Toxicity studies

This presentation is about toxic effects of different drugs and also how to reduce to its effect.
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1. COURSE TITLE ENVIRONMENTAL TOXICOLOGY COURSE CODE ZOL- 610 CREDIT HOURS 3 (2-1) SEMESTER BS ZOOLOGY 8 TH M-A , E-A , E-B DEPARTMENT OF ZOOLOGY

2. COURSE CONTENTS ENVIRONMENTAL TOXICOLOGY ZOL 610 3(2-1 ) Toxicology: History , Terms and definitions, principles of Toxicology, Development and present scope of environmental toxicology, Framework of environmental toxicology, toxicological evaluation, Sources of environmental toxicants, / Pollutants: Gaseous chemicals and heavy metals, toxicity testing, Characteristics of exposure, spectrum of Toxic effects, Indices of toxicity, toxico - dynamics, toxico -kinetics (absorption, distribution and elimination of toxic agents) Biotransformation, Detoxification and biodegradation. Pollution and remediation, ecological risk assessment

3. Toxicity Studies

4. Source, Synthesis & chemistry Pre c linic a l Studi e s & Toxicology Studies Animal models, Efficacy and Safety Clinical Trials Drug Development

5. Preclinical Safety & Toxicity Testing Aims at discovering complications or sequelae arising from the pharmacological actions of drug and any unexpected side-effects .

6. Primary Goals  Estimate safe starting dose for clinical studies & subsequent dose escalation schemes in humans.  Identi f y pot e ntial targ et organ s fo r toxicit y and study whether such toxicity is reversible.  Assess dose dependence & relationship to exposure  Assess hazards that cannot be evaluated in clinical trials (e.g. carcinogenicity and teratogenicity) Identify hazards and estimate safe dose range

7. Necessity of toxicity studies Completely novel compound of unique action under consideration Any chemical modification made to a known drug Formulations of a compound are considered ( Separately as well as formulated form) Novel combination of drugs made available as a single formulation Veterinary drugs → if treatment is given to animals intended for human food

8. Requirements Chemical Criteria Substance used in toxicity studies should be as pure as material to be given to man

9. Materials: Animals Species: Species difference → single largest difficulty in interpretation of toxicity studies Species → health, behaviour, endemic disease and reaction to well studied toxic agent is familiar Two different species(Rodent & Non Rodent) used Should differ phylogenetically as widely as possible

10. Materials: Animals Species: Species in which test material is pharmacologically active due to presence of receptor should be used Ex. Avoid dogs for studying toxic effects of sulphonamides, monkeys are better Strains: Use either animals derived from random breeding in a closed colony or hybrids of two inbred lines

11. Materials: Animals Number, age and sex: Should be sufficient Long term experiments i.e. Carcinogen testing, large number may be necessary to detect relatively low incidence of the reactions Young immature animals are preferred generally → rapid growth, but for detection of actions on endocrine and reproductive systems, sexually mature animals are required Should include groups of both males and females

12. Route / Frequency of administration Two routes to be used, one should be which is intended for clinical use Aim should be to maintain the appropriate blood and tissue level of the compound under consideration for test duration

13. Durat i on Depends on type of the test (acute, sub-acute, chronic) All the important toxic effects of test substances can probably predicted from experiments lasting no more than three months In the absence of any rational basis for choosing a period, a duration of twice the maximum exposure likely for the human beings is convenient guide. 2 yrs. max

14. Toxicity Studies Systemic Toxicity studies Reproductive toxicology studies Local Toxicity studies Allergenicity/ Hypersensetivity studies Genotoxicity studies Carcinogenicity studies

15. Systemic Toxicity Studies

16. Single dose (Acute) Short term repeated dose ( Subacut e) Repeated dose: 10% life-span (Subchronic) Repeated dos e : > 10% life span (Chronic) Systemic Toxicity Studies

17. Single-dose (Acute) Toxicity Studies Dose – toxicity relationship Specific toxic effects Mode of toxic action Median Lethal dose (LD 50) Route dependent toxicity Sex dependent toxicity

18. Single-dose (Acute) Toxicity Studies Rodent species (mice and rats) Use the same route as intended for humans Use at least one more route in one of the species. This Ensures systemic absorption of the drug (unless the intended route in humans is only intravenous) Recommended limit for oral dosing : Higher value of either 2000 mg/kg or 10 times the normal dose that is intended in humans

