Introduction to toxicology, Acute, Sub-Acute, Chronic Toxicity, Organ-specific toxicity, Mutagenicity, teratogenicity and carcinogenicity, Effect on reproductive system,

1. Introduction to toxicology, Acute, Sub-Acute,
Chronic Toxicity, Organ specific toxicity,
Mutagenicity, teratogenicity and carcinogenicity,
Effect on reproductive system,
By: Dr. Manoj Kumar

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”
• Mathieu Orfila, determined the relationship between poisons and their biological
effect. 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. 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.

6. 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

7. 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

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

9. SINGLE DOSE STUDIES/ ACUTE TOXICITY
• 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.
• Minimum Lethal Dose (MLD) and Maximum Tolerated Dose (MTD) should be established

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

11. 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.

12. REPEATED DOSE STUDIES/SUB-ACUTE OR CHRONIC TOXICITY
• 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

13. • 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.

14. Chronic toxicity studies
These 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%.

15. • 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
• During the study period, the animals are observed for normal physiological
functions, behavioral variations and alterations in biochemical parameters at
regular intervals (at-least every 14 days).
• At the end of the study, tissues are collected from all parts of the animal and
subjected to histological analyses.

16. TYPES OF LOCAL TOXICITY STUDIES
Dermal toxicity studies -Rats & Rabbit
-Local signs (erythema, oedema)
-histological examination
Dermal photo-toxicity
Studies
-Guinea pig
-Used in treatment of leucoderma
-Examination of erythema & oedema formation
Vaginal toxicity studies -Rabbit or Dog
-Observation of swelling,
-histopathology of vaginal wall
Rectal tolerance studies -Rabbit or Dog
-Signs of pain, blood or mucous
-histology examination of rectal mucosa

17. Ocular toxicity studies -Albino Rabbit
-Changes in cornea ,Iris & aqueous humor,
histological examination of eye
Parenteral drugs -For intravenous/ intramuscular/ subcutaneous/
intra-dermal injection
-Sites of injection examined grossly and
Microscopically
Inhalation toxicity studies -One rodent and non rodent species
-Acute , sub-acute and chronic studies performed
-Observation of respiratory rate
-Histological examination of respiratory passages,
lung tissue

18. 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

19. Micronucleus Assay
• In vivo test for chromosomal damage using mammalian
hematopoietic cells.
• Chromosome damage in rodent hematopoietic cells

20. 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

21. Fertility Testing
Male fertility study
One rodent species (preferably rat) should be used.
Dose selection from previous 14 days and 28 days study
3 groups (6 adult male rats) with 3 different doses (test drug with intended clinical route)
Dosing for min 28 days and max 70 days
Male Rat then paired with female in 1:2 ratio
Drug treatment of the male animals should continue during pairing
Female rats are then examined for their fertility index after 13 days
All males are then sacrificed at the end of the study and their reproductive organs are then examined .

22. • Female reproduction and developmental toxicity studies
• One rodent species (rat preferred)
• Drug should be administered in both the sexes (28 days in males and 14 days in females) before
mating
• Three graded doses should be used (Highest dose should be maximum tolerated dose)
• 15 males and 15 females should be used per dose group (route is intended therapeutic dose).
• Observations on body weight, food intake, clinical signs of intoxication, mating behaviour, progress
of gestation or parturition periods, length of gestation, parturition, postpartum health and gross
pathology (and histopathology of affected organs) of dams should be recorded.

23. • Teratogenicity study
• One rodent (preferably rat) and one non-rodent (rabbit) species are to be used.
• The highest dose should cause minimum maternal toxicity and the lowest one should be proportional to the proposed
dose for clinical use in humans
• 20 pregnant rats (or mice) and 12 rabbits, on each dose level.
• Observation
• parameters should include: signs of intoxication, effect on body weight, effect on food intake,
• examination of uterus, ovaries and uterine contents, number of corpora lutea, implantation sites,
• resorptions (if any); and for the foetuses, the total number, gender, body length, weight and gross or
• visceral or skeletal abnormalities
• Observation parameters should include: (Dams) signs of intoxication, effect on body weight, effect on food intake,
examination of uterus, ovaries and uterine contents, number of corpora lutea, implantation sites, resorptions (if any);
and
• for the foetuses, the total number, gender, body length, weight and gross or visceral or skeletal abnormalities

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