ACUTE, SUB ACUTE & CHRONIC TOXICOLOGICAL STUDIES

Toxicological studies of drugs at acute, sub acute & chronic toxicity levels.

1. ANIMAL TOXICITY TEST FOR ACUTE, SUBACUTE & CHRONIC TOXICITY. Presented by Sindhu k Mvsc scholar, Dept of vpt , covas .

2. Toxicity studies - Introduction Toxicology classically has been defined as the study of poisons & concerned with the adverse effects of xenobiotics . Casarett 1996 defined it as a science that defines the limits of safety of chemical agents for human & animal populations. 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. History of Toxicity Studies 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.

4. Sources of toxic substances Classified based on their chemical nature mode of action class (exposure class and use class ) Exposure class: Food, air, water or soil . Use class: drugs as drug of abuse, therapeutic drugs, agriculture chemicals, food additives, pesticides, plant toxins and cosmetics

5. evidences The use of sheep brain for the production of rabies vaccine has been phased out in 1992 Classical swine fever virus vaccine is produced in PK-15 Cells instead using large number of rabits PPR , FMD and sheep pox vaccines are also produced using cell culture vero , BHK-21 and vero cell culture system respectively. The use of laboratory animals e.g., rabbit and G. pig has been now abandoned for the isolation or typing of mycobacetria with the availability of improved synthetic media.

6. Biomedical ethics Before conducting any toxicological testing in animals or collecting tissue/cell lines from animals, the study should be approved by the Institute Animal Ethics Committee (IAEC) or the protocol should satisfy the guidelines of the local governing body.

7. NECESSITIES OF TOXICOLOGICAL STUDIES 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.

8. Magnitude of use of animals It has been estimated that approximately 20 million animals are being used for testing and are killed annually; about 15 million of them are used to test for medication and five million for products. China has become one of the biggest countries using lab animals, as is evident in the higher numbers and quality of lab animals (e.g., specific pathogen free, or SPF1; genetically modified) increasingly used in scientific research—16 million a year, compared to 12 million in the 25 European Union countries in 2005 (FELASA 2007)—and the increased publication of animal experiment results in international journals .

9. , Japan was second only to the US, which used 17.3 million animals in 2005. The third largest use in 2005 was Great Britain, which used 1.9 million animals. People for the ethical treatment of animals (PETA) reported that the National Centre for Laboratory Animal Sciences (NCLAS) in Hyderabad, supplies approximately 50,000 animals to laboratories every year and to 175 institutions in India, including pharmaceutical companies and educational institutions.

10. , In India, among rodentia group of animals e.g. mice. rat, G.pig , rabbit, mice is predominantly used in most of cases followed by other rodentia . The number of monkeys used in research has now drastically reduced in institutions. Use of some other unique animals like hamster, cotton rat, gerbil is not much common. There are inbred strains of mice which are used for research and genetically modified laboratory animals are also imported into India by some of institutes. The information with regard to available laboratory animals resource in different institutions/universities in India is not precisely known .

11. Standard Methods Multiple methods have been standardized (certified) by multiple organizations American Society for Testing and Materials (ASTM) Organization for Economic Cooperation and Materials (OECD) – (Europe based) National Toxicology Program (NTP) All above standardized protocols available from US EPA, Federal Register and researchers that developed the programs

12. REGULATORY MECHANISMS IN INDIA Institute Animal Ethics Committee (IAEC) Committee for the Purpose of Control and Supervision of Experiments in Animals (CPCSEA) Drugs & Cosmetics Act, 1940, Appendix-I Department of Animal Husbandry, Dairying & Fisheries, Ministry of Agriculture, New Delhi-2001 Department of Biotechnology. The Prevention of Cruelty to Animals Act, 1960 as amended up to 30th July 1982 and Animal Welfare Board

13. Relevant Test Models Pharmacokinetic profile Pharmacodynamic response 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

14. In vivo toxicological models

15. In-vivo >< in-vitro 1.In Vivo Studies In vivo safety pharmacology studies should be designed to define the dose-response relationship of the adverse effect observed The time course of the adverse effect should be investigated e.g. onset and duration of response 2.In Vitro studies In vitro studies should be designed to establish a concentration-effect relationship

