animal toxicity studies.pptx

animal toxicity tests are useful in finding the right dose for drugs to be used in humans. this ppt contains the method of toxicity studies and different types of toxicity studies.

1. Animal toxicity studies (= NoN clinical toxicity studies)

2. Overview Introduction Requirements Classification of toxicity studies Methodology Conclusion

3. OBJECTIVES Identify toxic substances prior to clinical use Cumulative toxicity study Allow careful selection of doses Dose identification like MLD, lethal dose, MTD, etc Therapeutic index

4. REQUIREMENTS Follow Good Laborotary Practice (GLP) Trained and qualified staff. Instruments- properly calibrated and standardized. SOPs should be followed All documents and tissues preserved for 5 years.

5. Classification of Toxicity studies Systemic toxicity studies Male fertility studies Female reproduction and developmental studies Hypersensitivity studies Genotoxicity studies Carcinogenicity studies Local toxicity studies

6. Systemic toxicity studies Single dose toxicity studies Repeated – dose toxicity

7. Control Group 1 Group 2 Group 3 Group 4 Vehicle 1x 2x 3x 4x 5 5 5 5 5 ( atleast ) Observe for 14 days (signs/mode of death) Calculate LD50, MTD 1. Single dose toxicity studies

8. Calculation of LD50 Graphical method Arithmetic method

9. Example – Graphical method (Miller and Tainter ) Group Dose(mg/kg) Log dose Dead Dead % Probit score 1 64 1.81 0/10 3.04 2 71 1.85 2/10 20 4.16 3 81 1.91 4/10 40 4.75 4 90 1.95 9/10 90 6.28 5 100 2.00 10/10 100 6.96

10. Example – Arithmetic method ( karber’s method) Group Dose(mg/kg) Dose difference (a) Dead Mean mortality (b) Product ( a*b) 1 64 - 0/10 (A) - - 2 71 7 2/10 (B) 1 = A+B/2 7 3 81 10 4/10 (C) 3 = B+C/2 30 4 90 9 9/10 (D) 6.5 = C+D/2 58.5 5 100 10 10/10 (E) 9.5 =D+E/2 95 Total= 190.5 LD50= 100-(190.5/10)= 81mg/kg

11. 2. Repeated-dose toxicity studies Number of animals required 14-28 days 84-182 days Group Rodent (each sex) Non-rodent (each sex) Rodent (each sex) Non-rodent (each sex ) Control 6-10 2-3 15-30 4-6 Low dose 6-10 2-3 15-30 4-6 Intermediate dose 6-10 2-3 15-30 4-6 High dose 6-10 2-3 15-30 4-6

12. Control Group 1 Group 2 Group 3 Vehicle 1x 2x 3x Test substance daily for 14-28/90/180 days Monitor for mortality, clinical signs, body weights.

13. 3. Male fertility study Group GROUP MALE RATS 1 CONTROL 6 2 LOW DOSE 6 3 INTERMIGIATE DOSE 6 4 HIGH DOSE 6

14. Drug given for 28-70 days Paired with females of proven fertility (1:2) 10 days or detection on vaginal plug Fertility index after 13 days of gestation Sacrifice males ( weight of testis/ epididymis/ sperms examined)

15. Female reproduction and developmental studies 1. Segment 1: Female Fertility Studies GROUP MALE & FEMALE RATS CONTROL 15 + 15 LOW DOSE 15 + 15 INTERMIGIATE DOSE 15 + 15 HIGH DOSE 15 + 15

16. Drug given to both males and females (28 days in males and 14 days in females) before mating Continue during mating and gestation period Dams shoud be allowed to litter Continue drug till weaning Observe dams for pups for -body weight, -general signs -food intake, -sex-wise distribution -mating behaviour , -growth parameters -gestation and parturition period -survival

17. 2. Seg- II Teratogenecity study GROUP PREGNANT RATS PREGNANT RABBITS CONTROL 20 12 LOW DOSE 20 12 INTERMIGIATE DOSE 20 12 HIGH DOSE 20 12

18. Drug should be administered throughout the period of organogenesis (GD 6-17 ) All foetus for gross examination One half - skeletal abnormality Secon d half- visceral abnormality Observe ( for dams) for foetus -Effect on body weight -gender -Food intake -body length -Examine uterus, ovaries, uterine contents -weight -Implantation site -visceral/skeletal abnormality

