THE MAHARAJA SAYAJIRAO UNIVERSITY, VADODARA
Pharmacology Practical Exam
PRESENTED BY:
Nikunj Unadkat
Roll No. 25
Exam seat no. 601029
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Objective
Acute Toxicity Study
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What is Toxicology
• Toxicology is the scientific study of adverse effects that occurs in living organisms
due to chemicals.
• It involves observing and reporting symptoms, mechanism, detection and
treatment of toxic substance, in particular relation to the poisoning of humans.
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Adverse Drug Effects
• Any undesirable effects of Drug
1. Predictable
2. Non predictable
SECONDARY TOXIC EFFECTS
SIDE EFFECTS
EFFECTS Are results of
Unwanted but often excessive
Indirect
unavoidable effects pharmacological
consequences of
at therapeutics effects of drug due to
primary action of
doses. over dosage or
drug
prolonged use
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Toxicity Tests
Acute Toxicity 14 days
Sub-acute Toxicity 28 days
Sub-chronic Toxicity 3 months
Chronic Toxicity 6 to 12 months
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Acute toxicity (LD50) test
Definition
Acute systemic toxicity evaluates the adverse effects that occur following
exposure of organisms to a single or multiple doses of a test substance within 24
hours by a known route (oral, dermal or inhalation).
The LD50 (median lethal dose) test was introduced in 1927 by J. W. Trevan
to estimate the dose of a test substance that produces 50% death in a given
species of animals.
Substances with LD50 below 5 mg/ kg are classified to be highly toxic while
substances with LD50 above 15,000 mg/kg are termed relatively harmless
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Classification of LD50 based on dose
range
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History and timeline of acute toxicity
testing
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1920: The conventional LD50 test
• First acute toxicity test
• It was called “Classical LD50”
• Up to 100 animals for five dose-groups is used.
• Animals are dosed with the test chemical to determine the dose that would result
in 50 % deaths
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1931: Karbal method
• Involves the use of 30 animals which are divided into six groups of five animals
each.
• The animals are dosed with the test substance and observed for the first four
hours, 24 hours and daily for 14-days for signs of toxicity.
• At the end of 14 days the total number of death is recorded
• LD50 = LD 100 – {Σ [Dose difference × Mean dead]}/ Number of animals per group
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• Example: Result of lethal dose of thymoquinone for determination of LD50 after
intraperitoneal injection in rats (n=10)
Group Dose (mg/kg) Dose No. of Dead Mean Product
Difference (a) Mortality (b) (a*b)
1 25 ----- 0 ---- ----
2 50 25 4 2 50
3 75 25 7 5.5 137.5
4 100 25 9 8 200
5 150 50 10 9.5 475
Σ a*b=862.5
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• LD50=Maximum dose – {Σ [Dose difference × Mean mortality] } / Number of
animals
• LD50=150-(862.5/10)
• LD50=63.75 mg/kg
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1938: Arithmetical Method of Reed and
Muench
• In this test, animals are exposed to the chemical and the log dose, number of
death and survival, cumulative death and survival are calculated.
• In this approach, 40 test animals were divided into four groups of 10 animals each
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1940: Approximate lethal dose
• Due to the limitations characterized by high mortality rate in the conventional
LD50 test.
• An alternative procedure for the determination of approximate lethal dose (ALD)
was developed in the early 1940s
• The animals are administered with the dose of the test substance that increases
by 50 percent over the previous dose
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1944: Miller and Tainter method
• It involves the use of 50 animals which are divided into 5 groups of 10 animals
each.
• LD50 is calculated using probit analyses table.
• Probit values are plotted against log doses and the dose corresponding to probit
five becomes the LD50 value.
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Comparison of various conventional
methods used for LD50 determination.
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Limitations posed by the conventional LD50
methods and development of alternative methods
• The alternative to animal testing kicked off in the 1980s when animal rights
activists motivated the cosmetic industry to begin researching on unconventional
methods to animal tests.
• They recommended that proper experimental design should reflect on methods
that could reduce, refine and replace (3Rs) the current techniques.
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The 3Rs alternatives
1. Reduction
Reduction approach implies that the number of animals employed in a given test
should be minimized while still maintaining consistency and accuracy with scientific
practices that would yield convincing and valid results
2. Refinement
Refinement approach is geared towards providing better welfare to animals by
minimizing pain (by using appropriate anesthetic and analgesics), distress and
provision of a suitable environment for animals
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The 3Rs alternatives
3. Replacement
Replacement approach involves methods other than the use of animals . Such
methods include, in vitro and in silico approaches
Implementation of the 3Rs principles had significantly reduced the number of
animals as well as reduction in drug failure rate in the discovery and development
pipeline
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OECD
• The mission of organization for Economic Co-operation and Development is to
promote policies that will improve the economic and social well-being of people
around the world.
