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The nonclinical safety study recommendations for the marketing approval
of a pharmaceutical usually include single and repeated dose toxicity
studies, reproduction toxicity studies, genotoxicity studies, local tolerance
studies, and for drugs that have special cause for concern or are intended
for a long duration of use, an assessment of carcinogenic potential. Other
nonclinical studies include pharmacology studies for safety assessment
(safety pharmacology) and pharmacokinetic (absorption, distribution,
metabolism, and excretion (ADME)) studies. These types of studies and
their relation to the conduct of human clinical trials are presented in this

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  1. 1. Presented by Dr. Rahul G. Kadam Ph.D Scholar Roll NO. P1661
  2. 2.  Toxicology is a branch of science that deals with toxins and poisons and their effects and treatment.  Toxicological screening is very important for the development of new drugs and for the extension of the therapeutic potential of existing molecules.  The US-FDA states that it is essential to screen new molecules for pharmacological activity and toxicity potential in animals (21CFR Part 314).  Toxicity tests are mostly used to examine specific adverse events or specific end points such as cancer, cardiotoxicity, and skin/eye irritation.  Toxicity testing also helps calculate the No Observed Adverse Effect Level (NOAEL) dose and is helpful for clinical trails.
  3. 3.  Paracelsus (Father of Toxicology): determined specific chemicals responsible for the toxicity of plants and animals (dose-response relationship).  "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy” --Paracelsus  Mathieu Orfila, determined the relationship between poisons and their biological He is referred to as the father of modern toxicology. Paracelsus (1493-1541) Recent developments: after 1920 (introduced determine LD50
  4. 4. Benefit –risk ratio can be calculated Prediction of therapeutic index Therapeutic index= Maximum tolerated dose Minimum curative dose Smaller ratio, better safety of the drug
  5. 5.  Pharmacological effects are same in man as in animals  Toxic effect in species will predict adverse effects in man  Giving high doses in animals improves predictability to man  Risk assessment can be made by comparison of toxic doses in test species with predicted therapeutic dose in man
  6. 6. PHASES OF DRUG DEVELOPMENT (ANIMAL MAN) PHASE III PHASE IVPHASE I PHASE IPHASE I PRECLINICALPRECLINICAL PHASE II Product Approval (NDA/MAA) Patient studies Entry to man (IND / CTA) NoneNone Healthy subjects Safety and tolerability Healthy subjects Safety and tolerability Genetic toxicity (in vivo) Repeat dose toxicity testing + Bioanalysis / Toxicokinetics Drug Metabolism Reproductive Toxicity Testing (teratogenicity) Genetic toxicity (in vivo) Repeat dose toxicity testing + Bioanalysis / Toxicokinetics Drug Metabolism Reproductive Toxicity Testing (teratogenicity) Patients Small scale efficacy studies Patients Small scale efficacy studies Patients Large scale multicentre studies Patients Large scale multicentre studies Chronic (long term) toxicity testing + Bioanalysis / Toxicokinetics Reproductive Toxicity Testing (fertility and pre/post natal) Carcinogenicity studies Drug Metabolism Chronic (long term) toxicity testing + Bioanalysis / Toxicokinetics Reproductive Toxicity Testing (fertility and pre/post natal) Carcinogenicity studies Drug Metabolism Patients Large scale post-marketing studies Patients Large scale post-marketing studies As required As required Genetic toxicity (in vitro) Single / repeat dose toxicity studies + Bioanalysis / Toxicokinetics Safety Pharmacology Drug Metabolism Lead candidate ClinicalNon-clinical MOLECULE
  7. 7. Studies should comply with GLP Performed by trained and qualified staff Use of standardized and calibrated equipment SOP’s followed in laboratory tasks All documents should be preserved for minimum 5 years after marketing of the drug
  8. 8.  OECD Guideline  EPA Guideline  FDA Guideline  GAITONDE Guideline
  9. 9. TOXICOKINETIC STUDIES Generation of Pharmacokinetic data to access systemic exposure achieved in animals Relation to dose level and the time course of toxicity study To support choice of species & Treatment regimen Design on clinical studies accordingly
  10. 10.  Pharmacodynamic responses  Pharmacokinetic profile  Species, sex, age of experimental animals  Susceptibility, sensitivity and reproducibility of test system  In vitro: Isolated organs, tissues cell-cultures  Mechanism of effect in vivo
  11. 11. Systemic toxicology studies Single dose studies Repeated dose studies Reproductive toxicology studies Male fertility Female reproduction & Developmental studies Local toxicity studies Hypersensitivity studies Genotoxicity studies Carcinogenicity studies