16. TYPES OF TOXICITY STUDIES Single dose studies/ Acute toxicity studies Repeated dose studies / sub-acute or Chronic studies Local toxicity studies Allergenicity / Hypersensitivity toxicology studies Genotoxicity studies Carcinogenicity / Oncogenicity studies

17. Systemic toxicology studies Preliminary Definitive Maximum Non Lethal dose (MNLD) determined MTD and MLD determined Evaluate effects Target organ of toxicity may be determined SINGLE DOSE STUDIES/ ACUTE TOXICITY

18. , Acute toxicity testing- study the effect of a single dose on a particular animal species. Acute toxicity testing be carried out with two different animal species (one rodent and one non-rodent ). In acute toxicological testing, the investigational product is administered at different dose levels, and the effect is observed for 14 days. All mortalities caused by the investigational product during the experimental period are recorded and morphological, biochemical, pathological, and histological changes in the dead animals are investigated.

19. , The LD 50 was used as an indicator of acute toxicity previously. The determination of the LD 50 involves large numbers of animals, and the mortality ratio is high. (24 h testing) Graphical method Arithmetical method ( karbers’s ) method. when number of animal is small) Because of these limitations, modified methods were developed: 1. The fixed dose procedure 2. The acute toxic category method 3. The up-and-down method

20. Test report The test report should include the following information: Test substance : Physical nature, Purity Physicochemical properties; Identification data; Source of substance; Batch number

21. CONTD Vehicle (if appropriate ): Justification for choice of vehicle (if other than water ). Test animals: Species/strain used and justification for choice made; Number, age, and sex of animals at start of test; Source, housing conditions, diet, etc.; Individual weights of animals at the start of the test.

22. OECD Organization for Economic Cooperation and Development (OECD) Test Guidelines (TGs 402, 403, 420, 423, and 425) describe acute systemic testing. Fixed Dose Procedure (OECD TG 420) Acute Toxic Class method (OECD TG 423) Up‐and‐Down Procedure (OECD TG 425) Acute Dermal Toxicity (OECD TG 402) Acute inhalation toxicity (OECD TG 403)

23. Six OECD Test Guidelines describe short‐term repeat‐dose toxicity testing Repeated Dose 28‐day Oral Toxicity Study in Rodents (TG407) Repeated Dose 90‐Day Oral Toxicity Study in Rodents (TG 408) Repeated Dose Dermal Toxicity: 21/28‐day Study (TG 410) Sub-chronic Dermal Toxicity: 90‐day Study (TG 411) Repeated Dose Inhalation Toxicity: 28‐day or 14‐day Study (TG 412) Sub-chronic Inhalation Toxicity: 90‐day Study (TG 413)

24. 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

25. . Rodents and non-rodents are used to study the sub-chronic toxicity of a substance. Dose: Expected therapeutic level (daily ) or expected therapeutic level to increasing dose phase-wise manner. The test substance is administered orally for =/>90 days, and regular body weight variations, biochemical and cardiovascular parameters changes, and behavioral changes are observed. At the end of the study, the experimental animals are sacrificed. Gross pathological changes are observed, and all the tissues are subjected to histopathological analyses. There should be little individual variation between the animals, and the allowed weight variation range is ±20%. Used to determine the maximum tolerable dose and nature of toxicity .

26. . Chronic toxicity studies are conducted with a minimum of one rodent and one non-rodent species. The test compound is administered over more than 90 days, and the animals are observed periodically. A chronic toxicology study provides inferences about the long-term effect of a test substance in animals, and it may be extrapolated to the human safety of the test substance . The report on chronic oral toxicity is essential for new drug entities. There should be little individual variation between the animals, and the allowable weight variation range is ±20%.

27. Dose High dose: Produce significant retardation of growth or some pathological changes (10 times the expected maximum clinical dose). The low dose is about twice the expected maximum clinical dose Third dose is medium dose fixed midway between the high and low dose

28. . During the study period, the animals are observed for normal physiological functions, behavioral variations and alterations in biochemical parameters at regular intervals ( atleast every 14 days ). At the end of the study, tissues are collected from all parts of the animal and subjected to histological analyses.

