THE MEASUREMENT OF LD50

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• THE MEASUREMENT OF LD50

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Definition

• An LD50 represents the individual dose required
  to kill 50 percent of a population of test
  animals. It is an index determination of medicine
  and poisons virulence. The lower the LD50 dose,
  the more toxic the pesticide.

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LD50 Median lethal dose

• LD50 (median lethal dose)
• The dose of a drug that produces death in 50 of
  the animal population tested

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Drug variability and toxicity assessment

• ED50 Effective dose for 50 of subjects
• LD50 Lethal dose for 50 of subjects
• The therapeutic index
• TI LD50 / ED50
• No drug is 100 safe

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Definition

• Historically, the LD50 was determined with a high
  degree of precision and was used to compare
  toxicities of compounds relative that high
  precision is not necessary to compare toxities.
  Therefore, the median lethal dose is now
  estimated from the smallest number of animals
  possible.

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Definition

• Toxicity test can be divided into three parts
  acute toxicity testing, chronic toxicity testing,
  and special testing .

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Definition

• Acute toxicity testing single dosage everyday,
  to observe its toxic reaction after 7 days and 14
  days.

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• Acute toxicity test that is lethal in
  approximately 50 of animals. Determine maximum
  toleterated closes. Usually two species, two
  routes, single close (compare with therapeutic
  dose).

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• Chronic toxicity test 1 to 2 years. Required
  when drug is intended to be used in humans for
  prolonged periods. Usually run concurrently with
  clinical trial. Goals of chronic tests are to
  show which organs are susceptible to drug
  toxicity. Clinical chemistry physiologic signs,
  autopsy studies, hematology, histology, electron
  microscopy studies. Identify target organ of
  toxicity.

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Experimental purpose

• To grasp the basic metered method of LD50 and the
  suitable condition of the Karbar's method.
• To be familiar with the general group method for
  animals.
• To comprehend the deduction procedure of the
  Karbar's method.

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Experimental method

• Karbar's method
• There are many methods in determining LD50 such
  as Bliss, sequential method, grouping method ect.
  The most classic and precise method is Bliss
  method which is used in this experiment.But it
  needs to use computer and corresponding software.
  It is more complicated,so we often use Karbar's
  method.

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Experimental condition

• The dose must be increased with geometric
  proportion series. (The logarithm of Dose with
  arithmetic series)
• The number of every group must be equal.
• The response should satisfy the Gaussian
  distribution roughly.

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Experimental animals

• mice

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Experimental drugs

• the different concentration of Dipterex 2,
  4,6-trinitrophenol

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• Dipterex

It can irreversibly inhibits AChE
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Mechanism of intoxication

• Organophosphates are irre-versible
  anticholinesterase drugs. Their phosphor atom
  combines with AChE by covalent bond and forms
  phosphated AChE that is uneasy to be hydrolyzed.
• The activity of AChE diminishes markedly and the
  concentration of ACh increases.
• Administering as soon as possible in order to
  prevent enzyme from aging.

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Experimental apparatus

• mice cage, animals equi-armbalance, 1ml
  injection syring, and electronic calculator

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Experimental procedure

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How to choose and divide the experimental animals

• Principle of choice We should choose the animals
  sensitive to the trial agents. Considering about
  source, economic value, convenient handling to
  the animals, we often choose mice for our
  experiment.
• Principle of group The number of every group
  should be more than the number of groups. We
  divide the animals into 7 groups in this
  experiment, and there are 10 animals in each
  group.

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Group

• How to group animals Principal We divide these
  animals into different groups according to their
  weight this time, and then group them in random.
  Their weight of each group must be almost the
  same, and the different of the sum of weight in
  each group must be less than 5g
• How to group students Divide the students into
  10 groups in order to decrease errors which may
  be caused by handling of different classmates.

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Dosage

• Administration route intraperitoneal injection
  method.
• The concrete method as follows raise mouses
  tail root with the right hand, slip to cervical
  part from back to the head with left hand, knead
  its scalp with thumb and forefinger, turn over it
  and fix its tail, hold the injection with the
  right hand, prick to left intraperitoneal
  (trumpet) and pumpback to make sure that there is
  not the blood.
• We should thrust and pluck quickly when inject.

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Observe the death number and calculate the death
rate

• observing time one hour
• fill the death rate and data in the table

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Calculation formula should be applied by these
conditions

• When the death rate(P n)of the minimum dosage
  equals to 0 and the death rate(P m)of the
  maximal dosage equals to 100, we use the basic
  formulas.
• When the P n less than 20 or the P m more than
  80, we use the correction formula.
• When the P n more than 20 or the P m less than
  80, we can not use any formula.

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Basic formula

• ( P n 0 and P m 100 )
• LogLD50XK-d (?p-0.5)
• quorum XKdeath rate 100logarithmic
  dose
• d grouping interval
• ?pthe sum of all groups

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Corrected formula

• ( P nlt20 or P mgt80 )
• LogLD50 XK d (?p - )
• LD50Log-1 LogLD50

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95 confidence level LD504.5S Log LD50 LD50

• S Log LD50 d
• S Log LD50 the standard error of Log LD50
• p the every groups death rate
• n the animals number of every group

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Attention

• All of us should give drugs at the same time.
• Dipterex can volatilize easily, so we should be
  attention to cover bottle at any time, and dont
  touch it with your hand directly.
• Use the right way of intraperitoneal injection to
  the mice in order to avoid being griped by the
  mice.

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Questions

• How to group the mice and the students?
• Whats the symptom of the mice when they are
  injected with metrifonate?
• Whats the meaning of the 95 confidence level?
• How to prepare the drugs?

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