ROLE OF THE LD50 TEST IN THE PHARMACEUTICAL INDUSTRY

555 for classifying the "toxicity" of a wide range of chemicals and for regulating their use and the exposure of the public to them (eg, in the control of industrial chemicals8). Increasing legal reliance on the LD50, and the demand for precise measurement of that value, has led to mandatory use of large numbers of animals to minimise statistical variability. In assessment of the effects of a single dose of a substance, it is more useful to know its general actions over a range of doses than just to have a seemingly precise estimate of one effect, lethality, quantified as the LD50. In an acute toxicity test a range of single

Memorandum ROLE OF THE LD50 TEST IN THE PHARMACEUTICAL INDUSTRY A. D. DAYAN

B. CLARK H. MORGAN

M. JACKSON

F. A. CHARLESWORTH Association

of the British Pharmaceutical Industry,

12

Whitehall,

London SW1

SOME measure of the potential toxic effect of a single dose of a medicine has become an accepted part of the information demanded by governments for the regulation of new medicines.’ The requirement, the extent of which differs widely between countries, has grown from practices common in the pharmaceutical industry as the development of new medicines has become a formalised process. It has been a regrettable consequence of the increasing regulation of drug development that certain tests, including the measurement of acute toxicity by determination of the LD50, now form a mandatory part of every application to license a new medicine in the UK. Determination of the "formal" LD50 is of limited importance in the development of medicines, and the pharmaceutical industry has long since largely adopted other methods for assessing acute toxicity.4 However, as a result of the interest focused by antivivisectionists the LD50 determination has become an emotive issue, which obscures the general need for acute toxicity studies within the industry. What are the LD50

test

and the Acute

Toxicity

Test?

The LD50 test, as originally defined by Trevan,5 was an elegant application of statistical principles to overcome the innate variability of measurements made by biological assay of substances (such as insulin and digitalis) that could not then be analysed chemically. He showed that, for any given end-point, the greatest accuracy of measurement was obtained by calculation of the dose at which the chosen effect occurred in 50% of the test population-hence designation of that end-point as the "lethal dose 50". The concept has since become widely used-eg, the effective dose in 50% of the population (ED50)-and disseminated throughout works on quantitative bioassay. The simplicity and apparent accuracy of the LD50 brought it into general use very quickly, particularly as increasingly rigorous mathematical techniques were devised for accurate ’calculation of the notional 50% and its variance.6,7 Finally, the LD50 became an inherent part of regulatory and industrial practice P. M. DUNN AND G. M. STIRRAT: REFERENCES Health Organisation. Definitions and recommendations. International Classification of Disease, 9th ed. Geneva: WHO, 1979: vol 1, 763-68. 2. Webster’s collegiate dictionary, 5th ed. London: G. Bell, 1938. 3. Williams G. Textbook of criminal law. London: Stevens, 1983: 291, 303. 4. Stewart AL, Reynolds EOR, Lipscomb AP. Outcome for infants of very low birthweight: Survey of the world literature. Lancet 1981; i: 1038. 5. Koppe JG. Evaluating intensive care of the very immature infant. Lancet 1983; ii: 49. 6. Dunn PM, Lashford A. In: South Western Regional Perinatal Survey, 1980-82, volI. 1. World

1983: 145-50. 7. Dunn PM. Definition of perinatal mortality. Lancet 1977; i: 1254-55. 8. International Federation of Gynecology and Obstetrics Standing Committee

on

Perinatal Mortality and Morbidity. Report following a workshop on monitoring and reporting perinatal mortality and morbidity. Heidelberg, 1982. 9. Kloosterman JG. Birthweight and maturity. WHO report of a seminar on the prevention of perinatal morbidity and mortality, Tours, April 22-26, 1969. WHO Public Health Papers no 42, 1972; 38. 10. Alberman E, Dennis KJ, eds. Late Abortion in England and Wales. RCOG Report of a national confidential study. London: Royal College of Obstetricians and Gynaecologists, 1984. 11. Gosden JR, Mitchell AR, Gosden CM, Rodeck CH, Morsman JM. Direct-vision chorion biopsy and chromosome-specific DNA probes for determination offetal sex in first trimester prenatal diagnosis. Lancet 1982; ii: 1416-19. 12. Robertson of Oakridge. Times Jan 19, 1984. 13. Munir AE. Perinatal rights. Med Sci Law 1984; 24: 31-40.