29. Types of local toxicity studies 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

30. 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

31. D. allergenicity /hypersensitivity toxicology studies 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

32. E. Genotoxicity studies 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

33. In vivo tests In vivo test for chromosomal damage using mammalian hematopoietic cells. Chromosome damage in rodent hematopoietic cells E.g .; Micronucleus Assay

34. f. Carcinogenicity/ oncogenicity studies 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

35. Note on stem cells STEM CELLS NOW EMERGING AS AN ALTERNATIVE TO LABORATORY ANIMALS Drug company interest in stem cell drug testing was demonstrated in July 2008, when GlaxoSmithKline entered into a $25 million-plus agreement with the Harvard Stem Cell Institute. By testing drugs on specific cells and tissues created from iPS cells, we can even predict a patients individual response to a treatment realizing the vision of personalized medicine. In the current issue of Stem Cells and Development (2007) , Cezar and her colleagues revealed a novel way to test drug toxicity: by monitoring the behaviour of embryonic stem cells exposed to a drug-candidate compound. Studying how potential drugs affect embryonic stem cells could provide a far more accurate prediction of a drug's potential toxicity than conventional animal models can. Currently , the most successful development of stem cells as in vitro models for toxicology testing is in human cardiac tissue.

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37. Oecd guidelines for acute, sub-chronic & dermal toxicity studies ACUTE 401 ~ Acute Oral Toxicity 402 ~ Acute Dermal Toxicity

38. Sub chronic 407 ~ Repeated Dose Oral Toxicity ( Rodents : 28 days / 14 days study ) 408 ~ Sub-chronic Oral Toxicity Test ( rodents : 90 days study ) 409 ~ Sub-chronic Oral Toxicity Test ( Non-rodents : 90 days study ) 410 ~ Repeated Dose Dermal Toxicity ( 21/28 days study ) 411 ~ Sub-chronic Dermal Toxicity (90 days study )

39. chronic 452 ~ Chronic Toxicity Studies 453 ~ Combined Chronic Toxicity/Carcinogenicity studies

40. Definitions relative to doses ACUTE STUDIES ED 50 : The median effective dose = the dose for which half(50%) of the animals exhibit an effect (E) and half of animals exhibit no effect LD 50 : The median lethal dose = the effect may be defined as specific toxic event (tremors) & sometimes defined as lethality ED10 & ED90 are doses at which 10% & 90% of the animals respectively demonstrate effect

41. General studies LIMIT DOSE : a dose which is considered high enough that if no mortality / significant toxicity is seen in animals receiving this dose, no higher doses are required eg : Limit doses(EPA/OECD) 1.Acute Oral T oxicity 5000mg/kg 2.Acute Dermal Toxicity 2000mg/kg 3.21-days Dermal Toxicity 1000mg/kg 4.Chronic Studies of Pesticides 1000mg/kg Note: the limit dose of a non-nutritive material added to diet is generally considered to be 5% (50,000ppm)

42. Sub-chronic & chronic studies ADI: Acceptable Daily Intake (established for food additives/ residues & published by EPA) NOEL: No Observed Effect Level. Dose at which no effect is seen NOAEL: No Observed Adverse Effect Level. Dose at which no adverse/toxic effect is seen

43. Chronic studies MTD: Maximum Tolerated Dose. Is the highest dose that can be tolerated without significant lethality from causes other than tumors. (for EPA studies MTD for chronic studies with pesticides is a dose which produces an approximate 10-15% decrement in body weight gain) HTD: Highest dose tested. Highest dose that can be expected to yield results relevant to humans. This is a proposed new dose which would be selected based on evaluation of results of sub-chronic studies.

