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Determinants of Repeated exposure Toxicity

*If acute toxicity studies show a potential for cumulative local or systemic toxicity.

*Exposure of humans to xenobiotics at levels much lower than those are acutely fatal, but they are exposed over longer periods of time.

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Factors associated with likelihood for repeated exposure toxicity

C-Kinetic parameters and biotransformation:

1-Bioavailability of the xenobiotic.

2-Biotransformation.

A- Repeated exposure duration:

1-Latent period.

2-Number of exposures.

B-Dosing Characteristics.

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Duration of the repeated exposure study

Depend on the reasons for conducting the study which include:

1-Determination of potential carcinogenicity or non-carcinogenicity of the test substance.

2-Determine threshold on No Observed Adverse Effect dosages.

3-To meet requirements of regulatory authorities for activities such as product registration.

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Accordingly

*Repeated exposure studies vary in duration from a few days to a lifetime of exposure.

*It can be subdivided into

1-Short term toxicity studies.

2-Subchronic studies.

3-Chronic studies.

*Repeated exposure studies should be carried in sequence, to give guidance on the types of adverse effects to be anticipated, and also to allow the choice of appropriate dosing for the longer studies.

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Short Term Studies

*Vary in length from a few days (7-9) up to 28 days.

*Shorter studies (around 7 days), have sometimes referred to subacute studies,

*The daily dosages less than the acute doses and total duration is much longer (and not below “sub”) than for the acute studies. So the term subacute is not a preferred description.

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Subchronic Studies*Aim:

1-Determine the potential for non-carcinogenic or long-term toxicity effects.

2-The Lowest Observed Adverse Effect Level LOAEL.

3-The No Observed Adverse Effect Level NOAEL

*Involve exposing the test species for 15-20% of their lifespan to the test substance.

*The duration in rats is 90 days while in dogs; the duration extends to 6 months or even one year

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Chronic studies

*Aim: Determine the in-vivo carcinogenic potential of the test substance.

*Exposure of the test species to the test substance for their life span, or greater part there of. The duration in rats is 24 months and 7 years or more in dogs.

*Because of high cost of such studies, the majorities are usually conducted as a combined chronic and carcinogenicity studies.

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Study Protocol (Experimental Design)

1-Animals: species and number.

2-Rout of administration of the test substance.

3-Dose of the test substance.

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Animals

*Animals should be of comparable age and within a restricted weight range.

*Randomly selected subgroups from the test batch of animals should be checked for health status before the start of the study, including:

1-     Clinical veterinarian examination

2-     Blood chemistry

3-    Viral antibodies

4- Necropsy for growth pathology and histology of selected tissues and organs (lungs, GIT, liver and kidney)

*two or more species of animals are used (the rat and the dog are usually selected)

*Equal number of male and female animals is used (usually 50 animals in each dose group).

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Route of administration

*For most, the common route is oral

*The preferred procedure is to incorporate the test substance in diet, although the drinking water is sometimes used

*Dermal application, inhalation and parental routes are used for special purposes such as industrial products and drugs

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Dosage and grouping

*Three dose levels are used to determine the nature and site of the toxic effects as well as the NOAEL.

*The first dose that is high enough to elicit definite signs of toxicity but not high enough to kill many of the animals

*The second low dose that is expected to induce no toxic effect

*The third intermediate dose

*The fourth group (the control group) will receive any vehicle as a control

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Monitoring for toxicity

Specific techniques to assess sites of toxic response in particular tissues are implied include:

1-Clinical observation for signs of toxicity

2-Periodic measurement of body weight

3-Food and water consumption

4-Peripheral blood hematology

5-Blood chemistry

6-Urine analysis

7-Necropsy

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Special Toxicity Studies

Teratogenicity

Mutagenicity

Carcinogenicity

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A-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

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Effect on the mother

1-Effects on lactation.

2-Acceptance of offspring.

3-Effects on the offspring as regards:

1-Growth and development

2-Sexual maturation

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Effects on fertility

1-Gonadal function

2-Estrous cycle

3-Mating behavior

4-Conception rates

5-Early stages of gestation (implantation of fertilized ovum)

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Effects on the development of the fetus

1-Degree of normality

2-Teratogenicity and mutagenic effects

3-Intra-uterine mortality

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Methodology

*Animals used: (Rats)

*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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B-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.

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Types of Mutation

1-Point mutations:

*Alteration in a single nucleotide pair in the DNA molecule and usually leads to a change in one biochemical function.

2-Chromosome aberrations:

*Include breaks and rearrangement of chromosomes as well as changes in chromosome number.

*Can be detected by cytological examination of the chromosomes.

*Detected by changes in phenotypes and their location.

*Can be determined by genetic mapping.

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Mutation Tests

(1)In-Vitro Test:

*Organisms used: sub mammalian systems including bacteria, yeast, plants and insects.

*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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(2)In-Vivo Tests

Dominant Lethal Assay

*Indicate that genetic damage has occurred in the form of structural or numerical chromosome aberrations.

*Advantages:Tests the sensitivity of mammalian germ cells in-vivo at different stages of development.

*Disadvantages: Cannot detect point mutation.

*Methodology:

Male mice or rats are treated with a dose 1/5 of LD50 .

-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

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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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In-vivo Cytogenes

*Methodology:

-Cytological examination of mammalian cells (animal or human) to detect chromosomal abnormalities.

-Tissues used: B.M., 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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C-Carcinogenicity Tests

*A substance that produce any type of tumor whether the tumor is benign or Malignant in test species is called Carcinogenic.

*Chemical induced carcinogenesis in man is substantiated only through epidemiological studies from occupational, environmental or medical exposure.

*If a compound is known to be a carcinogen in man the carcinogenicity has to be confirmed in experimental animals.

*Although certain compounds are not carcinogenic in test species, they can biotransformed in suitable species to active carcinogen.

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Types of Carcinogens

A-Genotoxic (Initiators):

-Primary: acting directly on DNA.

-Secondary (Commoner): Reactive metabolites act on DNA.

B-Epigenetics: They increase the likelihood of tumor development i.e. not true carcinogens.

1- Promotors: e.g. Saccharin in large doses and cigarette smoking

2- Co-carcinogens: enhance the effect of genotoxic substances (same agents as promotors).

3- Hormones: in hormones dependent tumors e.g. cancer breast and prostate.

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*Animals:

Rats and Dogs.

*Test duration:

-Rats: 2-3 years.

-Dogs: 7 y ears.

*Route of administration: Oral.

*Dose: 3 dose concentrations

-Maximally tolerated dose(MTD).

-1/3 of MTD.

-1/9 of MTD.

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*All animals that become seriously ill during the course of the test are killed and complete clinical, chemical and pathological examination is done.

*If tumors become evident during the course of the test, the time of occurrence, characteristics and type of the tumor should be recorded.

*At the end of the test the animals are killed and complete pathological examination of all tissues is done.

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