19. Single-dose (Acute) Toxicity Studies At least 5 animals / sex / group At least 4 graded doses (4 dose-groups) Observation for toxic effects (if any) : 14 days Obsevation for mortality : 7 days [parenteral administration] 14 days [oral administration]

20. Reduction of the number of animals, use of single sex Preliminary studies, followed by main test (Limit test) UFpixaAencddudtdeoostweoxnpircporccoleacdsesudr(ueGr(euGi(duGeiuldiinedleeinl4ien24e32)402)5) (En(dEpnodinpto:inEtv:immdeonrttatolixtyic)ity) P3Siragenhliitminiagnlsasrtouyfdsoytnu:ed1ysae:nx1i/masnatilempealr psteerpstep SISntaitritailndgodsoes:eex: pjeuexscptteibncetgilnomgwoserotxmapl e itcyttoeixndicsLioDtmy 5e0 FDiox a se n ed im sdt a eo l ps s epsedofu5p, /50d,o3w0n0baynad f2a0ct0o0r omfg3/.k2g F CD i o x s e et d ins d tue o ep s d e p s etid o ll f uo 5 pu , t/ 5 c od , mo 3 we n ret a ivl n el r d esne 2 dd -, pi o.ei. m nmt g ao / c k rht g aielivteyd Do<se=>stesuprpveidvaulp / down till end point achieved Modifications (OECD guidelines: 420, 423, 425; yr 2000)

21. Other parameters : Symptoms, signs and mode of death LD 10 and LD 50 values, preferably with 95 % CI Genetic effects if any Establish the: Minimum lethal dose (MLD) Maximum tolerated dose (MTD) Target organ of toxicity (if possible)

22. Cytotoxic anticancer agents MTD determined first in Mice Findings in Rat confirmed to establish a linear relationship between toxicity and body surface area MTD can be established in non-rodent species, if: Predictability is known to be poor in Rodents (e.g. Antifolates) Drug has a novel mechanism of action

23. Toxicokinetic studies Generation of pharmacokinetic data (ADME): A part of non-clinical toxicity studies or A separate supportive study Purpose: Assessment of systemic exposure of test substance Relationship of toxicology to dose and time course Choose species & regimen in subsequent toxicity studies Designing of subsequent non-clinical toxicity studies

24. Rodents • One rodent species (preferably rat) • At least 4 graded doses including control • Minimum of 5 animals of each sex • Proposed clinical route of administration • Test substance given daily for 10 consecutive days Dose-ranging study Establishment of MTD for repeated dose studies Identification of Target organ of toxicity Non-Rodents One male and one female (Dogs > Primates) Starting dose (3 or 5 x extrapolated effective dose) or MTD (whichever is less) Dose escalation every 3 rd day, lowered if toxicity seen Then test substance given daily for 10 consecutive days

25. Repeated-dose Toxicity Studies Duration depends on proposed clinical trial At least 2 species, one non-rodent Species-specific pharmacokinetics, if any, should preferably resemble human beings Route intended for human clinical use

26. Repeated-dose Toxicity Studies Wherever applicable, include a Control group 3 other groups are formed, as: Highest dose Lowest dose Intermediate dose Observable toxicity [MTD] No observable toxicity [NOAEL] intended therapeutic dose or multiple of it Some symptoms ; not gross toxicity or death placed logarithmically betn doses

27. Rodent Non-rodent : 6-10/sex/group : 2-3/sex/group 14-28 Day repeated-dose (Subacute toxicity studies) Rodent Non-rodent : 15-30/sex/group : 4-6/sex/group 90-Day repeated-dose (Subchronic toxicity studies) Rodent Non-rodent : 15-30/sex/group : 4-6/sex/group 180-Day repeated-dose (9 months for non rodents?) (Chronic toxicity studies)

28. In all cases:  Behavioural : General appearance, activity, behaviour  Physiological : Body weight, food intake  Biochemical : Hematology, serum and urine analysis  Pathological : Organ weights, gross & microscopic study of viscera & tissues Parameters to be monitored In j ec t io n sit e : Gross and Microscopic examination In non-rodent species: Electrocardiogram : Initial and Final Fundus examination If parenteral drug administration:

29. Repr o ductive Toxicology

30. Reproductive Toxicity Male Fertility Female Reproduction & Developmental toxicity Female Fertility Teratogenicity Perinatal development

31. Male Fertility Study One rodent species (preferably rat) 3 dose groups and a control group : Dose selection : results of previous 14 or 28-day toxicity study Highest dose : showing minimal toxicity in systemic studies 6 adult male animals per group

