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- 1. Chapter 3 Principles &methods of testing for toxicity
- 2. Learning Objectives Discussthe studies on general toxicity Describe developmental & reproductive toxicity studies Explain the different methods of mutagenicity studies Describe the tests used to study carcinogenic effect of chemicals 2
- 3. Chapter Outline Studieson general toxicity: Acute, sub-acute, sub-chronic & chronic toxicity studies Studies on various types of specific adverse effects: Developmental & reproductive toxicity Mutagenicity Tests for carcinogens Immunotoxicity assessment Irritation & sensitization - local tolerance studies 3
- 4. I. Studies onGeneral Toxicity Toxicological testing is best performed with pure substances since examination with chemical mixtures is practically impossible & may give rise to erroneous conclusions due to possible chemical interactions If it is necessary to determine the toxicity of a cpx mixture, investigation should start by establishing the composition of the mixture & determining which components of the mixture are bioactive 4 Principles of descriptive animal toxicity tests Two main principles underlie all descriptive animal toxicity testing A. The effects produced by a compound in lab animals , when properly qu alified, are applicable to humans • O n the basis of dose per unit of body surface, toxic effects in humans are usually in the same range as those in experimental animals • O n a body weight basis, humans are generally more vulnerable than are experimental anim als 1
- 5. Studies on GeneralToxicity… Each substance then must be classified & characterized with respect to its physicochemical properties The stability of a given substance determines how it should be administered for testing, particularly if it is inactivated in the GIT In general, the starting point for toxicological testing of new substances is testing in lab animals A chemical may be administrated to animals by oral, dermal, IV, IM or IP routes 5
- 6. Studies on GeneralToxicity… The ideal experimental animal is one that metabolizes & secretes a substance in the same manner as humans do Thus, rodents, hamsters, rabbits, cats & dogs are the most frequent human surrogates used for toxicological testing Mainly b/c they are economical, amenable to frequent handling & lacking in the vomiting reflex Other animals, such as monkeys, are also used 6
- 7. Studies on GeneralToxicity… Metabolic differences b/n humans & surrogate test animals can result in erroneous conclusions about the activity of a substance in humans Use of younger animals provides the advantage of obtaining faster results due to their higher rate of metabolism, as well as their being cheaper to purchase & house Age & gender of animals can influence the outcome of the investigation 7
- 8. Studies on GeneralToxicity… The purity of the substance is very important, since impurities may significantly influence toxicological findings Before animal testing is started, a literature search must be done to collect available results of any previous testing If the test substance is administrated to experimental animal by mixing it with food or water, it may be affected by oxidation (i.e., through contact with air), or by temperature variations or by the feeding habits of the experimental animals 8
- 9. Studies on GeneralToxicity… Gastric intubation (gavage), an artificial means of substance introduction, has advantages with respect to dosing exactness & reduced variation The gavage is performed after the animals have fasted in order to avoid mixing chemical test substances with stomach contents In general, the chemical dose should not exceed 5% of the test animal’s diet 9
- 10. Studies on GeneralToxicity… Alternative test methods: The use of lab-cultured single-cell organisms Isolated organs or mammalian cell lines NB: The advantage of animal testing: The possibility to examine the results of different routes of exposure under controlled conditions 10
- 11. Studies on GeneralToxicity… However, differences in genetic pool, lifespan, susceptibility, metabolic pathways & other parameters of organic life, present serious limitations when extrapolating the results of animal testing to human populations Animal experiments are usually classified according to their duration as: Acute, sub-acute, chronic or sub-chronic testing Chronic & acute toxic effects for a specific toxicant can differ both qualitatively & quantitatively 11
- 12. 12 Fig. Typical tieredtesting scheme for the toxicological evaluation of new chemicals
- 13. A. Acute toxicitystudies Objectives: To determine the Median Lethal Dose (LD50) after a single dose administered through one or more routes, one of which is the intended route of administration in humans To determine Maximum Tolerated Dose (MTD) & No Observable Effect Level (NOEL) To identify potential target organs for toxicity, determine reversibility of toxicity, & identify parameters for clinical monitoring To help select doses for repeated-dose toxicity tests 13
- 14. Acute toxicity studies… Duration: A few days to 2 wks after a single dose Test system/animal system: 2 species required: mice, rats, or rabbits or dogs Dose administration: Oral (by gavage or with food), SC, IP, Intradermal, Inhalation, Intranasal, Topical (epicutaneous), IV Parameters: Mortality, clinical pathology, gross necropsy, weight change, signs of toxicity 14
