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ALTERNATIVE METHODS FOR LD50 STUDY

ALTERNATIVE METHODS FOR LD50 STUDY   :-

Objective:-

To give an idea about alternative methods of LD50 and how to reduce the use of animals in LD50 study as much as possible.   

LD50:- 

It means if we administer any dose of drug to animal group for experimental purpose  for the estimation of therapeutic effectiveness of that drug, and if 50% of animal get died than it means that particular dose of drug is lethal dose 50 (LD50).

The smaller the LD50 value, the more toxic the chemical is. The opposite is also true:                        the larger the LD50 value, the lower the toxicity. It was developed in 1920’s and called “classical LD50” involved 100 animals for 5 dose-groups, later  in 1981 it was modified by the Organization for Economic Co-operation and Development (OECD) and reduced number upto 30 for 3 dose-groups. In 1987 further reduced to 20 animals. Changing any of these variables (e.g., type animal or age) could result in finding a different LD50 value.¹

Toxicity tests :-

It examine toxic effects when a chemical is absorbed into the body, via mouth, skin,  lungs. The most common test of acute (short-term) toxicity is the LD50 test. Many different substances are tested in this way, including all drugs, agricultural chemicals, cleaners, and some cosmetics and their ingredients.²

Animals :- Mice, rats, rabbits, guinea pigs, cats, dogs, fish, monkeys and birds.

                    

                                     

                            Fig:- Shows use of animals in Europe in 2005.¹¹

Rout of administration :-

The LD50 values of a new drug are determined by various rout of administration (intravenous, intraperitoneal , subcutaneous and oral). ³

Methods:-

There are various methods to calculate LD50 values; like the graphical method, arithmetical method and statistical approach.

For research purpose, the most widely used method is Litchfield and Wilcoxson.

For routine practical class work ; Reed-Muench, Miller-Tainter and Karber’s method.

Procedure:-

For calculating LD50 by any one method, find out the least tolerated (smallest) dose (100% mortality) and most tolerated (highest) dose (0% mortality) by hit and trial method. Once these two doses are determined, select at least 5 doses in between the least tolerated and most tolerated doses, and observe mortality due to these doses. Then apply correction factor to 0% and 100% mortality group[for 0% dead = 100(0.25/n) and for 100% dead = 100x(n-0.25/n), where n = number of death]. The percentage mortality values are converted to probit values by reading the corresponding probit units from the probit table.

Plot the probit value against log doses and read LD50 value as the dose that corresponds to probit 5.⁴

Probit values: -

Bliss proposed transforming the percentage-killed into a "probability unit" (or "probit"). He defined it arbitrarily as equal to 0 for 0.0001 and 10 for 0.9999). He included a table to aid other researchers to convert their kill percentages to his probit, which they could then plot against the logarithm of the dose and thereby, it was hoped, obtain a more or less straight line. Such a so-called probit model is still important in toxicology, as well as other fields.

Table for calculation of probit value: ³

%	0	1	2	3	4	5	6	7	8	9
0		2.67	2.95	3.12	3.25	3.36	3.45	3.52	3.59	3.66
10	3.72	3.77	3.82	3.87	3.92	3.96	4.01	4.05	4.08	4.12
20	4.16	4.19	4.23	4.26	4.29	4.33	4.36	4.39	4.42	4.45
30	4.48	4.50	4.53	4.56	4.59	4.61	4.64	4.67	4.69	4.72
40	4.75	4.77	4.80	4.82	4.85	4.87	4.89	4.92	4.95	4.97
50	5.00	5.03	5.05	5.08	5.10	5.13	5.15	5.18	5.20	5.23
60	5.25	5.28	5.31	5.33	5.36	5.39	5.41	5.44	5.47	5.50
70	5.52	5.55	5.58	5.61	5.64	5.67	5.71	5.74	5.77	5.81
80	5.84	5.88	5.92	5.95	5.99	6.04	6.08	6.13	6.18	6.23
90	6.28	6.34	6.41	6.48	6.55	6.64	6.75	6.88	7.05	7.33

               

(1) Arithmetical method of Karber method ⁵

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.

