24 hrs. Animals should be observed for 14 days. observations:- 1. signs of intoxication 2. effect on body weight 3. gross pathological changes 4. histopathology of grossly affected organs(if any) All mortalities caused during experimental period are recorded and morphological, biochemical, pathological and histological changes in the dead animals are recorded.

11. 11 Acute toxicity studies/ single dose studies in animals are essential for any pharmaceutical product intended for human use. Information obtained from these studies is useful in choosing doses for repeated dose studies Providing preliminary identification of target organs of toxicity, These studies may also aid in the selection of starting doses for phase 1 human studies Provide information relevant to acute overdosing in humans. Death is considered as a single end point and other behavioural parameters are not taken under consideration. LD 50 is calculated (dose required to kill 50% of the population)

12. 12 Lethality testing and calculation :- This is an essential regulatory requirement for a drug before clinical study. Lethal dose – “dose of a given drug which produces mortality in the treated animal, especially in most sensitive species model.” LD 50 is defined as “dose of a given drug which produces mortality in 50% of total treated animals preferably in the most sensitive species model.” This is preferred over sub chronic or chronic testing. Lethality testing is done mainly on 3doses of selection Toxicity is varied according to route of administration. There is a rule of thumb, i.e. LD 50 is roughly 30%of oral, I.V is 10% of that of the oral. Duration of testing ranges from 7 days to 14 days. One dose which has 100% lethality Second –produces marginal lethality Third is taken in between of first 2 doses.

13. 13 There are 2 methods of calculating LD 50 Mathematical method Graphical method Mathematical method :- K arber’s method is very commonly used because it is simple and does not need to plot the dose response curve. CALCULATION:

14. 14 1 Exp grpc Dose(mg/kg) Dose diff(DD) No of animals(n) No of dead animals Mean mortality DD X MM 1 3 - 10 0 (A) -- -- 2 5 2 10 2 (B) 1 2 3 10 5 10 3 2.5 12.5 4 12 2 10 5 4 8 5 15 3 10 7 6.5 19.5 6 20 5 10 10 8.5 42.5 84.5 Σ DD MM Mean mortality= (A+B)/2 =(0+2)/2 =1 Σ (DD MM) = 84.5;then value is divided by the no. of animals in the group (here n =10), hence, value obtained is 84.5/10 = 8.45 Finally, this value is subtracted from the minimum dose which produces the 100 % mortality, i.e. 20mg/kg So, LD 50 = 20 – 8.45 =11.55 mg/kg

15. 15 GRAPHICAL METHOD :- This method well described by the Miller and Tainter (1944) as well as Litchfield and Wilcoxon (1949). Depends on toxic log dose response curve and interpretation of the result is done by the curve. First convert the percentage response into the probit , then plot the curve log dose on x- axis and y-axis contains probit scale. The LD 50 is the antilog (1d) value which falls on x-axis.

16. 16 REPEATED DOSE TOXICITY STUDIES :- Subacute/ subchronic or chronic toxicity study falls under these category. SUBACUTE/SUB CHRONIC STUDY : Aim is to identify target organ toxicity and establishment of MTD for subsequent studies. Atleast 2 mammalian species are used (1 rodent and 1 non rodent) Rodent – 6 to 8 weeks of age, non rodent 4 to 9 months age. Treatment & duration : 14,28,90 or 180 days (depends on therapeutic indication) Route of administration : same as intended for humans. Doses : At least 3 graded doses. Group 1 --- Control group – normal or vehicle group. Group 2 --- Treatment group – drug treatment group.

17. 17 CHRONIC TOXICITY STUDIES :- Repeated dose study from 6 months to 12 months or even upto 2 years . Primary goal of these studies is to characterize the toxicological profile of the test compound following repeated administration. this includes identification of potential target organs of toxicity and exposure/response relationships, & may include potential reversibility of toxic effects. This information should be part of the safety assessment to support the conduct of human clinical trials and the approval of a marketing authorization.

18. 18 CHRONIC TOXICITY STUDIES : are conducted with a minimum of one rodent and one non rodent species. In rodents, studies are usually for 6 months to 2 years In rodents, studies are usually for 1year . Dose selection :- Highest dose – should produce observable toxicity. Intermediate dose – should cause some symptoms but not gross toxicity or death. Should be placed logarithmically between the highest and lowest doses. Lowest dose – should not cause observable toxicity. Observation Site of injection should be subjected to gross and microscopic examination. Non rodent species : Electrocardiogram and fundus examination. Other parameters include changes in body weight, food/water intake and …

19. 19 1 Liver function tests : total bilirubin, direct or indirect bilirubin, bile acids, ammonia. 2 Hepatocellular enzymes : AST, ALT, SDH, LDH 3 Muscle parameters : Creatinine kinase (CK), AST, ALT, LDH 4 Pancreatic parameters : Amylase, lipase 5 Lipid parameters : cholesterol, triglycerides cholestatic enzymes – AP, GGT 6 Calcium, potassium : often influenced by kidney function, kidney parameters should be evaluated concurrently. 7 Kidney parameters : urea nitrogen, creatinine urine specific gravity should be evaluated concurrently when evaluating renal function. Analytical parameters which should be assessed after subacute/sub chronic/chronic toxicity studies :

20. 20 CONCLUSION Drug discovery and development of a new drug undergoes numerous procedures in order to get the safe and effective drug. The principle of the toxicity study is to assess all the relative toxic effects of the drug regarding its dose, duration and its effect on the organs. These studies not only provide adequate therapeutic margin of initial dose levels and duration of dosing , but also give guidance for any special measures to be taken in initial clinical trials.

21. 21 SK Gupta, New Drug Development, Drug Discovery and Clinical Research,1st edition, pg : 18 to 21. Bikash Medhi , Ajay Prakash, Practical Manual of Experimental and Clinical Pharmacology, 2 nd edition, pg 136 to 140. REFERENCES

22. 22