32. Test substance by intended route of use for : minimum 28 days maximum 70 days Paired with female animals of proven fertility in a ratio of 1:2 Drug treatment of male animals continues during pairing Pairing continued till detection of Sperm in vagina or 10 days, whichever is earlier

33. Pregnant females examined after day 13 of gestation Males sacrificed at the end of the study Weights of each testis and epididymis recorded Sperms from one epididymis examined for motility and morphology Other epididymis and both testes examined for histology

34. Female Reproduction and Developmental Toxicity Studies For all drugs proposed for women of child bearing age Segment I : Female fertility Segment II : Teratogenicity Segment III : Perinatal development Segment I, II and III studies in albino mice or rats , and Segment II study also in albino rabbits as a second test species

35. Female Fertility Study (Segment I) One rodent species (rat preferred) 3 graded doses Highest dose : doesn’t affect general health of parent animals (usually the MTD from previous systemic studies) At least 15 males and 15 females per dose group Route of administration same as intended for therapeutic use

36. Pups : Physiology, Behaviour, Pathology (sex-wise distribution noted) Body weight Food intake Clinical signs of intoxication R e pro d uction and Parturition Pa t hol o gy Gross & Micro Drug treatment continued during mating and gestation period Females allowed to litter, medication continued till weaning of pups Drug administration : 28 days (males) & 14 days (females) before mating

37. Teratogenicity Study (Segment II) Rodent (preferably rat) and Non-rodent (rabbit) Drug administered throughout organogenesis 3 dose levels and a Control group: Highest dose : minimum maternal toxicity Lowest dose : as proposed clinical dose or its multiple Route of administration : same as intended for humans At least 20 pregnant rats (or mice) and 12 rabbits , for each dose

38. Observation parameters Females General Signs of intoxication Body weight Food inta k e Reproductive Uterus, Ovar i es Products of conception Foetuses General N u mb e r Gend e r L e ng t h Weight Pa t ho l ogy Gross (all) Visceral (half) Skeletal (half)

39. Perinatal Study (Segment III) Specially if → Drug to pregnant / nursing mothers for long periods → Indications of possible adverse effects on foetus One rodent species (preferably rat) Dosing comparable to multiples of human dose and route At least 4 groups (including control), 15 females / group Drug throughout last trimester (from day 15 of gestation) Dose causing low foetal loss continued throughout weaning

40. F1 litter 1 male and 1 female from each group Test substance given Throughout growth to sexual life Mating performance and fertility of F 1 : F 2 generation F2 generation growth parameters monitored till weaning

41. Local Toxicity

42. Local Toxicity Dermal Toxicity Ocular Toxicity Vaginal Toxicity Rectal tolerance test Parenteral Drugs Tolerance Inhalation toxicity

43. Local Toxicity If intended for special route (other than oral) in humans Appropriate site (e.g., skin or vaginal mucous membrane) in a suitable species Preferably use of 2 species 3 dose levels and untreated and / or vehicle Control If the drug is absorbed systemically, appropriate systemic toxicity studies also required

44. Dermal Toxicity As cutaneous contamination is always a possibility Rabbit and Rat Applied on shaved skin Concentrations 7 fold higher than the clinical doses Period of application : 7 to 90 days Evaluation › Local signs (erythema, oedema and eschar formation) › Histological examination of sites of application

45. Ocular toxicity studies 2 species, including an albino rabbit with a large conjunctival sac Initial single dose application: To decide exposure concentrations for repeated-dose studies Repeated dose study : Duration subject to clinical use (Maximum of 90 days) 2 different concentrations exceeding human dose In acute studies, one eye kept as control. A separate control group should be included in repeated-dose studies.

46. Ocular toxicity studies Evaluation Slit-lamp examination To detect the changes in cornea, iris and aqueous humor. Fluorescent dyes (sodium fluorescein, 0.25 to 1.0%) To detect defects in surface epithelium Intra-ocular tension monitored by a tonometer Histological examination (fixation in Davidson’s or Zenker’s fluid)

47. Vaginal Toxicity Test Rabbit or Dog Topical application (vaginal mucosa) as pessary, cream or ointment 6-10 animals per dose group Higher concentrations / several daily applications (in multiples of daily human dose) 7-30 days, as per clinical use Observation parameters : General : swelling, closure of introitus Histopathology of vaginal wall