  12. 12. Preliminary Definitive • Maximum Non Lethal dose (MNLD) determined • MTD and MLD determined • Evaluate effects • Target organ of toxicity may be determined a) SINGLE DOSE STUDIES/ ACUTE TOXICITY
  13. 13. METHOD  Single dose tested in 2 rodent species  2 routes of administration  Oral dosing of 2g/kg or 10 times of normal human dose  Observation for 14 days after dosing  MNLD established  Symptoms , signs reported  Microscopic and Macroscopic evaluation
  14. 14. METHOD  Group of 20 animals of either sex dosed at MNLD  5 animals of each sex are observed for 48 hr and conduct autopsy for early pathological changes  Remaining 5 of each sex are observed for 14 days  MTD and MLD established  Signs of intoxication or recovery, changes in body weight, pathological changes  Complete macroscopic and microscopic examination  Target organs can be identified
  15. 15.  Two mammalian species(one should be non-rodent)  Long duration studies (30-180 days)  Dose is dependent on dose-escalating studies  Drug administered by clinical route  Parameters monitored and recorded are:  Behavioral  Physiological  Biochemical  Microscopic observations b) REPEATED DOSE STUDIES/SUB-ACUTE OR CHRONIC TOXICITY
  16. 16. a) MALE FERTILITY METHOD One rodent species(rat) 3 dose groups taken (each with 6 adult males), 1 control Drug treatment by clinical route for 28-72 days
  17. 17. Mated with females in 1:2 ratio Females getting pregnant should be examined After 13 days of gestation All male animals sacrificed •Weights of testis, epididymus recorded & examined for their histology •Sperms examined for motility & morphology
  18. 18. Segment I 19 Fertility and general reproductive performance study Segment II Teratogenicity Segment III Peri and post-natal study Fertility and early embryonic development (rat) Embryo- foetal development (rat & rabbit) Post natal development (rat) (post natal survival of offspring), growth parameters, vital senses, behavioral effects b) FEMALE FETILITY Drug administered to both males (28days) and females (14 days) before mating Implantation Embryogenesis
  19. 19.  Required when drug is administered by special route (other than oral) in humans  Study design:  2 species along with control used  Dose dependent on dose escalating studies  3 dose levels
  20. 20. Dermal toxicity studies Dermal photo-toxicity studies Vaginal toxicity studies Rectal tolerance studies Rats & Rabbit Local signs (erythema, oedema), histological examination Guinea pig Used in treatment of leucoderma Examination of erythema & oedema formation Rabbit or Dog Observation of swelling, histopathology of vaginal wall Rabbit or Dog Signs of pain, blood or mucous, histology examination of rectal mucosa
  21. 21. Ocular toxicity studies Parenteral drugs Inhalation toxicity studies Albino Rabbit Changes in cornea ,Iris & aqueous humor, histological examination of eye For intravenous/ intramuscular/ subcutaneous/ intra-dermal injection Sites of injection examined grossly and microscopically One rodent and non rodent species Acute , sub-acute and chronic studies performed Observation of respiratory rate Histological examination of respiratory passages, lung tissue
  22. 22. Guinea Pig Maximization test Local lymph node assay Determination of Maximum non irritant or minimum irritant dose Evaluation of Erythema and oedema Mice of one sex(either male or female) Drug treatment given on ear skin Auricular lymph node dissection after 5 days Increase in 3h-thymidine used for evaluation
  23. 23. To detect early tumorigenic effects in cases of chronic illness In vitro tests: Test for gene mutation in Bacteria Cytogenetic evaluation of chromosomal damage in mammalian cells E.g.; Ames’s Salmonella Assay detects increased number of aberrations in metaphase chromosomes DNA strand breaks, DNA repair or recombination, Measurements of DNA adducts In vivo tests: Chromosome damage in rodent hematopoietic cells E.g.; Micronucleus Assay
  24. 24.  Life-time Bioassays  Carcinogenicity studies are performed on:  Drug used for >6 months or frequent intermittent use for chronic diseases  Chemical structure of drug indicates carcinogenic potential  Therapeutic class of drugs which have produced positive carcinogenicity
  25. 25. Group sizes of 50 animals/sex at each of 3 dose levels Control group is of double size Record for onset of tumor development Usually carried out for 24 months in rats and 18 months in mice (life span studies) CONDUCT OF STUDY
  26. 26. EVALUATION OF RESULT Incidence of cancers in control and test  Trend towards increasing incidence with increasing doses Number of animals with single/multiple tumors Macroscopic changes observed by autopsy  Histopathology of organs and tissues