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Toxicity Test

Toxicity Test. Wongwiwat Tassaneeyakul Department of Toxicology Khon Kaen University. Objective of Learning. To understand the important role of toxicity test in drug/chemical development, Can describe the component of toxicity test commonly used,

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Toxicity Test

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  1. Toxicity Test Wongwiwat Tassaneeyakul Department of Toxicology Khon Kaen University 614 351

  2. Objective of Learning • To understand the important role of toxicity test in drug/chemical development, • Can describe the component of toxicity test commonly used, • Understand the relationship between these tests. 614 351

  3. ~100 Discovery Approaches Millions of Compounds Screened Preclinical Pharmacology Preclinical Safety 1 - 2 Products Clinical Pharmacology & Safety Discovery Exploratory Development Full Development Phase I Phase II Phase III 0 15 10 5 Idea Drug 11 - 15 Years High Risk Process: 11-15 Years, $800MM+ 614 351

  4. Frame work for Systems Toxicology Waters & Fostel, 2004 614 351

  5. Procedures on Living Animals : Great Britain 2000 • Total number of Procedures 2,714,726 • Total number of animals used 2,642,993 • Total number of toxicology procedures 454,904 (16.8%) • Total number of acute lethal tests in the rat (LD50/LC50) 2292 • Total number of Procedures in Dogs 7,632 (0.3%) • Total number of procedures in primates 3,690 (0.1%) 614 351

  6. Toxicity Tests • Acute toxicity test (single dose) • Subchronic toxicity test (repeated dose) • Chronic toxicity test (repeated dose) • Special test • 4.1 Mutagenicity test • 4.2 Development and reproductive test • 4.3 Carcinogenic test • 4.4 Neurotoxicity, • 4.5 etc…………………. 614 351

  7. Animals Toxicity Tests Assumption 1) Extrapolation from animal to human is possible 1.1 Appropriate dosage adjustment 1.2 Known human carcinogen are carcinogenic in (some) animals 1.3 Species differences mostly due to different metabolic pathways 2) High dose exposure is a necessary (to reduce a less number of animal used) 614 351

  8. Acute Toxicity Test • It involves lethal dose/concentrations and short-term exposures, • Appear immediately after exposure, • The end point is usually death, hence it is used to derive LD50 /LC50 • An LD50 is a dose of a toxic chemical that kills half of the population. • LD50 is obtained by plotting, for a given dose the proportion of the population that responded to that dose and all lower doses 614 351

  9. Acute Toxicity Tests OBJECTIVES • Estimate LD50 or LC50 for comparison • Identify target organ of intoxication to predict toxicity effect in human • Establish reversibility of toxicity • Calculate dose range guiding for further repeated-dose test COMPONENTS Acute lethality + Eye irritation + Skin test 614 351

  10. Acute Lethality Tests (LD50 test) METHOD Route: intended route (e.g. p.o. or parenteral) Species: 1 rodent + 1 non-rodent Dose : > 5 level Observed period:up to 14 days INDICATORS LD50 + 95% confidence interval Functional toxicity Histo/pathology, hematology, autopsy, etc. 614 351

  11. Alternative acute toxicity tests • Approximate lethal dose (ALD) “1.5” • Up and down method • The British Society of Toxicology (BST) protocol • 5, 50, 500 mg/kg then up and down adjust • 4. The “Fixed Dose” procedure • 5, 50, 500, 2000 mg/kg • very toxic, toxic, harmful, nontoxic 614 351

  12. Up and down method 614 351

  13. Aquatic toxicity testing Daphnia (water flea) “White rat of aquatic toxicity testing” Daphnia toxicity test 614 351

  14. Example problem Dose% alive 0 mg/L (control) 100 1 100 3 90 10 30 30 20 100 0 614 351

  15. Eye Irritation (Draize) Test METHOD Exception of test : pH <2 or >12 Route: eye Species: Rabbit (New Zealand White) Dose : 0.01- 0.1 ml or 100 mg Control :contralateral eye Measurement :cornea, iris, conjunctiva 614 351