- 15. B. Sub-acute toxicitystudies Objectives: To determine toxicity after repeated administration of the test material To help establish doses for subchronic studies Duration: 14 days Test system/animal system 2 species required: mice, rats, rabbits, guinea pigs, dogs 15
- 16. Sub-acutetoxicity studies… Doseadministration: 3 to 4 doses given by the same routes as previous toxicity tests Parameters: Mortality Signs of toxicity Pathology & histopathology Weight change Clinical pathology 16
- 17. C. Sub-chronic toxicitytesting Objectives: To establish a “no observable effect level" (NOEL) To characterize D-R r/ships following repeated doses To further identify & characterize specific organs affected after repeated administration To predict a reasonable & appropriate dose for chronic exposure studies (MTD) 17
- 18. Sub-chronic toxicity testing… Duration: Commonly 90 days, but varies from 2 wks to 6 months or up to 10% of species’ lifespan Test system/animal system: 2 species required: rodents, dogs Dose administration: At least 3 doses given by the same routes as previous toxicity tests; the lowest producing no apparent toxicity & the highest producing toxicity but less than or equal to 10% mortality 18
- 19. Sub-chronic toxicity testing… Parameters Mortality Weight change Signs of toxicity Clinical pathology Pathology and histopathology 19
- 20. D. Chronic toxicitytesting Objectives: To evaluate the cumulative toxicity of chemicals To assess carcinogenic potential Duration: Rodents - 6 to 24 months; non-rodents - 12 months or longer or up to 10% of species’ lifespan Length depends on intended period of human exposure 20 Chronic toxicity studies Long-term or chronic exposure studies are performed similarly to subchronic studies except that the period of exposure is longer than 3 months In rodents, chronic exposures are usually for 6 months to 2 years Chronic studies in non-rodent species are usually for 1 year but may be longer The length of exposure is somewhat dependent on the intended period of exposure in humans
- 21. Chronic toxicity testing… Test system/animal system: 2 species required: Rodents, dogs Dose administration: As in sub-acute/ subchronic toxicity studies Parameters: Mortality Pathology & histopathology Weight change Clinical pathology of all animals (mortalities & survivors) 21
- 22. II. Studies onvarious types of specific adverse effects Studies on various types of specific adverse effects Developmental & reproductive toxicity Mutagenicity Tests for carcinogens Immuno-toxicity assessment Irritation & sensitization - local tolerance studies 22
- 23. A. Developmental &reproductive toxicity Developmental toxicology: The study of adverse effects on the developing organism occurring anytime during the life span of the organism that may result from exposure to chemical or physical agents • Before conception (either parent) • During prenatal development, or • Postnatally until the time of puberty 23
- 24. Developmental & reproductive toxicity… Teratology: The study of defects induced during development b/n conception & birth Reproductive toxicology: The study of the occurrence of adverse effects on the male or female reproductive system that may result from exposure to chemical or physical agents 24
- 25. Developmental & reproductive toxicity… a)General fertility & reproductive performance (segment I) studies: They are usually performed in rats with 2 or 3 doses (20 rats per sex per dose) of the test chemical (neither produces maternal toxicity) Males are given the chemical 60 days & females 14 days before mating The animals are given the chemical throughout gestation & lactation 25
- 26. Developmental & reproductive toxicity… Segment I studies… Typical observations made include: • The % of females that become pregnant • The number of stillborn & live offspring, • The wt, growth, survival, & general condition of the offspring during the first 3 wks of life 26
- 27. Developmental & reproductive toxicity… b)Teratogenic effects (segment II) studies: Teratogens are most effective when administered during the 1st trimester, the period of organogenesis Thus, the animals (usually 12 rabbits & 24 rats or mice per group) are usually exposed to one of three dosages during organogenesis (day 7 to 17 in rodents & days 7 to 19 in rabbits), & the fetuses are removed by cesarean section a day before the estimated time of delivery (gestational days 29 for rabbit, 20 for rat, & 18 for mouse) 27
- 28. Developmental & reproductive toxicity… Segment II studies.. The uterus is excised & weighed & then examined for the number of live, dead, & resorbed fetuses Live fetuses are weighed Half of each litter is examined for skeletal abnormalities & the remaining half for soft tissue anomalies 28
- 29. Developmental & reproductive toxicity… c)Perinatal & postnatal toxicities of chemicals (segment III) studies: This test is performed by administering the test compound to rats from the 15th day of gestation throughout delivery & lactation Then determining its effect on the birth- weight, survival, & growth of the offspring during the first 3 wks of life 29
- 30. B. Mutagenicity Mutagenesisis the ability of chemicals to cause changes in the genetic material in the nucleus of cells in ways that allow the changes to be transmitted during cell division Mutations can occur in either of 2 cell types, with substantially different consequences Germinal mutations Somatic mutations 30