LD50 = The apparent least dose lethal to all in a group – å (a ´ b) /N

Where N = number of animals in each group, a = dose difference and b = mean mortality.

(2) Graphical method of Miller and Tainter⁵

The observed percentage mortality was converted into probit referring to the probit table. The values thus obtained were plotted against log dose. The LD50 value and its standard error were determined from the graph, if the line was straight enough.

Table 1 :-  Comparison of different methods

	Method Karber 5	Method of Miller and Tainter 5	Method of Lorke 6
No. of rodents used	More than necessary	More than necessary	Appropriate
Expenditure	High	High	Average
Accuracy of results	Inaccurate	Inaccurate	Doubtful

Factors :-Which affects the results of LD50 are -

(1) Species, (2) Age, (3) Sex, (4) Amount of food, (5) Social environment, (6) Route of exposure* (oral, dermal, inhalation) and (7) Physical environment such as temperature and humidity.

*Rout of exposure (example, some LD50s for Dichlorvos, an insecticide commonly used in household pesticide strips) :-

• Oral LD50 (rat): 56 mg/kg
• Dermal LD50 (rat): 75 mg/kg
• Intraperitoneal LD50: (rat) 15 mg/kg
• Inhalation LC50 (rat): 1.7 ppm (15 mg/m3); 4-hour exposure

Limitations:- 

(1)The LD50 gives a measure of the immediate or acute toxicity.

(2)Results may vary greatly.

(3)LD50 is not tested on humans.

(4)All relation to humans are only a guess.

(5)The LD50 test is neither reliable nor useful. Because the human lethal dose can't be predicted from animal studies :

SUBSTANCE	HUMAN LETHAL DOSE	RAT   LD50	MOUSE LD50	RABBIT LD50
Lindane	840mg/kg	125mg/kg	-	130mg/kg
Caffeine	192mg/kg	192mg/kg	620mg/kg	-
Boric acid	640mg/kg	2660mg/kg	3450mg/kg	-
Amytal	43mg/kg	560mg/kg	-	575mg/kg

FRAME (Fund for the Replacement of Animals in Medical Experiment) believes that the lethal dose test is unnecessarily cruel and scientifically invalid. The test involves giving groups of animal doses of a test substance until it kills half of them.

Several countries, including the UK, have taken steps to ban the oral LD₅₀, and the Organization for Economic Co-operation and Development (OECD), the international governments’ advisory body abolished the requirement for the oral test in 2001.

European Union and by the USA, making the use of alternatives to the oral LD₅₀ test mandatory.

The deletion of the LD50 test from the OECD guidelines was due to three alternative methods being adopted which all involve more humane treatment of the animals and use fewer animals than the LD50 test.⁷

These three alternative tests are:

(1)   Fixed Dose Procedure (FDP) — OECD TG 420.

This method does not use death as an end point, instead it uses the observation of clear signs of toxicity developed at one of a series of fixed dose levels to estimate the LD50.⁷

(2)   Acute Toxic Class method (ATC) — OECD TG 423.

This method does not use death as the only end point, it also uses signs of toxicity in its stepwise approach to estimating the LD50.

Principle: - It is based on the Probit model.

Procedure: -

The ATC method is a sequential testing procedure using only three animals of one sex per step. Depending on the mortality rate three but never more than six animals are used per dose level. This approach results in the reduction of numbers of animals used in comparison to the LD₅₀ test by 40–70%.

The oral ATC method was adopted as an official test guideline by OECD in 1996 and was slightly amended in 2001. The ATC method has been successfully used in Germany and in 2003 >85% of all tests on acute oral toxicity testing was conducted as oral ATC tests. In member states of the European Union the ATC method is used in the range of 50% of all tests conducted.⁸

(3)   Up-and-Down Procedure (UDP) — OECD TG 425.