• Work with governments to understand what drives economic, social and
environmental change.
• Set international standards on a wide range of things, from agriculture and tax to
safety of chemicals.
• The OECD was officially born on 30th September 1961, when the convention
entered into force.
• The OECG has been co-operating with India since 1995.
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1981: Incorporation of the LD50 tests into
the OECD guideline
• In 1981, the Organization for Economic Cooperation and Development (OECD)
incorporated the LD50 test into its new test guidelines.
• It involves the use of 30 animals for 3 doses
• In 1987 the OECD further modified this method by reducing the number from 30 to
20 animals where 5 animals per dose level are selected based on sighting studies
or from historical data of the chemical to be tested.
• The limit test is usually employed whenever a test substance is suspected to be
non-toxic based on historical information about the test substance.
• This involves the exposure of few numbers of animals to large dose (5,000mg/kg)
of the test chemical. If animals survive, the LD50 is estimated to be above 5,000
mg/kg and no further acute toxicity testing is required
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1983: Lorke’s method
• Use of 13 animals in 2 phases.
• In the first phase, 9 animals are divided into 3 groups of 3 animals each and are
administered 10, 100 and 1,000 mg/kg body weight of the test substance in order
to establish the dose range producing any toxic effect.
• The number of deaths in each group is recorded after 24-hours.
• In the second phase, 4 doses of the test substance are selected based on the result
of phase 1 and are administered to 4 groups of 1 animal each. After 24 hours, the
number of deaths is recorded and the LD50 is calculated as the geometric mean of
the highest non-lethal dose (a) and the least toxic dose (b).
• LD50 = √a×b
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1992: Fixed dose procedure (FDP)
• The test substance is given at one of the four fixed-doses (5, 50, 500, and 2,000
mg/kg) to 5 male and 5 female animals of the same species.
• The objective of the FDP is to identify a dose that produces clear signs of toxicity
but no mortality.
• If mortality occurs, retesting at a lower dose level is necessary except if the original
dose chosen is 5 mg/kg
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1996: Acute toxic class (ATC) method
• It is based on a sequential dosing
• One dose group 5, 50, 300 or 2,000 mg/kg body weight is used at a time.
• Procedure uses 3 animals of one sex per step at any of the defined dose levels.
• 3 but not exceeding 6 animals is used per dose level.
• The result of this approach is reproducible and the number of animals used is
reduced by 40–70% compared to the traditional methods
• In this method, death is not used as the only end point, but signs of toxicity in its
stepwise approach are also used for estimating the LD50
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1998: Up and down procedure (UDP)
• Test substance is estimated by testing individual animals sequentially, with the
dose for each animal being regulated up or down based on the results of the
preceding tests.
• Animals are dosed one at a time
• The dose for the next animal is increased by a factor of 3.2 if the preceding animal
survives, while the dose is decreased by a factor of 3.2 if the animal dies.
• It takes 1 or 2 days to observe each animal before dosing the next animal.
• Thereafter, animals that survive the test are monitored for delayed toxicity for 7
days
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2013: Proposed acute toxicity test method
of Enegide et al.
• The test method is divided into 3 stages
Stage 1
• Requires four animals which are divided into 4 groups of one animal each at 10, 100,
300 and 600 mg/kg or 50, 200, 400 and 800 mg/kg of the test substance.
• If mortality is not observed in this stage, the testing proceeds to stage 2.
Stage 2
• Involves three animals which are divided into 3 groups of one animal each receiving
different doses higher than those used in the first stage.
• If no mortality occurs, testing proceeds to stage 3.
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Contd..
Stage 3
• Requires the use of 3 animals which are distributed into 3 groups of one animal
each.
• Higher doses (not exceeding 5,000 mg/kg) of the test substance are administered
to the different animals. When no signs of toxicity and mortality are recorded at
this final stage of testing, the LD50 of the test substance is said to be greater than
5,000 mg/kg
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Confirmatory test
• A confirmatory test is usually carried out whenever death of an animal is recorded
at any stage by administering the lowest dose that cause mortality to 2 animals,
followed by observation.
• Where at least a single animal from the 2 animals dies, the confirmatory test is
validated. Also, if no mortality is still recorded at 5,000 mg/kg, a confirmatory test
is also carried-out by administering 5,000 mg/kg to 2 animals.
• LD50=[M0+M1]/2
• where M0 = highest dose of test substance that produced no mortality,
• M1 = lowest dose of test substance that produced mortality.
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Comparison of various alternative
methods used for LD50 estimation:
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Reference
• Review Article Advances in acute toxicity testing: strengths, weaknesses and
regulatory acceptance by sciendo
• [Link]
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Thank You
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