doses is administered to small numbers of animals and observations are made of the effects, at various times, sometimes including lethality. The numbers of animals required, the doses employed, and the observations made are all empirically adapted to the nature of the substance and the purpose for which the acutetoxicity test is being done, including foreknowledge of its biological activities and post hoc plans for other types of test. The numerical value of the "formal" LD50 is subject to wide variation arising from quite minor changes in experimental conditions. This has long been known to scientists, but it has taken a surprising time for it to be publicly expressed as a reason to distrust simple use of the LD50 for regulatory purposes.9 Recently, the LD50 has also become the focus of a highly emotional campaign against experiments in animals, which has confused the scientific, ethical, and political issues and has ignored the fact that acute toxicity can be evaluated without a formal, precise measurement of the LD50 using many animals. Smaller numbers of animals are commonly used for acute toxicity testing. One method is to dose just 2 animals at a time and to increase subsequent doses until an effect is produced or a predetermined limit is reached. Animals are followed to death or until a period (normally 14 has elapsed such that no new effect is expected. Many variants on this procedure exist (some known as the limit test), designed to limit the numbers of animals required to give an assessment of acute toxicity. The essential differences between the two types of technique are that the formal LD50 determination is a means of precise measurement of lethality under a defined set of experimental conditions, and an acute toxicity test is a more general method for evaluating acute effects, commonly including (but not limited to) lethality, and in which any required degree of statistical precision can be obtained by adjusting the numbers of animals employed. A formal LD50 may require 80-100 each of two rodent species, while an acute toxicity study will in general require fewer. A limit test would require 10-20 of a rodent species. The LD50 determination is one of a class of comprehensive rather than focused procedures more common in the biological than in the exact sciences, and has strong links with

days)

general pharmacological screening techniques.

II

Role of Acute Toxicity Tests in Drug Development The effects of various single high doses of medicines are examined for several scientific and clinical reasons, as well as to fulfil regulatory demands. i. As part of the preclinical exploration of the effects of a compound it will be administered to animals so that physiological variables can be measured, such as blood-pressure, heart rate, respiration, and autonomic responses. Any effects will be taken into account in considering whether and under what circumstances it should be administered to man. These tests of an essentially pharmacodynamic nature are conventionally done in a range of species depending on the compound and the knowledge of those

species. ii. It is also customary to conduct acute toxicity tests in rats and mice the lethal dose, or the effect up to a predetermined limit (eg, 2 g/kg). Animals are observed for 14 days, and any signs are noted. Any animals that die are examined at necropsy. Ifthree or more dose levels are administered it is possible to calculate an LD50 value. The degree of statistical precision depends on the number of animals and dose levels chosen. A "formal" LD50 is not normally necessary except for regulatory purposes and in most cases an adequate estimate of lethality will require few animals. It is necessary to know the circumstances, time-course, and nature of acute poisoning, particularly because of the risk of poisoning from overdosage, especially in children.12 This information is also useful in situations to assess

556 where exposure to the drug substance may be greater-for example, in the workplace. iii. To a more limited extent, knowledge of single dose phenomena can be helpful in defining the nature and dose levels of more prolonged toxicity tests, but these are also planned with the aid of more detailed knowledge of pharmacokinetics and the likely therapeutic dose. iv. Under certain circumstances (eg, for determining safe limits for impurities or assessing possible interactions between drugs simultaneously present in the body) it may be simplest and most economical in animals to evaluate combination effects in some form of acute toxicity test. v. For certain types of products it may still be necessary to conduct a single dose test to show freedom from abnormal toxicity on each batch manufactured. Vaccines, or other products the potency of which can only be checked in laboratory animals, may also require screening in acute studies. The use of such procedures is kept to the minimum but the need still remains in certain cases.

Clinical

,

of Acute Toxicity Data are Worthwhile? Those professionally concerned with drug development in the pharmaceutical industry have statedl3 that in general they see no need for estimation of the "formal LD50", with its requirement for large numbers of animals. Limited acute toxicity studies would satisfy scientific and practical needs, except in special instances, for example, where certain biological medicines are involved;’4see (v) above. Academic toxicologists share this view and support the use of some form of limited acute toxicity test.9 Regulatory authorities responsible for public safety have been slower to move, although there are now encouraging indications of changing attitudes in various countries. 15-17 What Sort

It appears therefore that, as far as new medicines are concerned, industry sees no general need for the LD50 value except in special circumstances, and some regulatory

authorities are moving away from their former reliance on it. It is hoped that this trend will continue so that there will be general acknowledgement that the "formal" LD50 normally has no part to play in development of new medicines, and that there will be greater regulatory flexibility in the assessment of acute toxicity in the future. REFERENCES

Janton C, Zbinden G, eds. Preclinical safety requirements in 1981. Institute of Toxicology, Swiss Federal Institute of Technology, and University of Zurich, 1981. Department of Health and Social Security. Notes on applications for product licences

1. Alder S, 2.

(medicines for human use), Medicines Act leaflets 3

2 and

4, 1981.

