This document describes the process for determining the lethal dose at which 50% (LD50) of test animals die following administration of a drug. Mice are divided into groups that receive varying doses of the test drug, either procaine hydrochloride or pentobarbital sodium, via injection. Mortality rates are recorded and converted to probit values, which are then plotted against the log dose to determine the LD50 value, found to be 105.9 mg/kg for procaine hydrochloride in mice. The LD50 provides a measure of a drug's acute toxicity.

1. TO DETERMINE THE LD50 VALUE OF A GIVEN DRUG.
BINAL PATEL
SHREE NARANJIBHAI LALBHAI PATEL COLLEGE OF PHARMACY, UMARAKH.

2. REQUIREMENTS
 Animal- Mice (20-25g)
 Drug- Procaine hydrochloride
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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3. PRINCIPLE
 The determination of ED50 (the
dose effective in producing certain
expected response in 50% of the
animal group) values helps in
ascertaining the potency of a drug
in terms of a reference standard.
 The calculation of ED50 value is
done when a drug is showing
graded response.
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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4.  But, when the response is quantal or all
or none, the ED50 value becomes LD50
(the dose lethal to 50% of the animal
group). Both these values i.e., ED50 and
LD50 are important for knowing the
safety of a drug.
 The ratio between LD50 and ED50
(LD50/ED50) represents therapeutic index.
Greater the therapeutic, safer is the drug.
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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5. METHODS
1. Graphical method
2. Arithmetical method and
3. Statistical approach.
For research purpose, the most widely used method is that of Litchfield and Wilcoxon.
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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6.  For calculating LD50 by either method, find out the least tolerated dose and most
tolerated dose by hit and trail method.
 Once these two doses are determined, select at least 5 doses in between the least
tolerated and most tolerated doses and observe the mortality due to these doses.
 Generally, mice are used for this purpose and each dose group should consist of 10
animals.
 One can determine the LD50 value by different routes of administration. The LD50
value of a new drug is determined by oral as well as by one of the parenteral routes of
administration.
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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7. PROCEDURE:
1. Use overnight fasted mice. Each dose group should consist of 10 mice.
2. Mice are divided into 6-8 group of 10 mice each. The drug under study is injected by
intraperitoneal route (or Oral route) in different doses to different groups.
3. The animals are then observed for signs of toxicity and the mortality for 2 hours and
then at 24 hours.
4. At the end of 24 hours number of animals dead are counted in each group percent
death is then calculated and is transferred to probits using probit table.
5. Probits are then plotted (y-axis) against log dose of the drug.
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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8.  Procaine Hydrochloride Toxicity Data in Mice
OBSERVATIONS:
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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9. TRANSFORMATION OF PERCENTAGES TO PROBITS
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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10. OBSERVATIONS:
 Procaine Hydrochloride Toxicity Data in Mice
Group Dose
mg/kg, ip
Log dose Dead/Total % Dead Corrected
%
Probit
1 68 1.83 0/10 0 2.5* 3.04
2 75 1.87 1/10 10 10 3.72
3 91 1.96 2/10 20 20 4.16
4 110 2.04 6/10 60 60 5.25
5 146 2.16 9/10 90 90 6.28
6 161 2.21 10/10 100 97.5* 6.96
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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11. 0
1
2
3
4
5
6
7
8
0 20 40 60 80 100 120 140 160 180
PROBIT
DOSE
105.9 MG/KG
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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12. RESULT
 THE LD50 VALUE OF PROCAINE HYDROCHLORIDE WAS FOUND TO BE 105.9 mg/kg, ip.
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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13. EXERCISE
 Pentobarbital sodium Toxicity Data in Mice
Group Dose mg/kg, ip Log dose Dead/Total % Dead Corrected % Probit
1 30
2 45
3 50
4 65
5 75
BINAL PATEL, DEPARTMENT OF PHARMACOLOGY, SNLPCP, UMARAKH.
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