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Hospice & Palliative Medicine International Journal

Research Article Volume 5 Issue 2

The diverse ways to determine experimental dose in animals 

Jyoti Arya,1 Renu Bist2

1Department of Zoology, Vaidik Balika PG College, Jaipur, India
2Department of Zoology, Centre of Advanced Studies, University of Rajasthan, Jaipur, India

Correspondence: Renu Bist, Associate Professor, Department of Zoology, Centre of Advanced Studies, University of Rajasthan, Jaipur, India, Tel 91 7424852622

Received: July 30, 2022 | Published: August 9, 2022

Citation: Bist R, Arya J. The diverse ways to determine experimental dose in animals. Hos Pal Med Int Jnl. 2022;5(2):21-24. DOI: 10.15406/hpmij.2022.05.00202

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Acute toxicity is manifested in terms of effects which occur after a single administration or a relatively brief exposure to a substance or mixture. The evidence for acute toxicity is usually obtained from animal testing so acute toxicity is usually characterised in terms of lethality and exposure times used in experimental protocols. Mainly two hazardous classes for acute toxicity are reported that are known as “Acute toxicity” and “STOT-SE (Specific Target Organ Toxicity-Single Exposure)”. This classification is based upon the evident lethality which is commonly reported as LD50/LC50 value. STOT-SE should be considered where there is clear evidence of toxicity to a specific organ, especially when it is observed in the absence of lethality. Once the LD50/LC50 value is determined, then acute toxicity estimate (ATE) could be decided therefore accurate measurement of LD50 is cardinal to understand the dose responses in animal experimentations. Current review entails a diverse method for determination of acute toxicity along with their merits and demerits. Also, it unfolds why three alternative methods i.e., fixed dose procedure (FDP), acute toxicity class (ATC) method and up and down method revealed a wide acceptance from scientific community even though the classical methods were present.

Keywords: LD50, acute toxicity, chronic toxicity, Karber’s method, LC50, ATE


Tests of toxicity are used to determine hazardous effects of a particular chemical which are is absorbed in our body by various routes such as through oral, cutaneous, inhalation and circulation. Acute (short-term) toxicity testing commonly done by LD50 test. Several drugs, agricultural chemicals, cleaners, some cosmetics and their ingredients are tested through this method.1 LD50 means that any dose of drug or chemical which is given to animal group for the estimation of medical effectiveness of that drug, and it gets 50% of animal’s death, then it means that particular dose of drug is lethal dose 50 (LD50). The hazzardicity is related inversely proportional to LD50 values of a drug means: less LD50 value, there will be more toxic effects and opposite is also true: the less toxicity at higher LD50 value. Initially it was developed in 1920’s and known as “classical LD50” in which involved 5 dose-groups were made for 100 animals but after some modifications in 1981 by the Organization for Economic Cooperation and Development (OECD) the experimental design has been changed as 30 animals for 3 dose-groups. In 1987, further number of experimental animals was reduced to 20 animals for LD50 determination.2

There are different types of toxicity classes based on their exposure duration viz acute toxicity (14 Days), sub-acute (repeated doses) toxicity (28 Days), sub-chronic toxicity (3 Months), chronic toxicity (6 Months to 2 Years) and special toxicity (Carcinogenicity). Prior approval by Institutional Animal Ethical Committee (IAEC) was required before conducting any kind of toxicity testing in animals or collecting any cell lines and a satisfactory protocol should be necessary by the local governing body of animal experimentation.

There are two basic values are important for toxicity determination first one is LD50 stands dose required for 50% mortality of test animals’ population. It is an index determination of medicine and poison’s virulence. Lower the LD50 dose, the more toxic the pesticide. The other one is LC50 value means concentrations of the chemical in air for 50% death of experimental animals during the observation period. Other durations of exposure (versus the traditional 4hours) may apply depending on specific laws. Higher LD50 value indicates the less toxic nature of substance whereas less LD50 or LC50 value reflects the higher toxicity.

The mission of Organization of Economic Cooperation and Development (OECD) is to promote policies that can serve the good socioeconomic health of people all over the world. It aims to work with governments to understand what drives economic, social and environmental change.3 It sets international standards on a wide range of things, from agriculture and tax to the safety of the chemicals.4 OECD strongly objected on the number of animals which were used in experiments of toxicity testing and mentioned it as a cruelty to have a large group for toxicity determination. So, in the perspectives of above-mentioned points, current review explains the classical methods which were globally used for toxicity determination namely, Karber’s method, Miller and Tainter method and Lorke’s5 method etc. The emphasis is

given upon three alternative methods which are widely accepted for acute toxicity determination and their merits.

Acute toxicity vs chronic toxicity

When a drug is administered at different dose levels that is it could be a single dose or in multiple doses for 24 h in two mammalian species (one non-rodent) it shows some short-term adverse effects, that are determined by acute toxicity. This type o toxicity only gives information about LD50, therapeutic index and the degree of safety of a pharmacological agent (Figure 1 & Table 1).6

Figure 1 A comparison of acute toxicity with chronic toxicity.



