Acute and Chronic Toxicity Studies in Animals

1. ACUTE AND CHRONIC TOXICITY STUDIES IN
ANIMALS
Dr.Swaroopa
MD Pharmacology, 1st year PG,
Rangaraya Medical College.
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1. Introduction
2. Objectives of toxicity studies
3. General principles
4. Types
5. Systemic toxicity studies
6. Lethality testing and calculation
7. Conclusion
8. References
CONTENTS

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• Father of Toxicology – Paracelsus
• Father of Modern Toxicology – Mathieu Orfila
All substances are poisons; there is none which is not a poison.
The right dose differentiate a poison and a remedy.

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TOXICITY STUDIES --- INTRODUCTION
• Casarett 1996 defined toxicology as a science that defines the limits of safety of
chemical agents for human and animal populations.
• Toxicity studies/screening (hyperpharmacology) is very important for the development
of new drugs & for the extension of the therapeutic potential of existing molecules apart
from pharmacodynamics and pharmacokinetic studies.
• During toxicity studies, always two or more species are preferred as there is species to
species variation in the pharmacological responses.

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Before conducting any toxicological studies in animals or collecting tissue/cell
lines from animals, the study should be approved by the Institutional Animal
Ethics Committee or CPCSEA ( Committee for the Purpose of Control and
Supervision of Experiments on Animals.
BIOMEDICAL ETHICS

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OBJECTIVES OF THE TOXICITY STUDY
 To identify any toxic substance prior to clinical use.
 Qualitative and Quantitative assessment of drug use.
 Mostly used to examine specific adverse events or specific end points such as cancer,
cardiotoxicity, skin/eye irritation.
 Different types of dose identification can be done. (MLD, LD50, MTD, ED50, NOAEL)
 Prediction of therapeutic index (LD50 / ED50 )
 Benefit-risk ratio can be calculated.
 To relate the toxicological findings to clinical safety.
 To support in selecting species, treatment regimen and designing subsequent non
clinical toxicity studies.

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GENERAL PRINCIPLES
In a large set-up, conducting toxicity studies should comply the following important
requirements:
 Should follow the Good Laboratory Practices(GLP).
 Studies should be performed by suitably trained and qualified staff.
 Instruments should be calibrated and standardized properly and of adequate capacity.
 Standard operating procedures(SOPs)should be followed.
 Documentation must be proper and anticipate the results.
 All documents, histology slides and paraffin tissue blocks should be preserved for a
minimum of 5years after marketing of the drug.

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TYPES OF TOXICITY STUDIES:
Systemic Toxicity studies
Male fertility studies.
Female Reproduction and Developmental Toxicity Studies.
Teratogenicity studies.
Perinatal studies.
Local toxicity studies
Geno toxicity studies.
Carcinogenicity studies.
• Single dose toxicity study(Acute toxicity studies)
• Repeated-dose toxicity study(subacute/chronic toxicity study).

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SYSTEMIC TOXICITY STUDIES in
animals

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ACUTE TOXICITY STUDY
 Acute toxicity/single dose study – study of the effect of a single dose on a particular
animal species.
 Testing carried out in 2 different animal species (one rodent and one non rodent)
 Route of administration : as intended for humans.
 Doses :administered at different dose levels, at least three-graded doses. Given in a
single bolus or by infusion within 24 hrs.
 Animals should be observed for 14 days.
observations:- 1. signs of intoxication
2. effect on body weight
3. gross pathological changes
4. histopathology of grossly affected organs(if any)
 All mortalities caused during experimental period are recorded and morphological,
biochemical, pathological and histological changes in the dead animals are recorded.

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Acute toxicity studies/ single dose studies in animals
 are essential for any pharmaceutical product intended for human use.
 Information obtained from these studies is useful in choosing doses for
repeated dose studies
 Providing preliminary identification of target organs of toxicity,
 These studies may also aid in the selection of starting doses for phase 1
human studies
 Provide information relevant to acute overdosing in humans.
 Death is considered as a single end point and other behavioural parameters
are not taken under consideration.
 LD50 is calculated (dose required to kill 50% of the population)

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Lethality testing and calculation :-
 This is an essential regulatory requirement for a drug before clinical study.
 Lethal dose – “dose of a given drug which produces mortality in the treated animal,
especially in most sensitive species model.”
 LD50 is defined as “dose of a given drug which produces mortality in 50% of total
treated animals preferably in the most sensitive species model.”
 This is preferred over sub chronic or chronic testing.
 Lethality testing is done mainly on 3doses of selection
 Toxicity is varied according to route of administration. There is a rule of thumb, i.e.
LD50 is roughly 30%of oral, I.V is 10% of that of the oral.
 Duration of testing ranges from 7 days to 14 days.
• One dose which has 100% lethality
• Second –produces marginal lethality
• Third is taken in between of first 2
doses.

