Toxicity studies


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5 TOXICITY STUDIES Prepared By: Gohel Rachana V. M. Pharm (QA) Sem-1 P. G. Department of Pharmaceutical Sciences Sardar Patel University V. V. Nagar 1




DEFINITIONS: Toxicity studies : It is the study of adverse effects of chemical and physical agents and the degree to which a substance can harm human or animals. Toxicity studies can be of, Acute toxicity : it involves harmful effects in an organism through a single or short term exposure. sub-acute toxicity: it is the ability of a toxic substance to cause effects for more than one year but less than the life time of exposed organism. chronic toxicity: it is the ability of the substance or mixture of substances to cause harmful effects over an extended period, usually upon repeated and continuous exposure. 3


Introduction TO TOXICITY STUDIES: It is essential to use at least two species (usually a rodent and a non- rodent) in the evaluation of the potential toxicity of a drug because species differ in their responses to toxic agents. A drug effect that is seen both in the rat and in the dog probably involves a common physiology mechanism that is likely to be present in the human, whereas an effect seen only in one of the two species indicates that the same is peculiar to that species, and is less likely to be present in the third species. 4


ACUTE TOXICITY STUDIES: The acute toxicity test in which a single dose is used in each animal on 1 occasion only for the determination of gross behavior and LD 50 or median lethal dose. It’s an initial step in the assessment and evaluation of the toxic characteristics of a substance. 5

Basic parameters of Acute toxicity tests:

Basic parameters of Acute toxicity tests Species Rats preferred for oral and inhalation tests; rabbits preferred for dermal tests Age Young adults Number of animal 5 of each sex per dose level Dosage Three dose levels recommended; exposures are single doses or fractionated doses up to 24 hours for oral and dermal studies; and 4 hour exposure for inhalation studies Observation period 14 days 6

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Data from the acute study may: (a) Serve as the basis for classification and labeling. (b) Provide initial information on the mode of toxic action of a substance. (c) Help arrive at a dose of a new compound. (d) Help in dose determination in animal studies. (e) Help determine LD 50 values that provide many indices of potential types of drug activity 7


AIM OF ACUTE TOXICITY TEST To determine the therapeutic index(LD 50 /ED 50 ) greater the index, safer is the compound. The greater the index, safer is the compound. LD 50 with confidence limits is to be established on one common laboratory species such as mouse/rat using the standard method . 8

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The LD 50 dose thus found was administered to guinea pigs, rabbits, cats or dogs on weight basis (on basis of relative surface area gives better results). To determine the absolute dose for a species in the column, the absolute dose given to the species in a row was multiplied by the factor given at intersection of the relevant row and column (Table 1). 9

Table 1 : Surface area ratios of some common laboratory species and man:

Table 1 : Surface area ratios of some common laboratory species and man To be determined 20g Mouse 200 g Rat 400g guinea pig 1.5 kg rabbit 2 kg cat 4kg monkey 12 kg dog 70 kg man 20g Mouse 1.0 7.0 12.25 27.8 29 .7 64 . 1 1 2 4 .2 3 8 7.9 200 g rat 0.14 1.0 1.74 3.9 4.2 9.2 17.8 56.0 400g guinea pig 0.08 0.57 1.0 2.25 2.4 5.2 10.2 31.5 1.5 kg rabbit 0.04 0.25 0.44 1.0 1.08 2.4 4.5 14.2 2 kg cat 0.03 0.23 0.41 0.92 1.0 2.2 4.1 13.0 4kg monkey 0.016 0.11 0.19 0.42 0.45 1.0 1.9 6.1 12 kg dog 0.008 0.06 0.10 0.22 0.24 0.52 1.0 3.1 70 kg man 0.0026 0.018 0.031 0.07 0.076 0.16 0.32 1.0 10


DESIGN OF ACUTE TOXICITY TEST Dose selection : is based on the results of a range finding test. Animals showing severe and enduring signs of distress & pain were killed after anaesthesia. Animal selection: Species and strain Number and sex of animals Age Assignment of animals Housing Administration 11

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Dose levels and dose selection Subs used in the toxicity tests should be pure. At least 3 to 4 dose levels were used, spaced appropriately to produce test groups a range of toxic effects & mortality rates. The data should be sufficient to produce a dose response curve and permit an acceptable estimation of LD50.If the lethality of the groups is such that only one group has a lethality falling between 4 and 6 probits, more groups may be required. Solvent: Volume: depends on size of the test animal. rodents NMT1 ml/100 g body wt max of 50 ml/kg. Injection was given slowly and uniformly to avoid undue killing by a drug having predominant action on the CNS/heart. 12

