Toxicity studies are important for developing new drugs and extending the use of existing drugs. They help determine the potential pharmacological effects and toxicity of new molecules in animals according to FDA guidelines. Toxicity tests examine adverse events like cancer, organ toxicity, and skin/eye irritation. They also help calculate the no observed adverse effect level dose for clinical trials. Acute, subchronic, and chronic toxicity studies in rodents and nonrodents are commonly used to identify target organs of toxicity and determine safe dosage levels over different time periods. Proper ethics approval is required before conducting any animal studies.

1. Toxicity studies
Dr. S. Parasuraman, M.Pharm., Ph.D.,
Senior Lecturer, Faculty of Pharmacy
AIMST University

2. Toxicity studies- Introduction
• Toxicology is a branch of science that deals with toxins and poisons
and their effects and treatment.
• 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”
– Paracelsus
• Mathieu Orfila, determined the relationship
between poisons and their biological He is referred
to as the father of modern toxicology.
Paracelsus
(1493-1541)
Recent developments: after 1920
(introduced determine LD50 by
USFDA )

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. Biomedical ethics
• Before conducting any toxicological testing in animals or collecting
tissue/cell lines from animals, the study should be approved by the
Institute Animal Ethics Committee (IAEC) or the protocol should
satisfy the guidelines of the local governing body.
• Malaysia:
– http://www.animalethics.org.au/animal-ethics-committees

6. Toxicity testing methods
• Acute toxicity testing
• Subchronic toxicity testing
(repeated dose =/ >90 day)
• Chronic toxicity testing
(repeated dose <90 day)
• Mutagenicity testing
• Carcinogenicity testing
• One-generation reproduction
toxicity testing
• Two-generation reproduction
toxicity studies
• Developmental
toxicity/embryotoxicity studies
• Genetic toxicity testing

7. Acute toxicity studies
• 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.

8. Acute toxicity studies
• The LD50 was used as an indicator of acute toxicity previously. The
determination of the LD50 involves large numbers of animals, and
the mortality ratio is high. (24 h testing)
– Graphical method
– Arithmetical method (karbers’s) method. when number of animal is
small)
• Because of these
limitations, modified methods
were developed:
1. The fixed dose procedure
2. The acute toxic category method
3. The up-and-down method
Cont.,

9. Acute toxicity studies
• Signs recorded during acute toxicity studies
Cont.,
• Increases motor activity
• Decreased motor activity
• Tremors and ataxia
• Tonic convulsion
• Catatonia
• loss of righting reflex
• Sedation
• muscle relaxation
• Piloerection/ straub tail phenomenon
• Cannibalism
• Straub reaction
• Hypnosis
• Analgesia
• Lacrimation
• Diarrahoea
• Salivation: viscid or watery
• Writhing
• Respiration:
depression, stimulation or
failure.
• Skin color
Piloerection PiloerectionNormal

10. Sub-acute toxicity studies
• Rodents and non-rodents are used to study the subchronic 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.

11. Chronic toxicity studies
• Chronic toxicity studies are conducted with a minimum of one
rodent and one nonrodent species.
• The test compound is administered over more than 90 days, and
the animals are observed periodically.
• Dose:
• 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%.

12. • 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
Chronic toxicity studies Cont.,

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

14. Parasuraman S. Toxicological screening. J Pharmacol Pharmacother 2011;2:74-9.
Available from: http://www.jpharmacol.com/text.asp?2011/2/2/74/81895