- 31. Mutagenicity… Germinal mutations: Damage DNA in sperm & ova, which can undergo meiotic division & therefore have the potential for transmission of the mutations to future generations If mutations are present at the time of fertilization in either the egg or the sperm, the resulting combination of genetic material may not be viable, & the death may occur in the early stages of embryonic cell division 31
- 32. Mutagenicity… Germinal mutations… Alternatively, the mutation in the genetic material may not affect early embryogenesis but may result in the death of the fetus at a later developmental period, resulting in abortion Congenital abnormalities may also result from mutations 32
- 33. Mutagenicity… Somatic mutations: Mutations in all other cell types & are not heritable but may result in cell death or transmission of a genetic defect to other cells in the same tissue through mitotic division 33
- 34. Mutagenicity… B/c theinitiating event of chemical carcinogenesis is thought to be a mutagenic one, mutagenic tests are often used to screen for potential carcinogens Numerous in vivo & in vitro procedures have been devised to test chemicals for their ability to cause mutations 34
- 35. Mutagenicity… Some geneticalterations are visible with the light microscope In this case, cytogenetic analysis of bone marrow smears is used after the animals have been exposed to the test agent B/c some mutations are incompatible with normal development, the mutagenic potential of a chemical can also be evaluated by the dominant lethal test This test is usually performed in rodents 35
- 36. Mutagenicity… The dominantlethal test… The male is exposed to a single dose of the test compound & then is mated with two untreated females weekly for 8 wks The females are killed before term, & the number of live embryos & the number of corpora lutea are determined 36
- 37. Mutagenicity… Salmonella/microsome/Ames test: The test for mutagens that has received the widest attention that is developed by Ames & colleagues This test uses several mutant strains of S. typhimurium that lack the enzyme phosphoribosyl ATP synthetase, which is required for histidine synthesis These strains are unable to grow in a histidine- deficient medium unless a reverse or back- mutation to the wild type has occurred 37
- 38. Mutagenicity… Ames test… Other mutations in these bacteria have been introduced to enhance the sensitivity of the strains to mutagenesis The 2 most significant additional mutations enhance penetration of substances into the bacteria & decrease the ability of the bacteria to repair DNA damage 38
- 39. Mutagenicity… Ames test… B/c many chemicals are not mutagenic or carcinogenic unless they are biotransformed to a toxic product by enzymes in the ER, rat liver microsomes are usually added to the medium containing the mutant strain & the test chemical The number of reverse mutations is then quantified by the number of bacterial colonies that grow in a histidine-deficient medium 39
- 40. C. Tests forcarcinogens a) The Standard Test: The currently accepted design for carcinogenicity testing is to expose rats & mice to the agent for about 2 years For each species, 50 male & 50 female animals per group are dosed with a vehicle or the test agent in that vehicle Daily observations are made & if any animals become moribund during the experiment, they are killed so that tissues are not lost due to autolysis 40
- 41. Tests for carcinogens… The Standard Test.. All animals, including those that survive to the scheduled end of the experiment, are subjected to autopsy & almost 40 different tissues are taken from each animal for histological examination All observations are recorded, summarized & analyzed 41
- 42. D. Immunotoxicity Assessment Objective: To determine the potential of a test material to induce immune suppression or immune enhancement Duration: Subacute (14 days) or sub-chronic (90 days) exposure Test system/animal system: Rodents Dose administration: Repeated doses administered as in sub-acute/sub-chronic toxicity studies 42
- 43. Immunotoxicity Assessment… Parameters: Level 1: • Hematology • Histopathology or lymphoid organs • Quantity of T- & B-cells (cellularity of lymphoid organs) • Blastogenesis (mitogen responsiveness; mixed lymphocyte reaction) • Quantitation and funciton of natural killer cells • Macrophage function • Cytokine production 43
- 44. Immunotoxicity Assessment… Parameters… Level 2: • Kinetics of antibody production to T- dependent antigens • Quantity of IgM/IgG-producing (plaque- forming) cells • Delayed hypersensitivity responses to known sensitizers • Immune response to infectious agents (e.g., Listeria, Streptococcus) • Immune response to transplantable tumors 44
- 45. E. Irritation &sensitization - local tolerance studies Objectives: To determine the potential of a test material to provoke ocular irritation, dermal irritation, or sensitization Duration: Irritation - 1 hr to 3 wks after a single topical or corneal administration Sensitization - intradermal or topical induction doses followed by topical challenges with a non- irritating dose (6 - 8 wks total) Test system/animal system: Rodents, rabbits 45
- 46. Irritation & sensitization- local tolerance studies… Test system/animal system: Rodents, rabbits Dose administration Single patch administration Multiple doses over 2—4 weeks Topical (epicutaneous), intradermal, or corneal 46
- 47. Irritation & sensitization- local tolerance studies… Parameters: Degree of pruritis, erythema, edema, papules, and vesicles Corneal irritation, swelling, or injury Microscopic integrity of corneal endothelium Other features of the eye (conjuctive, cornea, iris, lens, anterior portion of vitreous humor) 47