19. 3. Segment- III (perinatal study) GROUP DAMS CONTROL 15 LOW DOSE 15 INTERMIGIATE DOSE 15 HIGH DOSE 15

20. Drug given throughout the last trimester (from day 15 of gestation) continued throughout lactation and weaning. Sacrifice dams and examine F1- 1 male, 1 female selected at weaning. Drug given throughout period of growth to sexual maturity, pairing, gestation, parturition, lactation F2 – monitor till weaning

21. LOCAL TOXICITY STUDIES

22. 1.Dermal Toxicity study Study should be done in rabbit and rat Test material- applied on 10% surface area Three formulations of varius concentrations applied ( several fold higher than clinical) Duration- 7 to 90 days

23. 2. Dermal photo-toxicity study GROUP GUINEA PIG CONTROL 5 TEST 10 Test is called Armstrong/ Harber Pretest is done in 8 animals P atch application for 2hrs with and without UV exposure. 24hr and 48 hr recordings highest non-irritant dose is ascertained. Main test D one with dose selected from pretest . 0.3 ml/ patch for 2 hrs, followed by 10 J/cm sqr of UV exposure. Test should repeated on day 0,2,4,7,9 and 11, challenge on day 20 & 24

24. Other local studies Preferred animal Number Duration Vaginal toxicity studies Rabbit/dog 6-10/group Min. 7 days Rectal tolerance test Rabbit/dogs 6-10/group Min 7 days Max 30 days Ocular toxicity studies Albino rabbit At least 2 species Max 90 days Inhalational toxicity studies One rodent One non-rodent Not mentioned Max 6 hrs. /day 5 days/week Other local toxicity studies

25. 3. Genotoxicity studies In vitro test Chromosomal damage with mammalian cells In- vivo test Rodent hematopoetic cells Test for measurements of DNA adducts DNA strand break Test for gene mutation in bacteria

26. Ames test Earliest , most widely used. Uses bacteria – salmonella typhimurium strains, E.coli Result Micronucleus assay An increase in number of micronucleated cells in bone marrow biopsy– chromosomal damage . Others Mouse lymphoma assay Comet assay Sister chromatid exchange

27. 4. Carcinogenicity studies Ideal for - For drugs to be used for more than 6 months Drugs for intermittent use in chronic condition Concern about carcinogenicity Rodent (preferable rat). 3 dose levels Highest- sub-lethal Lowest- human intented dose 7 days /week for 24 months

28. Animal Toxicity requirements for CT Systemic Toxicity Studies Route of administration Duration of proposed human administration Phase of CT For which study is proposed Long term toxicity requirements Oral or parenteral or Transdermal Single dose or several doses in one day, Up to 1wk > 1wk but up to 2wk >2wk but up to 4wk Over 1 month I, II, III I, II, III I, II, III I, II, III 2sp,2wk 2sp;4wk 2sp;12wk 2sp;24wk Inhalational (anaesthetics and aerosols) Up to 2wk Up to 4wk >14wk I, II, III I, II, III I, II, III 2sp;1mo (exp 3h/d,5d/wk) 2sp;12wk (exp 6h/d,5d/wk 2sp;24wk,(exp 6h/d,5d/wk

29. Local toxicity study Route of administration Duration of proposed human administration Phase of CT For which study is proposed Long term toxicity requirements Dermal Ocular / Otic /nasal Up to 2 wk > 2 wk Up to 2wk > 2 wk I,II III I, II, III I, II I II I, II, III 1sp;4wk 2sp;4wk 2sp;12wk 1sp;4wk 2sp;4wk 2sp;12wk Vaginal or Rectal Up to 2wk >2wk I, II III I, II, III 1sp;4wk 2sp;4wk 2sp;12wk

30. Special toxicity study MALE FERTILITY FEMALE REPRODUCTION & DEVOLOPMENTAL ALLERGINISITY DERMAL PHOTO TOXICITY GENOTOXICITY CARCINOGENECITY Phase I, II, III in male volunteers Seg-II in 2sp,phase II, III involving female patients of child bearing age Seg-I ;phase III involving female patients of child bearing age Seg-III ; phase III for drugs to be given to pregnant or nursing mothers for long periods Phase I, II, III, when there is a cause of concern or for parenteral drugs Phase I, II, III -if the drug or a metabolite is related to an agent causing photosesitivity In-vitro studies- phase I Both in vitro & in vivo- phase II, III Phase III When there is a cause for concern, or when the drug is to be used for more than 6 months