This method does still use death as an endpoint, but doses animals one at a time to see if the dose needs to be put up or down to achieve an estimate of the LD50 therefore giving the minimum number of animals a lethal dose of the test substance.⁷

 In the up-and-down procedure, animals are dosed one at a time. If an animal survives, the dose for the next animal is increased; if it dies, the dose is decreased. Each animal is observed for 1 or 2 days before dosing the next animal. Surviving animals monitored for delayed death for a total of 7 days. ⁹

Signs recorded during acute toxicity studies:-

These are increased motor activity, anaesthesia, tremors, arching and rolling, clonic convulsions, tonic extension, lacrimation, Straub reaction, salivation, muscle spasm, writhing, hyperesthesia, loss of righting reflex, depression, ataxia, stimulation, sedation, blanching, hypnosis, cyanosis and analgesia.²

Some drugs and their LD50 value:-

SUBSTANCE	ANIMAL, ROUTE	LD50
Sucrose (table sugar)	rat, oral	29,700 mg/kg
Vitamin C (ascorbic acid)	rat, oral	11,900 mg/kg
Grain alcohol (ethanol)	rat, oral	7,060 mg/kg
Table Salt	rat, oral	3,000 mg/kg
Paracetamol (acetaminophen)	rat, oral	1,944 mg/kg
Aspirin (acetylsalicylic acid)	rat, oral	200 mg/kg
Caffeine	rat, oral	192 mg/kg

Some other terminology:-

ID₅₀ (Median infective dose) :- It is the number of organisms received by a person or test animal qualified by the route of administration (e.g., 1,200 org/man per oral).

LCt₅₀ :- which relates to lethal dosage from exposure, where C is concentration and t is time. It is often expressed in terms of mg-min/m³.

ICt₅₀ :- It is the dose which will cause incapacitation rather than death. These measures are commonly used to indicate the comparative efficacy of chemical warfare agents, and dosages are typically qualified by rates of breathing (e.g., resting = 10 l/min) for inhalation, or degree of clothing for skin penetration.

ED50:-

It means the dose effective in producing certain expected response in 50% of the animal group. It helps in ascertaining the potency of a drug.

If the response is all or none, the ED50 value becomes LD50.

Both (LD50 and ED50) values are important for knowing the safety of a drug.

Therapeutic index: -

The ratio between LD50 and ED50 (LD50/ED50) represents therapeutic index. Greater the therapeutic index, safer is the drug. Ideally one would like to determine a dose that is effective in most of the animals (ED99) and least toxic to most of the animals of the group (LD10). A high therapeutic index is preferable to a low one: this corresponds to a situation in which one would have to take a much higher dose of a drug to reach the lethal threshold than the dose taken to elicit the therapeutic effect.

                                        Therapeutic ratio = LD50 / ED50                                  

Drugs with a narrow therapeutic range requires drug monitoring eg. Dimercaprol, Theophylline, Warfarin and Lithium carbonate. Some antibiotics require monitoring to balance efficacy with minimizing adverse effects, including: Gentamicin, Vancomycin, Amphotericin B, and Polymyxin B.¹⁰

REFERENCES :-

1. Botham, P.A. (2004). Acute systemic toxicity—prospects for tiered testing strategies. Toxicology in Vitro 18:227-230.  

2.Turner, R., Acute toxicity: The determination of LD50. In Screening Methods in Pharmacology, Academic Press, New York, 1965, pp.300.

3. Ghosh, M. N., Toxicity studies. In Fundamentals of Experimental Pharmacology, Scientific Book Agency, Calcutta, 1984, pp. 153–158.

4. Kulkarni S. K. ,“ Hand book of experimental pharmacology”, third edition,1999, Page no. 168-170.

5.Turner, R., Quantal responses. Calculation of ED50. In Screening Methods in Pharmacology, Academic Press, New York, 1965, pp. 61–63.

6. Lorke, D., A new approach to practical acute toxicity testing. Arch. Toxicol., 1983, 53, 275–289.

7. www.frame.org.uk

8. Eva Schlede, Elke Genschow, Horst Spielmann, Gisela Stropp and Detlev Kayser

 “Oral acute toxic class method: A successful alternative to the oral LD₅₀ test”, Received 19 August 2004.

9. Robert D. Bruce, “An Up-and-Down Procedure for Acute Toxicity Testing”, research article. 

10. www.merckvetmanual.org

11.  Fifth Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member States of the European Union Commission of the European Communities, published November 2007