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Route of administration LD 50 value depends on the route of administration. INCREASING ORDER intravenous ,-Preferred to intraperitoneal route intraperitoneal , subcutaneous and oral. Signs recorded: increased motor activity, anesthesia , tremors, arching and rolling, clonic convulsions etc. 13

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Observation After the test the animal is the sole occupant of the cage, with free access to food and water during the observation period of 1–2 h, and there after at intervals. At the end of the test surviving animals were weighed and sacrificed. A gross necropsy was performed autolysis. Necropsies must be performed no later than 16 h after death. 14

Determination of acute lethality :

Determination of acute lethality Five animals in each group (inbred mice, 10-12 weeks old) were used for calculation of LD 50 value There are two following methods: Graphical method (Miller and Tainter) Arithmetic method of Karber 15

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Graphical method (Miller and Tainter ) :- This method is simple and accurate enough in most of the cases and should always be tried first. The observed percentage mortality was converted into probit referring to the probit table (table 2) The values thus obtained were plotted against log dose. The LD 50 value and its standard error were determined from the graph, if the line was straight enough. 16

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Graph between probit and Log dose 17

Arithmetical method of Karber:- :

Arithmetical method of Karber :- The LD 50 may be calculated by Karber’s method that not involve any plotting of dose-response curve. It is the simplest and rapid method The interval mean of the number dead in each group the interval mean of the number dead in each group of animals was used as well as the difference between doses for the same interval. The product of interval mean and dose difference was obtained. The sum of the product was divided by the no. of animals in a group and the resulting quotient was subtracted from the least lethal dose in order to obtain LD 50 value. 18

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19 x


CHRONIC TOXICITY STUDIES: It is the ability of the substance or mixture of substances to cause harmful effects over an extended period, usually upon repeated and continuous exposure. The result of chronic toxicity study in animals should suggest signs and symptoms of adverse reactions to look for in man. REASONS FOR CONDUCTING THE CHRONIC TOXICITY TESTS: to induce a toxic effect through chronic exposure to determine an apparent no observable effect level in the presence of such exposure . 20

Basic parameters:

Basic parameters Species Two species recommended; rodent and non rodent (rat and dog) Age Young adults Number of animal 20 of each sex for rodents, 4 of each sex for non rodents per dose level Dosage Three dose levels recommended; include a toxic dose level and NOAEL( no observed adverse effect level ); exposures generally for 12 months; FDA requests 24 months for food chemicals Observation period 12-24 months 21


DESIGN OF CHRONIC TOXICITY TESTS Test Animals Animal type and sex Age Number of animals Test Substance Method of administration Dosage Administration period Observation 22


SUBACUTE TOXICITY STUDIES: These are designed to examine the adverse effects resulting from repeated exposure over a portion of average lifespan of an experimental animal. Acute non toxic compound may be toxic after prolonged exposure event at low doses, due to accumulation, changes in enzyme levels,and disruption of physiologic and biochemical homeostasis. 23

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Species Rodents (usually rats) preferred for oral and inhalation studies; rabbits for dermal studies; non rodents (Usually dogs) recommended as a second species for oral tests. Age Young adults Number of animal 10 of each sex for rodents, 4 of each sex for non rodents per dose level Dosage Three dose levels plus a control group; include a toxic dose level plus NOAEL; exposure are 90 days Observation period 90 days (same as treatment period) Basic parameters of Sub acute toxicity tests 24

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Exposure period in subchronic studies may vary depending on the objective of the study, species selected for the study and route of administration employed. Subchronic toxicity studies should always attempt to expose the animals by same route that man is most likely to be exposed. 25

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Route of administration in subacute toxicity studies are oral, dermal, inhalation. oral and inhalation subacute studies are generally carried out for three months in shorter lived animals ( rodents ) and 1 year in longer lived animals. dermal studies are usually performed for 1 month or less. 26

Special Toxicity Studies :

Special Toxicity Studies Teratogenicity Mutagenicity 27

Teratogenicity tests :

Teratogenicity tests Tests for effects on reproduction should involve the study on animals which have been exposed to the test substance from the time of conception to the time they produce their own offspring plus a study of the offspring during growth and development 28


DESIGN OF TERATOGENICITY TESTS 29 * Animals : When selecting the animal type, species and breed, take into consideration knowledge concerning reproduction Number of animals: With rats and mice, use 20 or more animals With rabbits, use 12 or more * Route of administration : oral in diet or in drinking water * Duration of study : The three-generation reproductive study on parents, offspring and their sons and daughters. * Dose: At least three different doses are used; the largest of them represent a nearly maximum tolerated dose, which produces no significant effect.