  16. 614 351

  17. Skin Irritation Test 614 351

  18. Skin Sensitization Test 614 351

  19. Subchronic Toxicity Tests OBJECTIVES • Estimate NOAEL (No observed adverse effect level), MTD (maximal tolerable dose) • Identify target organ of intoxication after repeated dose exposure • Calculate dose range guiding for chronic toxicity test 614 351

  20. NOAEL , LOAEL, MTD MTD = highest dose that suppress <10% body wt gain in 90-days when compare to control group. 614 351

  21. Subchronic Toxicity Tests METHOD Route: intended route Species: 1 rodent + 1 non-rodent Dose : > 3 level + control high dose ………….. < 10% fatality ………… low dose ……………. No apparent toxicity Observed period:30-90 days 614 351

  22. Subchronic Toxicity Tests MEASUREMENT 1) Interim test : to establish baseline of appearance food consumption, body wt hematological/ biochemical test urinanalysi 2) Termination test : as interim + histopathological /autopsy 614 351

  23. Chronic Toxicity Tests • It involves Sub-lethal concentration and long-term exposure, • Effect could be anything (biochemical, physiological), but notdeath, • Chronic toxic response can last a long time or be permanent, • Under chronic conditions the organism survives but production or gene frequency could be affected, 614 351

  24. Chronic Toxicity Tests OBJECTIVES • Identify the spectrum of toxicity over a broad range of dose • Extrapolation of adverse effect to human • Prediction of safe level of exposure in human (Safety Factor, SF) 614 351

  25. Chronic Toxicity Tests METHOD Route: intended route Species: 1 rodent + 1 non-rodent Dose : > 3 level + control high dose ………….. MTD then 1/4, 1/8, ……….. Observed period:>90 days to 2 yrs 614 351

  26. Development and Reproductive Toxicity Test (DART) Objective: To evaluate potential toxicity on developmental animals and reproductive system. Study Type Multigenerational (2-3 G) study Three-segments single generation study 614 351

  27. Embryonic development and critical period for teratogenesis 614 351

  28. 614 351

  29. Phocomelia THALIDOMIDE 614 351

  30. Possible Targets in DART Reproductive system Key hormones Spermatogenesis/ Ovulation Mating function Sperm viability/ Fertilization Placentation Maternal behavior Developing offspring Implantation development Organ development & maturation Embryo / fetal growth Suckling behavior 614 351

  31. Indices used in DART 1. Fertility index = # females pregnant # females paired 2. Implantation index = # implantation sites # corpora lutea 3. Gestational (live birth) index = # lived pups born # pups delivered 4. Viability index = # pups surviving 4 days # lived pups at birth 5. Sex ratio = # male offspring X 100 # female offspring 614 351

  32. (A) Litter size after exposure to herbicide mixture; summary of groups (n = 275). Distribution of litter size in each dosing group: (B) control (n = 62); (C) very low dose (n = 31); (D) low dose (n = 58); (E) intermediate dose (n = 61); (F) high dose (n = 63). Interm, intermediate. *p < 0.05, ANOVA, Bonferroni. 614 351

  33. Multigenerational study F0 mating F1B mating F1A autopsy F2B mating F2A autopsy F3B F3A autopsy 614 351

  34. DNA DAMAGE DNA REPAIR MECHANISMS SHORT-TERM CONSEQUENCES Test of Genetic Damage PHYSIOLOGICAL DYSFUNCTION CELL DEATH ABNORMAL GROWTH & METABOLISM Decreased cellular proliferation Genomic instability Impaired protein/ gene expression Defective signalling pathways LONG-TERM CONSEQUENCES Cancer Disease Ageing 614 351

  35. The Ames/Salmonella Test “I know, let’s invent the Ames test” 614 351

  36. Prototrophic Bacteria Cell (His+/+) Energy Source 1 3 5 7 A B C D E F G Histidine+ Other amino acids Protein synthesis 2 4 6 Cell Growth Waste Product Auxotrophic Bacteria Cell (His-/-) Energy Source 1 3 5 7 A B C D No histidine+ Other amino acids Protein synthesis decrease 2 4 6 No Cell Growth Waste Product Mutation in enzyme 4 614 351

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  39. http://faculty.washington.edu/jclara/301/M301lecOut/MuRepair.htmlhttp://faculty.washington.edu/jclara/301/M301lecOut/MuRepair.html 614 351

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