48. Rectal Tolerance Test Preparations meant for rectal administration In rabbits or dogs 6-10 animals per dose group Volume comparable to human dose (or the maximum possible volume) to achieve administration of multiples of daily human dose 7-30 days, as per clinical use Observation parameters Clinical signs :- signs of pain, blood and/or mucus in faeces, condition of anal region/sphincter Gross examination and (if required) Histological examination of rectal mucosa

49. Parenteral Drugs Intravenous/ intramuscular/ subcutaneous/ intradermal inj. Injection sites in systemic toxicity studies examined grossly and microscopically If needed, reversibility of adverse effects may be determined on a case to case basis

50. Inhalation toxicity studies 1 rodent and 1 non-rodent species Acute, subacute and chronic toxicity studies according to the intended duration of human exposure Gases and vapors given in whole body exposure chambers ; aerosols are given by nose-only method Dose (limit dose of 5mg/l) in multiples of human exposure Particle size of 4 micron (especially for aerosols ) with not less that 25% being 1 micron

51. Inhalation toxicity studies 3 dose groups and a control Duration of exposure : maximum of 6 hr/day & 5 days/week Evaluation : Respiratory rate, BALF examination, histological examination of respiratory passages and lung tissue Regular parameters of systemic toxicity studies or assessment of margin of safety

52. Allergenicity / Hyper s ensitivity

53. Allergenicity / Hypersensitivity Any one of the standard tests: Guinea pig : Maximization test (GPMT) Mouse : Local lymph node assay (LLNA)

54. Guinea Pig Maximization Test Challenge (skin reaction appears) Induction : Minimum irritant dose (intradermal injection) Challenge : Maximum nonirritant dose (topical application) Doses are determined by a preliminary study Induction (immune response develops)

55. Main test A minimum of 6 male and 6 female animals per group One test group One control group One positive control group (preferable) If no response : re-challenge 7-30 days after primary challenge

56. Induction (Day 0) 3 pairs of intradermal injections on either shoulders : 0.1 ml Freund’s adjuvant alone 0.1 ml test material (lowest irritant dose) 0.1 ml test material in Freund’s adjuvant Day 7 : Topical patch at prepared shoulders (lowest irritant dose) Challenge (Day 21) Topical patch at prepared flanks (highest non-irritant dose) Left side: Test agent Right side: Vehicle Evaluation [Edema and Erythema] after 48 hr.

57. Local Lymph Node Assay Mice of the same sex, either only males or only females Drug treatment given on ear skin 3 graded doses (the highest being maximum nonirritant dose) plus vehicle control A minimum of 6 mice per group Test material applied on ear skin on 3 consecutive days On day 5, i.v. 3 H-thymidine / bromo-deoxy-uridine (BrdU) Draining auricular lymph nodes dissected after 5 hrs Evaluation : Increase in 3 H-thymidine or BrdU incorporation

58. Genot o xicity

59. Genotoxic compounds are presumed to be trans-species carcinogens, need not require long-term carcinogenicity studies If intended for chronic administration, a chronic toxicity study (up to one year) to detect early tumorigenic effects ICH Standard Tests are generally conducted In vitro test for gene mutation in bacteria In vitro cytogenetic evaluation of chromosomal damage : Mammalian cells or Mouse lymphoma tk assay In vivo test for chromosomal damage using rodent hematopoietic cells

60. Ames’ Test ( Bacterial Reverse Mutation Assay ) S. typhimurium tester strains TA98, TA100, TA102, TA1535, TA97 or Escherichia coli WP2 uvrA or Escherichia coli WP2 uvrA In-vitro exposure at a minimum of 5 log dose levels “Solvent” and “positive control” 2.5 fold (or more) increase in number of revertants in comparison to spontaneous revertants are considered positive

61. In-vitro cytogenetic assay Performed in CHO cells or on human lymphocyte in culture In-vitro exposure using a minimum of 3 log doses “Solvent” and “positive control” [Positive control : Cyclophosphamide / Mitomycin C gives a reproducible and detectable increase in clastogenic effect] > 50% inhibition of cells is considered significant

62. Mammalian cell test systems Division stimulated with phytohemagglutin Division arrested in metaphase using a spindle inhibitor Evaluation by light microscopy Increased number of aberrations in metaphase chromosomes

63. In Vitro Mouse Lymphoma TK Assay Mouse TK lymphoma cells Thymidine kinase (TK) enzyme involved in salvage pathway for incorporation of thymidine into cells via phosphorylation Trifluorothymidine (TFT) also phosphorylated by TK Cells containing TK are sensitive to toxic effects of TFT Forwar d m utation s from TK+ to T K - res u l t i n l os s o f TK activity Quntifiication of mutant cells : Cells with TFT resistance