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Observation Mother animal: measure weight and food intake. Do an autopsy approximately 1 day before the expected date of birth, study the establishment of pregnancy, count the no. of corpus lutea & implantations observe the internal organs Fetus: Determine whether the fetus has survived else note time of it’s death Measure the weight ,determine their sex. examination of the external and internal organs and study shape of bones also. 30

Mutagenicity test :

Mutagenicity test Mutagenesis is the induction of alterations in the information content (DNA) of an organism or cell that are not due to the normal process of recombination This alteration may occur in germ or somatic cells. Somatic mutations in a developing organism may lead to abnormal differentiation of its cells. Alterations in the duplicating somatic cells of an adult may lead to Cancer. Types of Mutation: 1-Point mutations- Alteration in a single nucleotide pair in the DNA molecule 2-Chromosome aberrations- breaks and rearrangement of chromosomes 31

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32 Mutation Tests (1)In-Vitro Test: *Methodology: AMES Test Strains and Mutations Salmonella typhimurium for mutations at at G-C pairs; Escherichia coli for mutations at A-T pairs. 2) Experimental endpoint: Reverse mutations in histidine (Salmonella) and tryptophane (Escherichia) operon A test for gene mutation in bacteria (AMES-Test)

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33 Bacteria Test item Plate incorporation: s. Typhimurium+ rat liver enzymes Preincubation: Mix, incubate 20-60 min, add top agar Plate on histidine free agar plates control mutation rate genotoxic Result The strain of Salmonella typhimurium used caries a defective (mutant) gene making it unable to synthesize the amino acid histidine (His) from the ingredients in its culture medium. However, some types of mutations (including this one) can be reversed, a back mutation, with the gene regaining its function. These revertants are able to grow on a medium lacking histidine.

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34 *Advantages: 1-Simple systems allowing detailed analysis of their genetic composition. 2-They are capable of characterizing the type of genetic damage caused by the test substance. *Disadvantages They lack the physiology and metabolism of mammals. This can be overcome partly by addition of the mammalian metabolizing system (as liver homogenates) to the growth medium.

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35 (2)In-Vivo Tests Dominant Lethal Assay *Indicate that genetic damage has occurred in the form of structural or numerical chromosome aberrations. *Methodology: Male mice or rats are treated with a dose 1/5 of LD 50 . -Males mated with groups of untreated females. -Females are killed 14 days after mating, dissected and scored for corpora lutea, early fatal deaths and total implantations * Advantages: Tests the sensitivity of mammalian germ cells in-vivo at different stages of development. *Disadvantages: Cannot detect point mutation

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36 Host Mediated Assay. *Detect substances which are not mutagenic in vitro but are converted to active mutagens in mammals OR substances may be mutagenic In-vitro but detoxified by mammalian system. *Methodology: -Salmonella are injected intraperitonealy into rat or a hamster. -The animal is treated with the test substance orally. -Afterwards sample is withdrawn from peritoneal cavity and mutation in salmonella is measured. *Advantages: 1-Provide information on the metabolism of mutagens. 2-Detect point mutation in mammalian system.

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37 In-vivo Cytogenes *Methodology: Cytological examination of mammalian cells (animal or human) to detect chromosomal abnormalities. Tissues used: lymphocytes, skin fibroblasts, gametocytes and amniotic fluid. *Of the three mammalian systems discussed, no single method is best. *A negative result in one test should be confirmed. *A positive result in any is enough evidence to classify the test substance as mutagenic

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REFERENCES: Ghosh MN, Toxicity studies. In Fundamentals of Experimental Pharmacology, Scientific Book Agency, Calcutta. P ower point presentation of toxicity studies by SHASHANK THAMMISHETTY 38

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Acute Vs chronic exposure: :

Acute Vs chronic exposure: Acute exposure occurs when a dose is delivered as a single event. Chronic exposure is likely to be small quantities of a substance over a long period of time which often results in the slow accumulation of compound in the body. Evaluation of cumulative toxic effects is receiving increased attention because of chronic exposure to low concentrations of various natural and synthetic chemical substances in the environment Thanq THANK U 40

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