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There are 2 methods of calculating LD50 1. Mathematical method
2. Graphical method
Mathematical method :-
Karber’s method is very commonly used because it is simple and does
not need to plot the dose response curve.
CALCULATION:

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1
Exp grpc Dose(mg/kg) Dose
diff(DD)
No of
animals(n)
No of dead
animals
Mean
mortality
DD X MM
1 3 - 10 0 (A) -- --
2 5 2 10 2 (B) 1 2
3 10 5 10 3 2.5 12.5
4 12 2 10 5 4 8
5 15 3 10 7 6.5 19.5
6 20 5 10 10 8.5 42.5
84.5
ΣDD MM
Mean mortality= (A+B)/2 =(0+2)/2 =1
Σ(DD MM) = 84.5;then value is divided by the no. of animals in the group
(here n =10), hence, value obtained is 84.5/10 = 8.45
Finally, this value is subtracted from the minimum dose which produces the
100 % mortality, i.e. 20mg/kg
So, LD50 = 20 – 8.45 =11.55 mg/kg

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GRAPHICAL METHOD :-
• This method well described by the Miller and Tainter(1944) as well as
Litchfield and Wilcoxon (1949).
• Depends on toxic log dose response curve and interpretation of the result is
done by the curve.
• First convert the percentage response into the probit, then plot the curve log
dose on x- axis and y-axis contains probit scale.
• The LD50 is the antilog (1d) value which falls on x-axis.

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REPEATED DOSE TOXICITY STUDIES :-
Subacute/subchronic or chronic toxicity study falls under these
category.
SUBACUTE/SUB CHRONIC STUDY :
• Aim is to identify target organ toxicity and establishment of MTD for
subsequent studies.
• Atleast 2 mammalian species are used (1 rodent and 1 non rodent)
• Rodent – 6 to 8 weeks of age, non rodent 4 to 9 months age.
• Treatment & duration : 14,28,90 or 180 days (depends on therapeutic
indication)
• Route of administration : same as intended for humans.
• Doses : At least 3 graded doses.
Group 1 --- Control group – normal or vehicle group.
Group 2 --- Treatment group – drug treatment group.

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CHRONIC TOXICITY STUDIES :-
• Repeated dose study from 6 months to 12 months or even upto 2 years.
• Primary goal of these studies is to characterize the toxicological profile
of the test compound following repeated administration.
this includes identification of potential target organs of toxicity and
exposure/response relationships, & may include potential reversibility of
toxic effects.
• This information should be part of the safety assessment to support the
conduct of human clinical trials and the approval of a marketing
authorization.

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CHRONIC TOXICITY STUDIES : are conducted with a minimum of one rodent
and one non rodent species.
In rodents, studies are usually for 6 months to 2 years
In rodents, studies are usually for 1year .
Dose selection :-
 Highest dose – should produce observable toxicity.
 Intermediate dose – should cause some symptoms but not gross toxicity or death.
Should be placed logarithmically between the highest and lowest doses.
 Lowest dose – should not cause observable toxicity.
Observation
 Site of injection should be subjected to gross and microscopic examination.
 Non rodent species : Electrocardiogram and fundus examination.
 Other parameters include changes in body weight, food/water intake and …

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1 Liver function tests : total bilirubin, direct or indirect bilirubin, bile
acids, ammonia.
2 Hepatocellular enzymes : AST, ALT, SDH, LDH
3 Muscle parameters : Creatinine kinase (CK), AST, ALT, LDH
4 Pancreatic parameters : Amylase, lipase
5 Lipid parameters : cholesterol, triglycerides cholestatic enzymes –
AP, GGT
6 Calcium, potassium : often influenced by kidney function, kidney
parameters should be evaluated concurrently.
7 Kidney parameters : urea nitrogen, creatinine urine specific gravity
should be evaluated concurrently when evaluating renal function.
Analytical parameters which should be assessed after subacute/sub chronic/chronic
toxicity studies :

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CONCLUSION
• Drug discovery and development of a new drug undergoes
numerous procedures in order to get the safe and effective drug.
• The principle of the toxicity study is to assess all the relative toxic
effects of the drug regarding its dose, duration and its effect on the
organs.
• These studies not only provide adequate therapeutic margin of
initial dose levels and duration of dosing , but also give guidance
for any special measures to be taken in initial clinical trials.

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• SK Gupta, New Drug Development, Drug Discovery and Clinical
Research,1st edition, pg : 18 to 21.
• Bikash Medhi, Ajay Prakash, Practical Manual of Experimental
and Clinical Pharmacology, 2nd edition, pg 136 to 140.
REFERENCES

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