Proposal for a Council Recommendation concerning tests relating to the placing on the market of proprietary medicinal products. Annex I, Single dose toxicity. Off J Europ

Pharmacology

HAS CLINICAL PHARMACOLOGY LOST ITS WAY? GIANNI TOGNONI MAURIZIO BONATI

Laboratory of Clinical Pharmacology, Istituto di Ricerche "Mario Negri", Via Eritrea, 62-20157 Milan, Italy

Farmacologiche

SOME fifteen years ago, a technical group convened by the World Health Organisation declared that, in the assessment of drug efficacy and safety, the discipline of clinical pharmacology has a key role.’The group was made up of investigators who, in the space of a few years, had progressed far from basic research on drug disposition in animals and man, and were now pursuing kinetic information of direct clinical importance.2,3 In their view, the basic mandate of this new discipline was to improve care of patients by promotion of safer and more effective use of drugs. Ten years later, when clinical pharmacology had its first world conference, early uncertainties of identity had largely resolved, and most people recognised a clear distinction from, on the one hand, pharmacology and, on the other, clinical medicine.4 Nonetheless, amidst all the reaffirmations of a clearly defined role,5-9 there were lingering doubts. Was the pace being set too much by pharmacology, with its emphasis on biochemistry, in-vivo and in-vitro drug metabolism, and kinetics at the expense of the clinical side? If other disciplines were being called upon increasingly (as they were) to assess the value of therapeutic interventions, would clinical pharmacology retain any special function of its own? The world scene has certainly changed since the original meeting of the WHO group. On the minus side, we might put the decline in the rate of therapeutic innovation and the purported depressant effects of some licensing regulations; 10-13 on the plus side, perhaps, are the increasing emphasis being given to published work on drug evaluation and utilisation, and the demands from developing countries for information that will enable them to formulate comprehensive drug policies based on need.14,15 After an earlier exercise in which clinical relevance was shown not to be the main focus of most work on drug kinetics" despite its constant growth,17 we thought it might be useful, on the occasion of the second world conference,l8 to see how far clinical pharmacologists accept the original basic mandate of the discipline-improvement of patient care.

Commun 1980; C355: 6-29.

METHODS

4 Association of the British Pharmaceutical

Industry. Guidelines for preclinical and clinical testing of new medicinal products, parts 1 & 2. London: ABPI, 1977. 5. TrevanJW The error of determination of toxicity. Proc Roy Soc 1927; 101B: 483-514. 6. Burn JH, Finney DJ, Goodwin LG. Biological standardisation, 2nd ed. Oxford: Oxford University Press, 1950. 7. Finney DJ Probit analysis. London. Cambridge University Press, 1971 8. Council Directive amending for the sixth time Directive 67/548/EEC on approximation of laws, regulations and administrative provisions relating to the 9

10. 11

12.

13

14. 15.

classification, packaging and labelling of dangeroussubstances (79/831/EEC). Off J. Europ Commun 1979; L259: 10-28. Zbinden G, Flury-Roversi M. Significance of the LD50 test for toxicological evaluation of chemical substances. Arch Toxicol 1981; 47: 77-99. Lorke D. A new approach to practical acute toxicity. Arch Toxicol 1983; 54: 275-87. Zbinden G, Gross F, eds Pharmacological methods in toxicology. International encyclopedia of pharmacology and therapeutics, Section 102 1-612 Oxford. Pergamon Press, 1979. Lawson GR, Craft AJ, Jackson RH Changing patterns of poisoning in children in Newcastle, 1974-1981. Br Med J 1983, 287: 15. Association of the British Pharmaceutical Industry. Report on the LD50 test for the Advisory Committee on the Administration of the Cruelty to Animals Act 1876, London ABPI, 1977. British Pharmacopoeia, 1980, addenda, 1981, 1982 and 1983, London: HMSO, 1981.

Griffin JP. Discussion of paper by Zbinden and Flury9. Arch Toxicol 1981; 49: 99-103.

16. Bass

R, Gunzel P, Henschler D, et al. LD50 versus toxicity Critical assessment of the Arch Toxicol 1982; 51: 183-86. 17. Le Beau JE. The role of the LD50 determination in drug safety evaluation. Reg Toxicol Pharmacol 1983; 3: 71

methodology currently in use.

To assess the priority given to clinical relevance we secured three kinds of information. Firstly, we asked selected groups what they were actually doing. Recipients of our questionnaire were those investigators unanimously indicated, in the response to our preliminary inquiry to members of the International Union of Pharmacology (IUPHAR) board of clinical pharmacology, as representative spokesmen of the discipline. Secondly, we tried to assess the extent to which clinical pharmacology journals concern themselves with the therapeutic use of drugs-what are the topics, and who writes about them?-and how far clinical pharmacologists are contributing the critical discoveries and key recommendations about drugs in clinical medicine. This we did by examining the content of four major clinical pharmacology journals and four general medical journals for the years 1980, 1981, and 1982. Thirdly, we tried to see how far clinical pharmacologists had contributed to the important debates, over the past five years, on efficacy and iatrogenic ill-effects. To this purpose, we examined the information available on "model cases" proposed, at our request, by the editors of two leading publications, Drzrgs and Side Effects

Anniial.