64. In Vivo Tests for Chromosomal Damage Mammalia n B o ne Marrow Chromosomal Aberration Assay Rodent Erythrocyte M i cronucl e us Assay

65. In-vivo cytogenetic assay One rodent species (preferably rat) Route of administration same as intended for humans 5 animals/sex/dose groups 3 dose levels, “solvent” and “positive” control (Cyclophosphamide) Dosing on day 1 followed by i.p. colchicine administration at 22 hours Sacrificed 2 hours after colchicine administration Bone marrow : Giemsa staining Clastogenic & Aneugenic effects in metaphase chromosomes (min 100)

66. In-vivo micronucleus assay 1 rodent species (preferably mouse) needed Route of administration of test substance same as humans 5 animals / sex / dose groups At least 3 dose levels, plus “solvent” and “positive” control Positive control : mitomycin C or cyclophosphamide Dosing : day 1 and 2 of study Sacrifice of animals 6 hours after the last injection Bone marrow : Smeared with May Gruenwald / Giemsa stain ↑ micronuclei in polychromatic erythrocytes [M-PCE] (min 1000)

67. Carcinogenicity

68. Carcinogenicity For expected clinical use more than 6 months For drugs used frequently in an intermittent manner If concern about the carcinogenic potential due to : Previous demonstration in the product class Structure-activity relationship suggests carcinogenic risk Preneoplastic lesions in repeated dose toxicity studies Long-term tissue retention results in reactions

69. Carcinogenicity Where life-expectancy in the indicated population is short (i.e., less than 2 - 3 years) - no long-term carcinogenicity studies Where therapy is generally successful, there may be later concerns regarding secondary cancers. So carcinogenicity studies needed A rodent species (preferably rat). Mouse only if justified Strain should not have normal incidence of spontaneous tumors

70. Carcinogenicity At least 3 dose levels : Highest dose : sub-lethal, should not reduce life span of animals by more than 10% of expected normal Lowest dose : should be comparable to the intended human therapeutic dose or a multiple of it, e.g. 2.5x; to make allowance for the sensitivity of the species Intermediate dose : placed logarithmically between the other 2 Untreated control and (if indicated) a Vehicle control group

71. Carcinogenicity Life span comparable to human’s over which drug use is intended Generally, 24 months for rats and 18 months for mice Atleast 50 animals of each sex Observation parameters Physiology, Behaviour, Biochemistry, Pathology Comprehensive descriptions of benign and malignant tumour development, time of their detection, site, dimensions, histological typing etc

72. Carcinogenicity

73. Requirements of Clinical Trials and Marketing

74. Clinical trials & marketing- Requirements Systemic Toxicity Studies -oral/parenteral/ transdermal route Duration of proposed human a d m inistr a ti o n Human phases for which study is proposed to be conducted Species Long term toxicity re q uir e ment Upto 1 week I, II, III 2 2 weeks 1-2 weeks ” ” 4 weeks 2-4 weeks ” ” 12 weeks 1 month ” ” 24 weeks

75. Systemic toxicity studies – Inhalational route Duration of proposed human a d m inistr a ti o n Human phases for which study is proposed to be conducted Sp e cies Long term toxicity r e q u irm e n t s Upto 2 wk I, II, III 2 1 month (exposure time 3hr/d, 5d/wk) Upto 4 wk I, II, III 2 3 months (exposure time 6hr/d, 5d/wk) 4 wks I, II, III 2 6 months (exposure time 6hr/d, 5d/wk)

76. Local Toxicity Studies Route Duration of Clinical trial P h ase Species Duration of Toxicity study Dermal Ocular Upto 2 weeks I, II 1 4 wk Otic Nasal Vaginal Rectal 2 weeks III 2 4 wk I,II,III 2 12 wk

77. REFRENCES Hughes, W. William. 2005. Essentials of environmental toxicology: The Effects of Environmentally Hazardous Substances on Human Health. Taylor & Francis, 325 Chestnut St., Suite 800, Philadelphia, Pa 19106. pp. 71-78 Shaw, I. and J. Chadwick. 2002. Principles of Environmental Toxicology. Taylor & Francis Ltd, 1 Gunpowder Square, London EC4A 3DF. Yu, Ming-Ho. 2005. Environmental toxicology: biological and health effects of pollutants. 2 nd edition. CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431