Attributes for short term and long-term exposure

 Acute toxicity

Chronic toxicity








Specific (where immune response is activated)



Neutrophils, Macrophages

Lymphocytes, plasma cells, macrophages, fibroblasts


Vascular changes

Active vasodilation, increased permeability

New vessel formation
(Granulation tissue)


Exudation and




Cardinal clinical





Generally absent
If present (Supportive and necrotizing inflammation)







Operative host

Plasma factors: complement, immunoglobulins, properdin, etc;
Neutrophils, nonimmune phagocytosis

Immune response,
Phagocytosis, repair



Fever, often high

Low-grade fever, weight loss,



Changes in

Neutrophil leucocytosis: (in viral infections)

Frequently none; variable leukocyte changes, increased plasma immunoglobulin

Table 1 A comparison of different traits of acute and chronic toxicity

Major acute toxicity symptoms

Acute toxicity is characterized by headache, nausea, vomiting, diarrhea, altered respiration, weight loss, muscle spasm, salvation, convulsion, loss of righting reflex, tremor lacrimation and somnolence(Table 2).

Toxicity category

Oral LD50(mg/kg)

Signal word

Approx. adult lethal dose(oral)




Few drops to 1 teaspoon




1 teaspoon to 1 ounce




1 ounce to 1 pound




More than 1 pound

Table 2 List of toxicity categories along with their specific LD50values and signal notation

Design of acute toxicity

It was developed in 1920’s and name was “classical LD50” in which 100 animals were taken for 5 dose-groups and later in 1981 some modifications were done by the Organization for Economic Co-operation and Development (OECD) and reduced number up to 30 animals for 3 dose-groups.

Methods to calculate LD50 values are - Litchfield and Wilcoxson, Reed-Muench, Miller-Tainter and Karber’s method. But large number of animals were required in all these methods and various factors like species, Age, Sex, Amount of food, social environment etc. affects the results of LD50. This type of toxicity evaluation method has some Limitations and results may vary greatly.

We should go to some other alternative methods in which minimum number of animals is required during toxicity testing methods to avoid sacrifice animals in excess. FRAME (Fund for the Replacement of Animals in Medical Experiment) believes that the lethal dose test is unnecessarily cruel and scientifically invalid. Several countries, including the UK, have taken steps to ban the oral LD50.6 The OECD, the international governments’ advisory body abolished the requirement for the oral test in 2001. Three alternative methods are used now namely Fixed Dose Procedure (FDP), Acute Toxic Class method (ATC), and Up-and-Down Procedure (UDP). In these methods, instead of animal death, only signs of toxicity were tested and recorded during studies like; increased motor activity, anaesthesia, tremors, arching and rolling. Alternative methods save numbers experimental animals.

A 14 days study

These studies include at least two species, one is rodent (mice/rat) and another is non-rodent (usually rabbit). Dose administration is orally and parenterally and groups of both sexes are treated by Various dose levels. Dose selection is such that causes less than 50% but not 0% and more than 50% but not 100% mortality. The main advantages of this study are as following:

  • Reproducible procedure.
  • Animal have less suffering.

Moderately toxic doses carry it in and it is expected that lethal can be avoided.

  • Few animals are used.

LD50 varies according to route

Route of exposure affects the dose of LD50. For example, some LD50 values are shown below along with their administration routes for dichlorvos which is a pesticide commonly used in household strips for eradication of pests:

Oral LD50 of dichlorvos in rat: 56 mg/kg

Dermal LD50 of dichlorvos in rat: 75 mg/kg

Intraperitoneal LD50 of dichlorvos in rat: 15 mg/kg

Inhalation LC50 of dichlorvos in rat: 1.7 ppm (15 mg/m3) in 4-hours exposure

Methods for the determination of LD50

Several methods have been used since a long period of time for ascertaining the LD50 values which is crucial for each particular compound in therapeutics. Few methods were developed earlier and used at a global scale but later on due to their practices of applying a large number of animals majority of them were discarded by scientific community and thus new alternative methods developed. All the methods try to find the least tolerated dose and most tolerated dose by hit and trial method. Once these two doses are determined, at least 5 doses are selected between them and a mortality rate is observed due to these doses. The percentage mortality values are converted to probit values by reading the corresponding probit units from the probit table. Finally, the probit values are plotted against log doses and LD50 value is read that corresponds to probit. Following are the few important methods which are of great significance in ascertaining LD50:

Karber's method

Miller and Tainter method

Lorke's method

Alternative methods

Fixed dose procedure (FDP)

Acute toxic class method (ATC)

UP and down Procedure (UDP)

Karber’s Method (Arithmetic method): The sum of the product was divided by the number of animals in a group and the resulting quotient was subtracted from the least lethal dose in order to obtain LD50 value.