44. Vehicles used for dosing ORAL: W ater, Methylcellulose or C arboxymethylcellulose (0.5-5% aqueous suspension), Oil( corn, peanut, sesame) DERMAL: Physiological saline, Water, Ethanol, Acetone, Mineral oil PARENTERAL: Physiological saline(sterile), sterile water for injection

45. Clinical signs of toxicity RESPIRATORY: blockage in the nostrils, changes in rate & depth of breathing, changes in color of body surfaces Signs like dyspnea, abdominal breathing, gasping, Apnea, Cyanosis, Tachypnea, nostril discharge = involvement of CNS respiratory center, pulmonary edema, cholinergic inhibition, pulmonary cardiac insufficiency.

46. MOTOR ACTIVITY Changes in frequency & nature of movements Signs like decrease/increase in spontaneous motor activity, Somnolence, Loss of righting reflex, Anesthesia, Catalepsy, Ataxia, Unusual locomotion, prostrations, Tremors & f asiculations = CNS, sensory, autonomic & neuromuscular system

47. convulsions Marked involuntary contractions or seizures of contraction of voluntary muscles Signs like Clonic convulsions, Tonic convulsions, Tonic- Clonic convulsions, Asphyxial convulsion, Ophisthotonos = CNS, respiratory failure, neuromusculsr & autonomic systems.

48. reflexes CORNEAL PINNAL RIGHTING MYOTACT LIGHT(pupillary) STARTLE REFLEX All signs indicates involvement of CNS, sensory, autonomic & neuromuscular systems

49. Ocular signs Lacrimation, Miosis , Mydriasis , Exophthalmos, Ptosis, Chromodacryorrhea ( red lacrimation ), relaxation of nictating membrane, corneal opacity, iritis , conjunctivitis. Mainly due to autonomic system involement

50. Cardio-vascular signs Bradycardia, tachycardia, vasodilation, vasoconstriction, arrhythmias Due to autonomic, CNS, cardiac-pulmonary insufficiency, myocardiac infarction, cold environment.

51. Other signs Salivation Piloerection Analgesia Muscle tone - hypotonia - hypertonia GIT signs: dropping feces, emesis, diuresis(hematuria & involuntary urination)

52. Autonomic signs Sympatomimetic – piloerection , partial mydriasis . Sympathetic block – ptosis, diagnostic if associated with sedation. Parasympathomimetic – salivation, miosis , diarrhoea . Parasympathomimetic block – mydriasis (maximum), excessive dryness of mouth.

53. Toxic signs of acetylcholinesterase inhibition MUSCARINIC EFFECTS NICOTINIC EFFECTS CNS EFFECTS Bronchoconstriction, Increased bronchosecreation . Muscular twitching Giddiness, anxiety, headache Nausia & Vomiting Fasciculation Apathy , confusion Diarrhea Cramping Depression, drowsiness, seizures, Bradycardia Muscular weakness Ataxia Hypotension Respiratory depression Miosis confusions Urinary incontenance coma

54. Effects of decreased body weights on relative organ weights of rats Decreases = liver No change = heart, kidneys, prostrate, spleen, ovaries. Increases =adrenal gland, brain, epididymides , pituitary, testes, thyroid, uterus.

55. Human studies General considerations for clinical studies Specific considerations for clinical studies Protocol design The study population Statistical analyses 3. Sequence of clinical studies Early clinical studies Further clinical studies 4. Submitting reports of clinical studies to CFSAN ( Center for Food Safety and Applied Nutrition)

56. PHASES OF DRUG DEVELOPMENT (ANIMAL MAN) PHASE III PHASE IV PHASE I PHASE I PRECLINICAL PHASE II Product Approval (NDA/MAA) Patient studies Entry to man (IND / CTA) None Healthy subjects Safety and tolerability Genetic toxicity (in vivo) Repeat dose toxicity testing + Bioanalysis / Toxicokinetics Drug Metabolism Reproductive Toxicity Testing (teratogenicity) Patients Small scale efficacy 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 Patients Large scale post-marketing studies As required Genetic toxicity (in vitro) Single / repeat dose toxicity studies + Bioanalysis / Toxicokinetics Safety Pharmacology Drug Metabolism Lead candidate Identified Clinical Non-clinical MOLECULE

57. Discussion session ,

58. references CRC hand book of toxicology Google images & Wikipedia Online Related materials from slideshare

59. Thank you