L D 50 =L D 100 ( a×b n ) MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqkY=Mj0xXdbba91rFfpec8Eeeu0xXdbba9frFj0=OqFf ea0dXdd9vqaq=JfrVkFHe9pgea0dXdar=Jb9hs0dXdbPYxe9vr0=vr 0=vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaiaadYeacaWGeb WaaSbaaSqaaiaaiwdacaaIWaaabeaakiabg2da9iaadYeacaWGebWa aSbaaSqaaiaaigdacaaIWaGaaGimaaqabaGccqGHsisldaaeabqaam aabmaabaWaaSaaaeaacaWGHbGaey41aqRaamOyaaqaaiaad6gaaaaa caGLOaGaayzkaaaaleqabeqdcqGHris5aaaa@48CD@

Where, LD50 = Median lethal dose

LD100 = Least dose required to kill 100%

a = Dose difference

b = Mean mortality

n = Group population.

Graphical method of Miller-Tainter: The Miller-Tainter method is the standard use in getting LD 50.

The dose is plotted against the probit value. Based on the graph, the LD50 will be estimated.

The experiment demonstrates the determination of LD50 of neostigmine on the experimental animals and its comparison to the standard LD50 of neostigmine.

Neostigmine (0.1 mg/kg,0.2 mg /kg,0.4 mg/kg, 0.8 mg/kg, 1.6 mg/kg) i.p.

Toxicological information

Oral LD50 Mouse:7500mg/kg IV LD50 Mouse:0.3mg/kg

 SC LD50 Mouse:0.54mg/kg IM LD50 Mouse:0.395mg/kg

 IV LD50 Rat:0.315mg/kg SC LD50 Rat: 0.445mg/kg

 IM LD50 Rat:0.423mg/kg

Lorke’s method: The method given by Lorke comprises of an initial examination in which a total of nine subjects are allocated into three groups. Each group of three animals obtains a different dose of the study compound. This process has two stages which are known as phases 1 and 2, respectively.

Phase 1

This phase requires nine animals. The nine animals are divided into three groups of three animals each. Each group of animals are administered different doses (10, 100 and 1000 mg/kg) of test substance. The animals are Placed under observation for 24 hours to monitor their behavior as well as if mortality will occur.

Phase 2

This phase involves the use of three animals, which are distributed into three groups of one animal each. The animals are administered higher doses (1600, 2900 and 5000 mg/kg) of test substance and then observed for 24 hours for behavior as well as mortality. Then the LD50 is calculated by the formula:

D= Highest dose that gave no mortality,

D100 = Lowest dose that produced mortality


Some limitations are noticed for LD50 analysis such as, the LD50 can provide a measure of the instantaneous, immediate or acute toxicity, but the results may fluctuate greatly. LD50 cannot be tested on humans so all data reported in relation to humans is only a conjecture. The LD50 test is neither steadfast nor expedient, because of the fact that human lethal dose can't be anticipated from animal investigations.8,9

Alternative methods

Fixed dose procedure (FDP): This method was given by British Toxicology Society in 1992. It is a process which can evaluate a constituent's acute oral toxicity.10,11 When compared to the earlier LD50 test which was given in 1927, this method gives alike results while using lesser animals and instigating less pain and suffering.12 1992 this test was proposed as an alternative to the LD50 test by the Organisation for Economic Co-operation and Developmentunder OECD Test Guideline 420.13–15 This method does not use death as an end point, instead an observation of well-defined insignias of 5 toxicity are developed at one of the series of fixed dose levels to evaluation the LD50.

Acute toxic class method (ATC): The ATC method is a substitute to the LD50trial, when we intend to decline the number of experimental animals under test. The use of considerably scarcer number of animals is actually needed for the classification of substances proposed by the German Federal Health Authority, and it provides alternatives to the LD50 test for grading substances by their acute oral toxicity confirmed by animal tryouts and biometric determination12 in a national German13 and international cooperative study.16 The benefit of this method is that death is not used here as an end point, rather it uses symbols of toxicity in stepwise manner for determining the LD50. The principle of ATC method is that it is based on the Probit model.

The ATC method is a serial assay method which uses only three animals of one sex in each stage. Based on the mortality rate usually three animals but never farther than six animals are used per dose level. This methodology consequents in the decrease of numbers of animals utilized in comparison to 9, by 40–70%.17

Up and down procedure: In this unique method, experimental animals are administered with the test compound one at a time and survivability is checked. If it is found that an animal endures tested concentration of compound, the quantity for the next experiment is amplified. Whereas if the animal dies, the dose amount is usually declined for the next animal. The duration of observation for each animal is generally between 1 to 2 days before treating the next animal. Dose enduring animals are continuously monitored for delayed death for a total 10 of 7 day.18



Conflicts of interest

The authors declared no conflicts of interest.


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